Toxicologic Pathology Forum: Tissue Evaluation in Nonclinical Toxicity Studies for Prophylactic Vaccines.

Nonclinical toxicity testing (GLP) of prophylactic vaccines to support human clinical trials is outlined in the World Health Organization nonclinical vaccine-development guidelines, which are followed by most regulatory agencies globally. Vaccine GLP toxicity studies include at least two groups: a buffer control (often phosphate-buffered saline) group and a highest anticipated clinical dose formulation group. However, studies may include additional groups, including lower-dose formulation groups and adjuvant-containing formulation control groups. World Health Organization guidelines touch upon expectations for dose group and tissue selection for microscopic evaluation, but there is variation in the interpretation of this aspect of these guidelines between vaccine developers. This opinion piece proposes a scientifically based approach for defining appropriate groups to evaluate in the dosing and recovery phases in nonclinical vaccine toxicity studies, as well as suggestions on selecting tissues for microscopic evaluation at the recovery phase of studies to promote alignment between vaccine manufacturers.

Comparison of ANP and BNP Granular Density in Atria of Rats After Physiological and Pathological Hypertrophy.

Atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP) are cardiac hormones located in atria granules. Both peptides respond to cardiac pressure and volume dynamics and accordingly serve as translation biomarkers for the clinical treatment of heart failure. Serum ANP and BNP play central secretary roles in blood pressure and cardiac output regulation and have proven utility as differential biomarkers of cardiovascular proficiency and drug-induced maladaptation, yet both peptides are impervious to exercise-induced hypertrophy. We employed immunoelectron microscopy to examine the effects of 28 days of chronic swim exercise or administration of a PPARγ agonist on atrial granules and their stored natriuretic peptides in Sprague Dawley rats. Chronic swimming and drug treatment both resulted in a 15% increase in heart weight compared with controls, with no treatment effects on perinuclear granule area in the left atria (LAs). Drug treatment resulted in larger size granules with greater BNP density in the right atria. Comparing swimming and PPARγ agonist treatment effects on ANP:BNP granule density ratios between atrial chambers revealed a shift toward a greater proportion of ANP than BNP in LAs of swim-trained rats. These data suggest a distinction in the population of ANP and BNP after chronic swim or PPARγ that makes it a novel metric for the differentiation of pathological and physiological hypertrophy.

Postnatal Hepatobiliary and Gastrointestinal Systems Development and Impact on ADME.

Toxicity can result from variable target organ sensitivity and exposure based on postnatal development. Changes in the gastrointestinal tract (GIT) in neonates are driven by initial enteral feedings. These are important for nutrient uptake as well as drug disposition and include motility, expansion of enzyme and transporter function, permeability, intestinal microbiome, and species-specific maturation. Some aspects of GIT function do not mature until driven by increased dietary complexity. As with the GIT, postnatal hepatic maturation in the rat includes a variety of anatomic and functional changes that include refinements in the activities or expression of drug transporters and drug-metabolizing enzymes. These changes may impact rodent pharmacokinetics, nonclinical toxicity profiles, and estimation of safe pediatric doses. Pilot or dose range finding studies can help characterize and mitigate toxicity related to drug disposition, especially in juvenile rodents. Interpretation of developmental toxicity requires knowledge of developing systems in humans and nonclinical models.

Modeling human tumor-immune environments in vivo for the preclinical assessment of immunotherapies.

Despite the significant contributions of immunocompetent mouse models to the development and assessment of cancer immunotherapies, they inadequately represent the genetic and biological complexity of corresponding human cancers. Immunocompromised mice reconstituted with a human immune system (HIS) and engrafted with patient-derived tumor xenografts are a promising novel preclinical model for the study of human tumor-immune interactions. Whilst overcoming limitations of immunocompetent models, HIS-tumor models often rely on reconstitution with allogeneic immune cells, making it difficult to distinguish between anti-tumor and alloantigen responses. Models that comprise of autologous human tumor and human immune cells provide a platform that is more representative of the patient immune-tumor interaction. However, limited access to autologous tissues, short experimental windows, and poor retention of tumor microenvironment and tumor infiltrating lymphocyte components are major challenges affecting the establishment and application of autologous models. This review outlines existing preclinical murine models for the study of immuno-oncology, and highlights innovations that can be applied to improve the feasibility and efficacy of autologous models.

Opinion on the Use of Animal Models in Nonclinical Safety Assessment: Pros and Cons.

Nonclinical evaluation of human safety risks for new chemical entities (NCEs) is primarily conducted in conventional healthy animals (CHAs); however, in certain instances, animal models of diseases (AMDs) can play a critical role in the understanding of human health risks. Animal models of diseases may be especially important when there is a need to understand how disease conditions associated with the intended indication might impact risk assessment of NCEs or when CHAs lack the human-specific target of interest (receptor, etc). Although AMDs have potential benefits over CHAs, they also have limitations. Understanding these limitations and optimizing the AMDs of interest should be done prior to proceeding with studies that will guide development of NCE. The purpose of this manuscript is to provide an overview of the major pros and cons of utilization of AMDs in nonclinical safety assessment.

Epithelial Alteration Associated With Recovery From Laryngeal Squamous Metaplasia in Rats: Kinetics of Recovery From a Test Item-Related Change.

Squamous metaplasia is a nonspecific adaptive response to chronic irritation in the larynx and is often diagnosed as a test item-related change in rat inhalation studies. Investigating scientists are frequently asked to assess the adversity of laryngeal squamous metaplasia and to interpret its relevance to human risk. One factor in predicting relevance to human risk is the kinetics (degree and speed) of recovery following the cessation of exposure to the test item. Most reports describing recovery from squamous metaplasia in the rat larynx discuss the more severe end of the spectrum of metaplastic change (moderate to severe) and include relatively long (6 weeks or more) recovery periods. We conducted 2 studies to evaluate the toxicity and recovery from any potential effects of 4-(Chloro-2-methylphenoxy) butyric (MCPB) acid, a herbicide, when administered by inhalation to young male Sprague Dawley rats for 3 to 4 weeks. The studies resulted in minimal to moderate laryngeal squamous metaplasia for which we describe the kinetics of recovery over 1 to 4 weeks. We found that the microscopic change epithelial alteration, which is normally considered to be a precursor in the development of squamous metaplasia, can occur as a transitional stage between squamous and normal epithelium during recovery.

Perspective on Adversity in Toxicology Evaluations.

Determining adversity of effects in toxicology studies continues to pose a dilemma to practicing toxicologists and pathologists. How this determination is made may follow either a focused or broad approach to assessing the study data. The choice of which approach is best is dependent on a variety of factors. Therefore, we present a philosophical perspective on the determination of adversity across toxicology studies that may be applied in inhalation studies and those conducted by other routes of exposure.

Nonclinical Safety Assessment of FHTR2163, An Antigen-Binding Fragment Against HTRA1 for the Treatment of Geographic Atrophy.

FHTR2163 is an antigen-binding fragment of a humanized immunoglobulin G1 monoclonal antibody directed against high-temperature requirement A serine peptidase 1 (HTRA1) that is being developed as a potential intravitreal (ITV) treatment for patients with geographic atrophy (GA), an advanced form of dry age-related macular degeneration. The nonclinical toxicology program was designed to assess the safety and tolerability of HTRA1 inhibition following ITV administration of FHTR2163 to support ITV administration in patients with GA. FHTR2163 was well tolerated in a single-dose ITV-administered 8-day toxicity study in cynomolgus monkeys following a 50 µL high (>700 mOsm/kg) osmolality formulation up to 12.5 mg/eye; however, 100 µL (2× 50 µL injections) of a high-osmolality formulation resulted in transient retinal detachment. Repeat-dose ITV administration every 2 weeks of FHTR2163 was well tolerated in 8- and 26-week studies with ITV injection of 100 µL (2× 50 µL) of iso-osmolar formulation up to 15 mg/eye, or 50 µL of the high-osmolality formulation up to 12.5 mg/eye. Observed transient and reversible ocular effects included inflammation and perivascular infiltrates, consistent with an immune response attributed to the administration of heterologous (humanized) protein. Overall, FHTR2163 was well tolerated, and the nonclinical package supported the continued clinical development of FHTR2163 in patients with GA.

Recent Advancements and Applications of Human Immune System Mice in Preclinical Immuno-Oncology.

Significant advances in immunotherapies have resulted in the increasing need of predictive preclinical models to improve immunotherapeutic drug development, treatment combination, and to prevent or minimize toxicity in clinical trials. Immunodeficient mice reconstituted with human immune system (HIS), termed humanized mice or HIS mice, permit detailed analysis of human immune biology, development, and function. Although this model constitutes a great translational model, some aspects need to be improved as the incomplete engraftment of immune cells, graft versus host disease and the lack of human cytokines and growth factors. In this review, we discuss current HIS platforms, their pathology, and recent advances in their development to improve the quality of human immune cell reconstitution. We also highlight new technologies that can be used to better understand these models and how improved characterization is needed for their application in immuno-oncology safety, efficacy, and new modalities therapy development.

Toxicologic Pathology Forum*: Commentary on "Opinion on Designation of Adverse and Nonadverse Histopathological Findings in Toxicity Studies: The Pathologist's Dilemma".

In the article "Opinion on Designation of Adverse and Nonadverse Histopathological Findings in Toxicity Studies: The Pathologist's Dilemma," the authors Gopinath and Mowat provide a framework for designation of adversity supplemented with photomicrographic examples. Given that adversity designation can significantly impact the no observed adverse effect level and clinical trial design, it is important to carefully consider all of the criteria by which such assignments are made. We highlight some of the specific assertions within the article that could benefit from a more detailed discussion. Our primary criticism surrounds the authors' primary reliance on histopathology in isolation for adversity designation, which in our opinion provides an overly simplified depiction of the process. We provide additional perspective on how context beyond histopathology often plays a critical role in adversity designation and highlight areas where inclusion of some of these scenarios would have provided the reader a more realistic view of the complex process of assigning adversity. * This is an opinion article submitted to the Toxicologic Pathology Forum. It represents the views of the authors. It does not constitute an official position of the Society of Toxicologic Pathology, British Society of Toxicological Pathology, or European Society of Toxicologic Pathology, and the views expressed might not reflect the best practices recommended by these Societies. This article should not be construed to represent the policies, positions, or opinions of their respective organizations, employers, or regulatory agencies.

Translational Research: The Cornerstone for Medical Technology Advancement.

Significant improvements in quality of life and mortality have occurred over the last century due to the giant advancements in medical innovation. Medical innovation continues to move forward, and it is expanding to areas never explored before. In particular, the advancement in big data analytics is now enabling the rapid progress in the understanding of gene influence in human diseases. The progress in medical innovation achieved until today is significant; however, the potential that future technologies have to modify patterns of disease thought to be incurable is mind-boggling. In the present issue of Toxicologic Pathology, a wide variety of devices and validation platforms are presented as a clear evidence of the multidisciplinary approach that is necessary for the progress of this field. As a clinician, scientist, and medical device innovator, I am confident that this special issue dedicated to the "Pathology of Medical Devices" will be of great value to the scientific and medical device innovation community.

Toxicologic Pathology Forum: Current Status on the Use of Animal Models of Human Disease in the Pharmaceutical Industry in Japan in Nonclinical Safety Assessment-Opinion Paper.

In nonclinical safety studies for new drug development, healthy animals have been commonly used. However, in some cases, the use of animal models of human disease is considered to be more favorable in evaluating risks in patients. To elucidate the current status of the use of animal models for nonclinical safety assessment, an internal questionnaire from the Japan Pharmaceutical Manufacturers Association and surveys (questionnaire period: August 27 to September 30, 2015) of both common technical documents and review reports of approved drugs (approval period: May 1999 to May 2017) disclosed by the Pharmaceutical and Medical Devices Agency were conducted. Although there were some concerns and limitations raised, the survey results revealed that animal models have been used in nonclinical safety assessment on a case-by-case basis and that nonclinical safety studies using animal models were included in the data packages of several approved drugs in Japan. The survey results also revealed that nonclinical safety studies using animal models have become more frequent in the past few years. In almost all cases, useful information, such as signs of toxicity under disease conditions and mechanisms of toxic change, was obtained from the results of nonclinical studies using animal models. Note: This is an opinion article submitted to the Toxicologic Pathology Forum. It represents the views of the author(s). It does not constitute an official position of the Society of Toxicologic Pathology, British Society of Toxicological Pathology, or European Society of Toxicologic Pathology, and the views expressed might not reflect the best practices recommended by these Societies. This article should not be construed to represent the policies, positions, or opinions of their respective organizations, employers, or regulatory agencies.

Refinement, Reduction, and Replacement (3R) Strategies in Preclinical Testing of Medical Devices.

The U.S. Food and Drug Administration Center for Devices and Radiological Health (FDA/CDRH) has recently published several in vivo test guidance documents that mention refinements, reductions, or replacement animal testing strategies to facilitate the leveraging of data from large animal safety tests for conventional rodent testing. In response to the recently enacted Food and Drug Administration Safety and Innovation Act Section 907, which facilitates expedited access to novel therapies commonly described as Breakthrough Therapy Designation, FDA/CDRH has discussed efficient regulatory strategies for first-in-human investigation, including early feasibility study guidance. Large gains in humane care and translational research could also be attained by examples in FDA's Guidance for the Use of International Organization for Standardization 10993-1, which states that large animal safety studies may be considered as replacement rodent tests if the scientific principles, methods, and end points (SPME) are considered and applied. This article discusses SPME for the replacement of conventional rodent testing by the inclusion and integration of clinical, diagnostic, and pathologic data obtained from well-designed large animal studies. The recommendations include consideration for study designs that utilize methods for an overall more comprehensive interrogation of animal systems.

The Emergence of Microphysiological Systems (Organs-on-chips) as Paradigm-changing Tools for Toxicologic Pathology.

Microphysiological systems (MPS), commonly known as organs-on-chips, are a rapidly advancing technology that promises to impact many areas of medical and toxicological pathology. In this minireview, the history of MPS and its potential utility in safety assessment are described with the toxicologic pathologist in mind. Several MPS development focus areas are defined, and recent progress in the area is highlighted. MPS will likely become an important tool for the toxicologic pathologist as part of our role in the safety assessment process within the pharmaceutical, biotechnology, medical device, and cosmetic and agrichemical industries.

The Nonclinical Safety Profile of GalNAc-conjugated RNAi Therapeutics in Subacute Studies.

Short interfering RNAs (siRNAs) and antisense oligonucleotides (ASOs) are the most clinically advanced oligonucleotide-based platforms. A number of N-acetylgalactosamine (GalNAc)-conjugated siRNAs (GalNAc-siRNAs), also referred to as RNA interference (RNAi) therapeutics, are currently in various stages of development, though none is yet approved. While the safety of ASOs has been the subject of extensive review, the nonclinical safety profiles of GalNAc-siRNAs have not been reported. With the exception of sequence differences that confer target RNA specificity, GalNAc-siRNAs are largely chemically uniform, containing limited number of phosphorothioate linkages, and 2'-O-methyl and 2'-deoxy-2'-fluoro ribose modifications. Here, we present the outcomes of short-term (3-5 week) rat and monkey weekly repeat-dose toxicology studies of six Enhanced Stabilization Chemistry GalNAc-siRNAs currently in clinical development. In nonclinical studies at supratherapeutic doses, these molecules share similar safety signals, with histologic findings in the organ of pharmacodynamic effect (liver), the organ of elimination (kidney), and the reticuloendothelial system (lymph nodes). The majority of these changes are nonadverse, partially to completely reversible, correlate well with pharmacokinetic parameters and tissue distribution, and often reflect drug accumulation. Furthermore, all GalNAc-siRNAs tested to date have been negative in genotoxicity and safety pharmacology studies.

Pathogenesis of Renal Injury and Gene Expression Changes in the Male CD-1 Mouse Associated with Exposure to Empagliflozin.

An increased incidence of renal tubular adenomas and carcinomas was identified in the 2-year CD-1 mouse carcinogenicity study with empagliflozin (sodium-glucose transporter 2 inhibitor) in high dose (1,000 mg/kg/day) male mice. A 13-week mouse renal investigative pathogenesis study was conducted with empagliflozin to evaluate dose dependency and temporal onset of nonneoplastic degenerative/regenerative renal tubular and molecular (genes, pathways) changes which precede neoplasia. Male and female CD-1 mice were given daily oral doses of 0, 100, 300, or 1,000 mg/kg/day (corresponding carcinogenicity study dose levels) for 1, 2, 4, 8, or 13 weeks. The maximum expected pharmacology with secondary osmotic diuresis was observed by week 1 at ≥100 mg/kg/day in both genders. Histopathologic kidney changes were first detected after 4 weeks of dosing in the male 1,000 mg/kg/day dose group, with progressive increases in the incidence and/or number of findings in this dose group so that they were more readily detected during weeks 8 and 13. Changes detected starting on week 4 consisted of minimal single-cell necrosis and minimal increases in mitotic figures. These changes persisted at an increased incidence at weeks 8 and 13 and were accompanied by minimal to mild tubular epithelial karyomegaly, minimal proximal convoluted tubular epithelial cell hyperplasia, and a corresponding increase in Ki-67-positive nuclei in epithelial cells of the proximal convoluted tubules. There were no corresponding changes in serum chemistry or urinalysis parameters indicative of any physiologically meaningful effect on renal function and thus these findings were not considered to be adverse. Similar changes were not identified in lower-dose groups in males nor were they present in females of any dose group. RNA-sequencing analysis revealed male mouse-specific changes in kidney over 13 weeks of dosing at 1,000 mg/kg/day. Treatment-related changes included genes and pathways related to p53-regulated cell cycle and proliferation, transforming growth factor ß, oxidative stress, and renal injury and the number of genes with significant expression change dramatically increased at week 13. These treatment-related changes in genes and pathways were predominant in high-dose males and complemented the observed temporal renal tubular changes. Overall, these mouse investigative study results support the role of early empagliflozin-related degenerative/regenerative changes only observed in high-dose male CD-1 mice as a key contributing feature to a nongenotoxic mode of renal tumor pathogenesis.

Göttingen Minipigs in Ocular Research.

Since their development in the 1960s, Göttingen minipigs have become a popular nonrodent animal model in biomedical research, especially for ocular studies, because of their ease of handling, size, well-monitored genetics, and ocular anatomy that is similar to humans. The purpose of this mini-review is to introduce the reader to the various ways in which this animal model is currently being utilized in ocular research and to provide an overview of the diagnostic modalities that aid in this research. To date, the Göttingen minipig has been used for a variety of ocular research endeavors, including studies evaluating the safety and efficacy of ocular therapeutics, glaucoma etiopathogenesis and treatment, novel biomaterials, surgical procedures, and implantable materials/devices. In addition to histopathologic evaluation of enucleated globes, the majority of these studies make use of advanced in vivo diagnostic techniques, including electroretinography, optical coherence tomography, fundoscopic imaging, and fluorescein angiography.

High Background Incidence of Spontaneous Subcapsular Adrenal Gland Hyperplasia of Tg.rasH2 Mice Used in 26-week Carcinogenicity Studies.

The Tg.rasH2 model was accepted by regulatory agencies worldwide for 26-week carcinogenicity assays as an alternative to the standard 2-year assays in conventional mice in 2003. Several references documenting spontaneous nonneoplastic findings and incidences of spontaneous tumors in the Tg.rasH2 mice have been published. The purpose of this publication is to add adrenal gland subcapsular hyperplasia to the database pertaining to spontaneous lesions noted in Tg.rasH2 mice, review physiology related to this finding, and discuss its significance. The incidence of spontaneous subcapsular adrenal gland hyperplasia was determined in control Tg.rasH2 mice from nine 26-week carcinogenicity studies carried out within the last 5 years at two contract research organizations. Incidence of this finding ranged from 56% to 79% in males and 88% to 100% in females, with an incidence average of 62% in males and 93% in females. Adrenal gland subcapsular hyperplasia is a common finding in male and female Tg.rasH2 mice that did not progress to neoplasia in Tg.rasH2 mice. In general, it tends to be more frequent and severe in females in comparison to males.

Middle Ear Histopathology Following Magnetic Delivery to the Cochlea of Prednisolone-loaded Iron Oxide Nanoparticles in Rats.

Delivery of therapy to the cochlea is a challenge and limits the efficacy of therapies meant to treat hearing loss, reverse tinnitus, and protect hearing from chemotherapy regimens. Magnetic injection is a technique that uses magnetic fields to inject nanoparticles from the middle ear into the cochlea, where they can then elute therapy to treat hearing disorders. To evaluate the safety of this treatment in the middle ear, 30 rats were subdivided into 6 groups and treated by single or multiple intratympanic injections of saline, prednisolone, nanoparticles, or nanoparticles loaded with prednisolone. A specially designed magnet array was used to magnetically inject the particles from the middle ear to the cochlea. Treatment began at study day 0, and animals were euthanized on study day 2, 30, or 90. Temporal bones were collected and prepared for histopathological examination. Intratympanic administration of magnetic nanoparticles and/or prednisolone resulted in minimal to mild inflammatory changes in all treated groups. The incidence and severity of the inflammatory changes observed appeared slightly increased in animals administered nanoparticles, with or without prednisolone, when compared to animals administered prednisolone alone. At study day 90, there was partial reversibility of the findings noted at study day 2 and 30. Repeat administration did not appear to cause greater inflammatory changes.

Regulatory Forum.

Revision of the International Council for Harmonization (ICH) S1 guidance for rat carcinogenicity studies to be more selective of compounds requiring a 2-year rat carcinogenicity study has been proposed following extensive evaluation of rat carcinogenicity and chronic toxicity studies by industry and drug regulatory authorities. To inform the ICH S1 expert working group in their potential revision of ICH S1, a prospective evaluation study was initiated in 2013, in which sponsors would assess the pharmacologic and toxicologic findings present in the chronic toxicity studies and predict a positive or negative carcinogenicity outcome using a weight of evidence argument (a carcinogenicity assessment document [CAD]). The Scientific and Regulatory Policy Committee was asked by the Society of Toxicology Pathology (STP) executive committee to track these changes with ICH S1 and inform the STP membership of status changes. This commentary is intended to provide a brief summary of recent changes to the CAD guidance and highlight the importance of STP membership participation in the process of CAD submissions.