Search

The delivery of biotherapeutic molecules (antibodies, proteins, peptides) and nucleic acids via the respiratory route has presented challenges for regulatory approval, due in part to a lack of understanding of the expected pathology, mechanisms of toxicity, and immunogenicity induced by the inhalation route. Although the first inhaled biotherapeutic was approved some time ago (Dornase Alfa, Pulmozyme; Genetech, 1993), no other inhaled biotherapeutics have been marketed for the treatment of human disease other than the inhaled insulins (Exubera; Pfizer, 2006 and Afrezza; Mannkind Corporation, 2014). As a result, scientific knowledge within the toxicologic pathology community is fragmented with precious little publicly available data. Therefore, one of the aims of this special edition was to generate a collection of manuscripts that pathologists and toxicologists could refer in order to understand the pathology, mechanisms of toxicity, immunogenicity, and challenges associated with the development of inhaled biotherapeutics.

In animal studies, light microscopic examination remains an important tool in the detection and characterization of effects on the skeleton. In the case of both anticipated and unanticipated effects on bone histology, pathologists must carefully select terminology that accurately conveys skeletal morphology without overstating what information can be derived from a standard decalcified paraffin-embedded section. The International Harmonization of Nomenclature and Diagnostic Criteria (INHAND) project issued standardized nomenclature for toxicologic pathologists to use with respect to the skeleton. Consistent with general INHAND principles, the nomenclature emphasizes a descriptive approach to classifying lesions, rather than using terms that imply a specific pathogenesis or disease process. This article, which is based on a presentation at the Society of Toxicologic Pathology’s 36th Annual Symposium, will discuss the role of anatomic pathology evaluation in the context of skeletal evaluation in toxicity studies. The integration of anatomic pathology data with quantitative bone end points is reviewed. An overview of the INHAND diagnostic scheme is provided and accompanied by case examples in which the INHAND terminology has been applied. In addition, this review summarizes key considerations for toxicologists and pathologists assigning adversity designations when considering bone end points.

This article summarizes a continuing education presentation on immunogenicity that was part of a continuing education course entitled, “Clinical Pathology of Biotherapeutics.” Immunogenicity of a biotherapeutic can have diverse impacts including altered systemic exposure and pharmacologic responses and, in a fraction of the cases, safety concerns including cross-reactive neutralization of endogenous proteins or sequela related to immune complex disease (ICD). In most cases, immune complexes are readily cleared from circulation; however, based on physiochemical properties, insoluble complexes form, activate complement, and deposit in tissues. Using published information and personal experience, a set of repeat-dose monkey toxicity studies with manifestations suggestive of ICD was reviewed to summarize the spectrum of clinical and pathology findings. The most common live-phase observation linked to ICD was an acute postdosing reaction following multiple dose administrations characterized by generalized collapse and attributed to acute complement activation. Less common live-phase observations were related to syndromes such as a consumptive coagulopathy or a protein losing nephropathy. The most common histologic change attributed to ICD was multi-organ vascular/perivascular inflammation followed by glomerulonephritis. The presentation concluded with a description of the challenges in assessing the relevance of immunogenicity-related reaction in monkey to human clinical use.

Inhibitors of Bruton’s tyrosine kinase (BTK) are under development as potential therapies for various autoimmune diseases. In repeat-dose toxicity studies, small-molecule BTK inhibitors (BTKi) have been reported to cause a constellation of histologic effects at the pancreatic endocrine–exocrine interface in male rats; however, similar findings were not reported in other species. Since the BTKi-induced pancreatic effect is morphologically similar to well-documented spontaneous changes (predominantly characterized by insular/peri-insular hemorrhage, pigment deposition, chronic inflammation, and fibrosis) that are known to vary by rat strain, we investigated potential strain-dependent differences in the pancreatic effects of a small-molecule BTKi, LY3337641. Following 13 weeks of LY3337641 treatment, Crl:CD(SD) rats were most sensitive, Crl:WI(Han) rats were of intermediate sensitivity, and Hsd:SD rats were least sensitive. These strain differences appear to be related to differences in rate of weight gain across strains and sexes; however, a definitive mechanism was not determined. This study demonstrated that BTKi-induced pancreatic effects were highly dependent on rat strain and correlated with differences in the incidence and severity of the spontaneous background change. When considered with the lack of pancreas effects in nonrat species, these changes in rats are unlikely predictive of similar changes in humans administered a BTK inhibitor.

Clinical and nonclinical studies have implicated glucagon-like peptide-1 (GLP-1) receptor agonist therapy as a risk factor for acute pancreatitis in patients with type 2 diabetes. Therefore, it is critical to understand the effect that dulaglutide, an approved GLP-1 receptor agonist, has on the exocrine pancreas. Dulaglutide 8.15 mg/kg (approximately 500 times the maximum recommended human dose based on plasma exposure) was administered twice weekly for 12 months to cynomolgus monkeys. Serum amylase and lipase activities were measured and 6 sections of each pancreas were examined microscopically. Ductal epithelial cell proliferation was estimated using Ki67 labeling. Dulaglutide administration did not alter serum amylase or lipase activities measured at the end of treatment compared to control values. An extensive histologic evaluation of the pancreas revealed no changes in the acinar or endocrine portions and no evidence of pancreatitis, necrosis, or pancreatic intraepithelial neoplasia. An increase in goblet cells noted in 4 of the 19 treated monkeys was considered an effect of dulaglutide but was not associated with dilation, blockage, or accumulation of mucin in the pancreatic duct. There was no difference in cell proliferation in ductal epithelium between control and dulaglutide-treated monkeys. These data reveal that chronic dosing of nondiabetic primates with dulaglutide does not induce inflammatory or preneoplastic changes in exocrine pancreas.

The Society of Toxicologic Pathology (STP) and American Society for Veterinary Clinical Pathology (ASCVP) convened a Clinical Pathology in Carcinogenicity Studies Working Group to recommend best practices for inclusion of clinical pathology testing in carcinogenicity studies. Regulatory guidance documents and literature were reviewed, and veterinary pathologists from North America, Japan, and Europe were surveyed regarding current practices, perceived value, and recommendations for clinical pathology testing in carcinogenicity studies. For two-year rodent carcinogenicity studies, the Working Group recommends that clinical pathology testing be limited to collection of blood smears at scheduled and unscheduled sacrifices to be examined only if indicated to aid in the diagnosis of possible hematopoietic neoplasia following histopathologic evaluation. Additional clinical pathology testing is most appropriately used to address specific issues from prior toxicity studies or known test article–related class effects. Inadequate data were available to make a recommendation concerning clinical pathology testing for alternative six-month carcinogenicity assays using genetically modified mice, although the Working Group suggests that it may be appropriate to use the same approach as for two-year carcinogenicity studies since the study goal is the same.

The National Toxicology Program (NTP) Satellite Symposium is a one-day meeting that is held in conjunction with the annual Society of Toxicologic Pathology (STP) meeting. The topic of the 2009 Symposium was ‘‘Tumor Pathology and INHAND (International Harmonization of Nomenclature and Diagnostic Criteria for Lesions in Rats and Mice) Nomenclature.’’ The goal of this article is to provide summaries of each speaker’s presentation, including the diagnostic or nomenclature issues that were presented, along with a few select images that were used for voting. The results of the voting process and interesting points of discussion that were raised during the presentation are also provided. A supplemental file with voting choices and voting results for each case presented at the symposium is available at http://tpx.sagepub.com/supplemental.

One of the challenges for toxicologic pathologists is to translate the diverse range of morphologic changes that are observed in toxicity studies into a consistent and readily understood set of diagnostic terms to be tabulated and summarized in regulatory reports or peer-reviewed publications. Historically, there have been multiple efforts to provide uniform nomenclature for lesions observed in laboratory animals. These efforts began as early as 1973 and have culminated in various textbooks, workshop reports, and monographs. The efforts have typically focused on the rat and mouse and, in many cases, were limited to proliferative lesions. Many of the texts or documents that describe suggested nomenclature are either not widely available or are out of print. To address this issue, members of the major societies of toxicologic pathology (JSTP, BSTP, ESTP, and STP) have been engaged in an international collaborative effort to codify and publish uniform nomenclature for both proliferative and nonproliferative lesions in laboratory rodents. Several features unique to this effort include (1) a truly international scope, (2) implementation of an open comment period allowing a wide group of toxicologic pathologists the opportunity to provide input, and (3) availability in a Web-based format. The project goes under the acronym INHAND (International Harmonization of Nomenclature and Diagnostic Criteria for Lesions in Rats and Mice). Project oversight is provided by the Global Editorial and Steering Committee (GESC), which consists of members from each of the major societies of toxicologic pathology. The most important components of the INHAND project are the Organ System Working Groups. Formation of these groups is coordinated by the GESC, and they consist of a global chairperson and members from each of the major societies of toxicologic pathology. The GESC actively seeks out organ system experts to serve on these groups. In addition, the GESC often accommodates requests from pathologists who volunteer for a specific organ working group. The Organ System Working Groups have the responsibility to prepare the nomenclature guidelines for both proliferative and nonproliferative lesions of rats and mice for their assigned organ system. In addition to lesions that occur spontaneously, the groups are asked to determine if there are common, xenobiotic-induced lesions for which standardized nomenclature might be needed. The working groups draw heavily from existing nomenclature documents, Web sites, and publications including prior work of the Registry of Industrial Toxicology Animal-Data (RITA) and the Standardized System of Nomenclature and Diagnostic Criteria (SSNDC). For each diagnostic entity, the working group selects a preferred diagnosis and acceptable alternative diagnoses, provides diagnostic criteria and differential diagnosis, and prepares representative photomicrographs. An important feature of the INHAND project is the access provided to the global open Registry Nomenclature Information System (goRENI; www.goreni.org) (Figure 1). Access is provided to all members of toxicologic pathology societies worldwide. Access is free but must be requested through the goRENI Web site (Figure 2). Once access is granted, pathologists can navigate by organ systems (Figure 3) and ultimately select a diagnosis they would like to view. Within the goRENI system, each diagnostic entity is referred to as a ‘‘manuscript.’’ An example is the written information, and photographic illustrations, provided for a bronchiolo-alveolar carcinoma as shown in Figure 4. Once an organ working group has prepared a draft document, the nomenclature is placed on the goRENI Web site for review and discussion. Members of the contributing STPs are notified of the dates of the review period through their respective societies. Following the designated comment period, the working group revises the nomenclature documents and, in consultation with the GESC, finalizes the nomenclature for that organ system. Finalized nomenclature will be available to toxicologic pathologists in two forms: (1) electronic access via the goRENI Web site and (2) print-based publication in the toxicologic pathology journals. Due to the substantial costs associated with print-based This is an opinion article submitted to the Regulatory Forum and does not constitute an official position of the Society of Toxicologic Pathology or the journal Toxicologic Pathology. The views expressed in this article are those of the authors and do not necessarily represent the policies, positions, or opinions of their respective agencies and organizations. Conflict of interest: The authors have not declared any conflict of interest.

Glucagon-like peptide-1 (GLP-1) receptor agonist therapy has been implicated as a possible risk factor for acute pancreatitis in patients with type 2 diabetes. Dulaglutide is a long-acting GLP-1 receptor agonist in development for treatment of type 2 diabetes. The effects of dulaglutide were evaluated in male Zucker diabetic fatty (ZDF) rats to examine whether dulaglutide may induce or modulate pancreatitis. Rats were randomized to dose groups receiving twice-weekly subcutaneously administered dulaglutide 0.5, 1.5, and 5.0 mg/kg/dose (corresponding human plasma exposures following twice-weekly dosing are 3-, 8-, and 30-fold, respectively) for 13 weeks or to vehicle control. Following termination, serially trimmed sections of pancreases were stained with hematoxylin and eosin or co-stained with an epithelial marker and a marker of either proliferation or apoptosis. Efficacious reductions in glucose and hemoglobin A1c occurred at all dulaglutide doses. Lipase activity was unaffected, and there were modest increases in total and pancreatic amylase activities at all doses without individual microscopic inflammatory correlates. Microscopic dulaglutide-related pancreatic changes included increased interlobular ductal epithelium without ductal cell proliferation (≥0.5 mg/kg), increased acinar atrophy with/without inflammation (≥1.5 mg/kg), and increased incidence/severity of neutrophilic acinar pancreatic inflammation (5.0 mg/kg). In summary, dulaglutide treatment was associated with mild alterations in ductal epithelium and modest exacerbation of spontaneous lesions of the exocrine pancreas typically found in the ZDF rat model.

Fischer 344 rats (60/sex/group) were given daily subcutaneou s injections of recombinant human parathyroi d hormone (PTH)(1-34) for 2 years at doses of 0, 5, 30, or 75 μg/kg. Treatment caused substantial increases in bone mass consistent with the known pharmacologic effects of once-daily administration. As determined by quantitative computed tomography (QCT) and histomorphometry, bone mass was markedly increased. Substantial new bone formation resulted in a large decrease in marrow space accompanied by altered bone architecture. Bone proliferative lesions were observed in all PTH(1-34)-treated groups. Osteosarcoma occurred in 3, 21, and 31 male rats and in 4, 12, and 23 female rats in the 5-, 30-, and 75- μg/kg treatment groups, respectively. Focal osteoblast hyperplasia, osteoma, and osteoblastoma were much less frequent. Although the specific cellular or molecular mechanisms responsible for the rat bone tumors have not been fully elucidated, the data suggest that these lesions resulted from the long duration of treatment and the exaggerated pharmacologic response of the rat skeleton to daily treatment with PTH(1-34). Important differences between the rat study and clinical use in adult humans suggest that the increased incidence of bone neoplasia in rats treated for 2 years is likely not predictive of an increased risk of bone cancer in skeletally mature adult humans being given PTH(1-34) for a limited period of time in the treatment of osteoporosis.

The INHAND Proposal (International Harmonization of Nomenclature and Diagnostic Criteria for Lesions in Rats and Mice) has been operational since 2005. A Global Editorial Steering Committee (GESC) manages the overall objectives of the project and the development of harmonized terminology for each organ system is the responsibility of the Organ Working Groups (OWG), drawing upon experts from North America, Europe and Japan.Great progress has been made with 9 systems published to date – Respiratory, Hepatobiliary, Urinary, Central/Peripheral Nervous Systems, Male Reproductive and Mammary, Zymbals, Clitoral and Preputial Glands in Toxicologic Pathology and the Integument and Soft Tissue and Female Reproductive System in the Journal of Toxicologic Pathology as supplements and on a web site – www.goreni.org. INHAND nomenclature guides offer diagnostic criteria and guidelines for recording lesions observed in rodent toxicity and carcinogenicity studies. The guides provide representative photo-micrographs of morphologic changes, information regarding pathogenesis, and key references. During 2012, INHAND GESC representatives attended meetings with representatives of the FDA Center for Drug Evaluation and Research (CDER), Clinical Data Interchange Standards Consortium (CDISC), and the National Cancer Institute (NCI) Enterprise Vocabulary Services (EVS) to begin incorporation of INHAND terminology as preferred terminology for SEND (Standard for Exchange of Nonclinical Data) submissions to the FDA. The interest in utilizing the INHAND nomenclature, based on input from industry and government toxicologists as well as information technology specialists, suggests that there will be wide acceptance of this nomenclature. The purpose of this publication is to provide an update on the progress of INHAND.

An important safety consideration for developing new therapeutics is assessing the potential that the therapy will increase the risk of cancer. For biotherapeutics, traditional two-year rodent bioassays are often not scientifically applicable or feasible. This paper is a collaborative effort of industry toxicologists to review past and current practice regarding carcinogenicity assessments of biotherapeutics and to provide recommendations. Publicly available information on eighty marketed protein biotherapeutics was reviewed. In this review, no assessments related to carcinogenicity or tumor growth promotion were identified for fifty-one of the eighty molecules. For the twenty-nine biotherapeutics in which assessments related to carcinogenicity were identified, various experimental approaches were employed. This review also discusses several key principles to aid in the assessment of carcinogenic potential, including (1) careful consideration of mechanism of action to identify theoretical risks, (2) careful investigation of existing data for indications of proliferative or immunosuppressive potential, and (3) characterization of any proliferative or immunosuppressive signals detected. Traditional two-year carcinogenicity assays should not be considered as the default method for assessing the carcinogenicity potential of biotherapeutics. If experimentation is considered warranted, it should be hypothesis driven and may include a variety of experimental models. Ultimately, it is important that preclinical data provide useful guidance in product labeling.

A long-term study was conducted in female F344 rats to determine the relative importance of dose, treatment duration, and age at initiation of treatment on the incidence of teriparatide [rhPTH[1-34)]-induced bone proliferative lesions. Treatment groups consisted of different combinations of dose (0, 5, or 30 μg/kg/d), treatment duration (6, 20, or 24 months) and age at initiation of treatment (2 or 6 months of age). The primary endpoints were the incidence of bone neoplasms and effects on bone mass and structure as evaluated by quantitative computed tomography and histomorphometery. Significant increases in the incidence of bone tumors (osteoma, osteoblastoma, and osteosarcoma) occurred in rats treated with 30 μg/kg for 20 or 24 months. No neoplasms were found when the 5 μg/kg treatment was initiated at 6 months of age and continued for either 6 or 20 months (up to 70% of life span). This treatment regimen defined a “no-effect” dose for neoplasm formation that nevertheless resulted in substantial increases in bone mass. These results demonstrate that treatment duration and administered dose are the most important factors in the teriparatide-induced bone tumors in rats.

An important component of the safety assessment of a pharmaceutical or environmental agent is determining if the agent has the potential to increase the risk of cancer in humans. In existing safety assessment paradigms, this concern has primarily been addressed by two-year carcinogenicity studies in both rats and mice (Jacobs and Jacobson-Kram 2004; Huff et al. 1998; International Conference on Harmonization 1995). For pharmaceutical agents, there has been increasing use in recent years of six-month studies in transgenic mice as an alternative to the twoyear mouse study (Jacobson-Kram et al. 2004). Despite the extensive use of these models, there remains considerable debate regarding the ability of these studies to predict human risk (Alison, Capen, and Prentice 1994; Jacobs 2005; Cohen 2004). Strategies to improve our ability to predict carcinogenic risk continue to evolve, and several of these focus on predicting carcinogenic outcomes with data from shorter-term studies. In this month’s edition of the Regulatory Forum, Dr. Sam Cohen, an opinion leader in the area of carcinogenicity assessments, presents his perspective on the ability to predict carcinogenic outcomes from short-term studies using hepatic carcinogenesis as a model. Following Dr. Cohen’s articles are commentaries representing alternative viewpoints: Dr. Gerald Long, whose expertise is in the area of carcinogenicity assessments of pharmaceuticals, and Dr. Ronald Lorentzen, an experienced regulatory scientist in the area of food safety. We hope the readership of Toxicologic Pathology finds these viewpoints of interest, and we invite you to submit additional commentary on this important and controversial area of safety assessment.