John E. Burkhardt

The prostaglandin E2 EP1 receptor mediates pain perception and regulates blood pressure

The lipid mediator prostaglandin E2 (PGE2) has diverse biological activity in a variety of tissues. Four different receptor subtypes (EP1-4) mediate these wide-ranging effects. The EP-receptor subtypes differ in tissue distribution, ligand-binding affinity, and coupling to intracellular signaling pathways. To identify the physiological roles for one of these receptors, the EP1 receptor, we generated EP1-deficient (EP1-/-) mice using homologous recombination in embryonic stem cells derived from the DBA/1lacJ strain of mice. The EP1-/- mice are healthy and fertile, without any overt physical defects. However, their pain-sensitivity responses, tested in two acute prostaglandin-dependent models, were reduced by approximately 50%. This reduction in the perception of pain was virtually identical to that achieved through pharmacological inhibition of prostaglandin synthesis in wild-type mice using a cyclooxygenase inhibitor. In addition, systolic blood pressure is significantly reduced in EP1 receptor-deficient mice and accompanied by increased renin-angiotensin activity, especially in males, suggesting a role for this receptor in cardiovascular homeostasis. Thus, the EP1 receptor for PGE2 plays a direct role in mediating algesia and in regulation of blood pressure.

Morphologic and Biochemical Changes in Articular Cartilages of Immature Beagle Dogs Dosed with Difloxacin

Quinolones are efficacious antibacterial compounds, but they have been associated with arthralgia in human patients; experimentally, they have caused lesions in articular cartilages of immature animals. The earliest morphologic and biochemical changes induced in articular-epiphyseal cartilage complexes by difloxacin, a fluoroquinolone, were investigated in 27 3-month-old Beagle dogs that were dosed orally with the drug at 300 mg/kg body weight per day. Paraffin-embedded sections of humeral and femoral heads that were stained with either hematoxylin and eosin or toluidine blue and fast green were evaluated histologically, and lesions were scored according to established criteria. Although morphologic changes were not observed in cartilages of the control dogs or of the treated dogs in the 24-hr group, the severity of lesions, as represented by mean scores for lesions, increased during the 36–48 hr after dosing. The initial morphologic change, observed in cartilages from the treated dogs of the 36-and the 48-hr groups, was necrosis of chondrocytes that was rapidly followed by disruption of extracellular matrix and formation of fissures. Although glycosaminoglycan was aggregated along the margins of fissures, its concentration was not reduced in cartilages of any group of treated dogs. Collagen, however, was depleted from the cartilages of the dogs that were euthanized 36 or 48 hr after the first dose of difloxacin. Because degenerative changes were observed ultrastructurally in chondrocytes by 24 hr in a previous study, it was concluded that collagen was lost from affected cartilages as an early sequel to the degeneration of chondrocytes.

Recommendations for the evaluation of pathology data in nonclinical safety biomarker qualification studies

A set of best practices for the conduct of histopathology evaluation in nonclinical safety studies was endorsed by the Society of Toxicologic Pathology (STP) in 2004. These best practices indicate that the study pathologist should have knowledge of the treatment group and access to all available study-related data for the animal from which the tissue was obtained. A new set of best practices for the conduct of histopathology review for safety biomarker qualification for nonclinical studies has been endorsed by the STP and is summarized in this document. These best practices are generally similar to those for nonclinical safety studies, specifically that the pathologist be “unblinded” or have access to study data. Although histopathology evaluation in biomarker qualification studies must be performed without knowledge of novel biomarker data, the study pathologist(s) should be involved in the attendant meta-analyses of these data. Blinded evaluation is an experimental tool in biomarker qualification studies that is appropriate only when well-defined criteria for specific histopathologic findings are identified prior to blinded review. Additionally, this paper also considers the management of bias, the use of a tiered evaluation approach, the importance of using qualified pathologists and standard reporting, and the management of spontaneous findings.

Scientific and Regulatory Policy Committee Recommended (“Best”) Practices for Determining, Communicating, and Using Adverse Effect Data from Nonclinical Studies

Recommendations (best practices) are provided by the Society of Toxicologic Pathology’s Adversity Working Group for making consistent interpretations of test article–related effects as “adverse” and assigning a “no observed adverse effect level” (NOAEL) in nonclinical toxicity studies. Adverse is a term indicating “harm” to the test animal, while nonadverse indicates lack of harm. Adverse findings in the study reports should be defined in relation to effects on the test species used and within the context of the given study. Test article–related effects should be described on their own merits, and decisions to consider them as adverse or nonadverse should be justified. Related effects may be discussed together; in particular, markers of toxicity that are not in and of themselves adverse ideally should be discussed in conjunction with the causal toxicity to determine adversity. Adverse findings should be identified in subreports (clinical data, pathology data, etc.) if sufficient information is available, and/or in the final study report as individual or grouped findings, but study NOAELs should be established at the level of the overall study report. Interpretations such as “not biologically relevant” or “not toxicologically important” should be avoided unless defined and supported by scientific rationale. Decisions defining adverse findings and the NOAEL in final study reports should combine the expertise of all contributing scientific disciplines. Where possible, use of NOAELs in data tables should be linked to explanatory text that places them in context. Ideally, in nonclinical summary documents, NOAELs from multiple studies are considered together in defining the most important adverse responses in the most sensitive species. These responses are then considered along with an understanding of their likely mechanisms, as well as other information such as variability in species sensitivity, comparative pathology, reversibility and progression, kinetics, and metabolism of the test substance to help assess human risk.

Immunohistochemistry of articular cartilage from immature beagle dogs dosed with difloxacin.

Effects of the fluoroquinolone difloxacin on articular-epiphyseal cartilage in growing beagle dogs have been described by light microscopic, electron microscopic, and biochemical methods. Here we present data from an immunohistochemistry study with humeral head cartilage from 3-mo-old beagle dogs after treatment with 1 or 2 oral doses of 300 mg difloxacin/kg body weight. Dogs were euthanatized either 24 hr (single dose) or 48 hr (2 doses) after onset of dosing, and cartilage tissue was stored at -90°C until it was studied by immunohistochemistry. Antibodies against matrix components (collagen II, fibronectin) as well as antibodies against cellular structures (integrins) were used. After single-dose treatment (24-hr group), cartilage lesions such as clefts were not observed, but increased staining for fibronectin was found in cartilage samples from 5 of 6 animals. Markedly increased staining for fibronectin was also demonstrated in the vicinity of clefts within cartilage of all animals of the 48-hr group. Collagen II staining was homogeneously distributed in cartilage from controls and was slightly reduced in territorial matrix in 2 of 6 dogs of the 48-hr group. Integrin staining on chondrocytes was not significantly affected by difloxacin under the given conditions with the exception of a slight reduction of the α v integrin chain in 1 of 5 dogs of the 48-hr group. Overall, the most important result is the finding that fibronectin was a sensitive immunohistochemical marker for change in cartilage samples due to difloxacin treatment in dogs.

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.

Topic of Histopathology Blinding in Nonclinical Safety Biomarker Qualification Studies

Various consortia and working groups, composed of professionals from industry, academia, and government institutions, have undertaken or are undertaking nonclinical work to qualify safety biomarkers of tissue injury and function. As this work has developed and voluntary data have been submitted to regulatory authorities, study practices have come under close scrutiny in an attempt to ensure that the best science is consistently being applied. One practice that has been discussed in a variety of venues is the process used to generate histopathology data as additional end points and/or correlates in these studies. Histopathologic evaluation plays a critical role in these biomarker studies, because microscopic demonstration of given cellular processes are commonly used as a reference standard to assess diagnostic performance of candidate new biomarkers using methods such as receiver operating characteristic (ROC) analyses. In this regard, it is acknowledged that pathologists should not perform the histopathologic evaluation with knowledge of the candidate biomarker data; however, it has been questioned whether pathologists should conduct the histopathology evaluation for these biomarker studies without knowledge of treatment or other study-related data (i.e., ‘‘blinded’’ evaluation). Research pathologists, and especially the Society of Toxicologic Pathology (STP), have closely examined regulatory study histopathology practices (Crissman et al. 2004; Wandall, Hansson and Ruden 2007). Recently, a set of Best Practices for the conduct of histopathology review within nonclinical safety studies was endorsed by the STP (Crissman et al. 2004). Key elements of these Best Practices indicate that the study pathologist should be informed as follows: (1) have knowledge of the treatment group from which the sample was obtained; and (2) have complete knowledge of all available study-related data that are associated with the animal from which the tissue was obtained. As these practices have been employed successfully for decades in the context of regulatory toxicology studies designed to assess human safety of new chemical entities with complete regulatory acceptance, the rationale for following a different process for biomarker validation and qualification is unclear to these authors. This informed type of analysis (described above) is often referred to as ‘‘unblinded,’’ because the study pathologist has knowledge of dose groups and other study-related data at the time that the review is being conducted. It is considered that ‘‘unblinded’’ evaluation is critical to discriminate treatment-related changes from background, especially when subtle treatment-related effects increase the incidence or severity of spontaneous background findings. As needed, the study pathologist may reexamine tissues using a procedure sometimes termed ‘‘targeted masked’’ evaluation. This procedure entails reexamination of selected or all treated dose groups, randomly combined with controls and without knowledge of animal or group identity, to determine whether a subtle or equivocal finding can be identified consistently from control tissues. This second evaluation is performed after the pathologist feels confident that each finding has been fully characterized, to ensure either that subtle findings are assessed for incidence and severity or that equivocal findings are assessed to discriminate a true change from spontaneous background (in an unbiased manner). Further to the nonclinical safety study Best Practices endorsed by STP, a peer-review process is generally used as a method for quality assurance, in which a second pathologist can corroborate the interpretations of the study pathologist. This second evaluation is typically conducted in an ‘‘unblinded’’ fashion, as this evaluation is not meant to generate new data but rather to provide a second party evaluation of the interpretations and conclusions of the primary pathologist. Taking into consideration all available historical data as well as current practices of histopathology data generation, the current authors were tasked by the Scientific and Regulatory Policy Committee of the STP to consider the development of either a Best Practices or a Points to Consider guideline for industry and regulators, with emphasis on histopathology practices related to biomarker qualification. Although the final document will not be available until later, we wished to present our preliminary opinion based on the aforementioned Best Practices (Crissman et al. 2004). The present authors recognize that histopathology is a special discipline in which considerations for best practices may differ from those of other disciplines used in biomarker evaluation. The present authors also acknowledge that some toxicities are either unique to humans or idiosyncratic in nature and thus undetectable in standard nonclinical studies. Therefore, the method of evaluating slides would not affect the sensitivity for detection of such changes. Conversely, the present authors agree that ‘‘unblinded’’ slide evaluation creates a high degree of fidelity and consistency in the ability to identify animal toxicities. We concur with

Effects of Nalidixic Acid on Hamster Knee Cartilage Morphology and Synovial Fluid Composition

Quinolone-induced changes were studied in the knee joints of 4-wk-old female hamsters given intraperitoneal doses of either nalidixic acid (400 mg/kg body weight) or vehicle on days 0 and 1. After euthanasia on day 4, synovial fluid was collected for cytologic evaluation and for analysis of concentrations of hyaluronan, proteoglycans, total protein, and collagen as hydroxyproline. Slides of formalin-fixed decalcified tissues were stained with hematoxylin-eosin or safranin O for histologic scoring of lesion severity. Nine of 10 hamsters treated with nalidixic acid had fissures within articular cartilage of the femur and reduced safranin O staining of matrix indicative of loss of proteoglycans. Synovial membranes from affected joints, however, were not inflamed. Synovial fluid cell counts and cytomorphology were unaffected by treatment. In synovial fluid from 5 of 10 treated hamsters, proteoglycans were elevated by more than 2 SDs above the control group, and individual animal levels correlated with the histologic severity score (r2 = 0.36; p = 0.02). The hyaluronan content of the synovial fluid from treated hamsters was mildly but significantly elevated (p = 0.005), and the histologic severity score again correlated with individual animal levels (r 2 = 0.42; p = 0.01). Hydroxyproline was unaffected by treatment. Although synovial fluid changes and histologic changes were correlated on a group basis, interanimal variability was significant and the magnitude of biochemical changes were far smaller than those that occur during inflammation. Changes in synovial fluid composition are not sufficiently robust to predict cartilage changes in individual animals.

Effects of cholestyramine and diet on small intestinal histomorphometry in rats.

Reports on the effects of cholestyramine on small intestinal structure of rats have produced contradictory data about changes in mucosal histomorphometry, perhaps due to interacting effects of dietary composition. In order to identify effects of cholestyramine and diet on structure of the small intestines, 40 male rats were divided into 4 groups of 10 and fed 1 of each of the following diets for 1 month: standard diet, purified fiber-free diet, standard diet + 2% cholestyramine, or purified fiber-free diet + 2% cholestyramine. Serum concentrations of cholesterol and triglycerides were moderately increased in rats fed the purified fiber-free diet versus the standard diet. Neither total length nor weight of small intestine were affected by either diet or cholestyramine. Mucosal weight was affected by interactions between cholestyramine and diet, indicating that outcome depended upon modulating effects of both variables. Significant interactions were similarly detected among the variables of anatomic site, diet, and cholestyramine for many histomorphometric parameters of intestinal mucosa. Cholestyramine reduced total mucosal thickness in both jejunum and ileum and reduced villus height and villus: crypt ratio in the ileum.

Practical Aspects of Discovery Pathology

Pathologists are uniquely qualified to play a central role in driving drug discovery and development programs by: 1) establishing disease models to assess potential therapies, 2) characterizing modifications in the disease state in response to therapies, 3) characterizing toxicologic mechanisms and responses to drug candidates, and 4) facilitating multidisciplinary efforts to monitor for the clinical occurrence, progression, and reversibility of adverse events. Such nontraditional deployment of resources must, to be viable, produce benefits to the pharmaceutical industry comparable to those of more conventional activities such as delivery of data in nonclinical safety studies. Additionally, benefits must be tangible from standpoints such as timesavings or improved quality of research decisions, manifesting as either program acceleration or improved candidate survival.