Interpretive Power and Responsibility of the Pathologist

1. Interpretive Power and Responsibility of the Pathologist Kevin McDorman, DVM, PhD, DACVP Amgen Inc. Thousand Oaks, CA

2. Interpretive Responsibility of the Pathologist • Focus on toxicologically-important and clinically-relevant findings • Attribute relationship to test article • Distinguish primary and secondary effects • Provide perspective on similar lesions induced by other compounds or natural occurrence • Present evidence concerning reversibility • Propose the most plausible pathogenesis for toxic changes related to the test article

3. Basics of Interpretive Duties • Natural disease vs. test article-induced • Understanding spontaneous lesions and concurrent diseases in test species • Influence of test article administration • De novo toxic injury • Primary test article effects • Secondary effects of direct injury • Evidence of reversible processes

4. Natural Disease in Test Species • Dogs • Spontaneous vasculitis in beagles • Concurrent infectious diseases • Rodents • Age-related degenerative changes • Spontaneous neoplasia • Concurrent infectious diseases • Nonhuman primates • Unspecified background and influences • Nutritional and parasitic • Genetic and age-related variation • Concurrent infectious diseases

5. Spontaneous Arterial Disease • Idiopathic necrotizing arteritis • Dogs (a.k.a. beagle pain syndrome) • Arterial wall necrosis with chronic-active inflammation • Coronary, meningeal and vertebral arteries most commonly effected • High doses of cardioactive drugs from several classes can increase the severity of lesions • Secondary changes in other organs (e.g., testicular necrosis) • Rodents (rat, mouse, and hamster) • Arterial wall necrosis with chronic-active inflammation • Vessels effected dependent on species and strain • Distribution and histopathological characteristics of treatment-related vasculitis or vascular necrosis need to be evaluated in the light of spontaneous vascular pathology

6. Mechanisms of Treatment-Related Vasculitis • Direct test article-induced toxicity to vasculature • Test article-induced exacerbation of spontaneous or concurrent disease • Test article-induced hypersensitivity or immune-mediated reactions • Test article-induced hemodynamic alterations (vasoactive molecules)

7. Useful Endpoints to Distinguish Test Article-Induced Vasculitis from Exacerbation of Natural Disease • Thorough assessment of clinical signs • Detailed histopathological characterization, including time course of lesion development • Evaluation after short-term exposure may help • Thorough assessment of cardiovascular function and hemodynamic status • Blood pressure, heart rate, flow rates

8. Potential Biomarkers to Distinguish Test Article-Induced Vasculitis from Exacerbation of Natural Disease • Potential biomarkers for endothelial injury • Circulating endothelial cells (CECs) • von Willibrand Factor (vWF) • Thrombomodulin • Potential biomarkers for smooth muscle injury • Smooth muscle alpha actin • Biomarkers for immune-mediated reactions • Presence of autoantibodies (e.g., those directed against cytoplasmic antigens of neutrophils – ANCA) • Immune complexes, peripheral eosinophilia

9. Concurrent Infectious Disease: Examples with the Ability to Confound Pathology Interpretation • Dogs • Cardiac inflammation and fibrosis caused by canine parvovirus • Hepatocellular necrosis and hemorrhage caused by canine hepatitis virus

10. Concurrent Infectious Disease: Examples with the Ability to Confound Pathology Interpretation • Rats • Upper airway inflammation, keratitis, lacrimal gland inflammation, and salivary gland inflammation, degeneration, and regeneration caused by sialodacryoadenitis virus • Upper and lower airway inflammation caused by microbial agents (e.g., Corynebacterium kutscheri, Mycoplasma pulmonis, Streptococcus pneumoniae, etc.) • Lymphomas and leukemias caused by oncogenic viruses • Liver necrosis and intestinal inflammation caused by microbial agents (e.g., Clostridium piliformis, Salmonella spp.)

11. Concurrent Infectious Disease: Examples with the Ability to Confound Pathology Interpretation • Mice • Liver necrosis and inflammation, and small intestinal necrosis, inflammation and regeneration caused by mouse hepatitis virus or microbial agents (e.g., Clostridium piliformis, Helicobacter hepaticus) • Mammary gland hyperplasia and neoplasia caused by mouse mammary tumor virus • Lymphomas and leukemias caused by oncogenic viruses • Necrosis of Beta islet cells and hyperglycemia caused by encephalomyelitis virus • Decreased synthesis of growth hormone caused by non-cytopathic lymphocytic choriomeningitis virus

12. Concurrent Infectious Disease:Examples with the Ability to Confound Pathology Interpretation • Nonhuman primates • Gastrointestinal disease (bacterial, protozoal, metazoal) • Peritoneal, mesenteric, and pancreatic inflammation caused by Oesophagostomum nematode larval migration • Nodular skin lesions caused by Yabapox

13. Influence of Test Article Treatment on Natural Disease • Primary and secondary mechanisms of tissue changes, as well as effects of the experimental treatment conditions, can all act to increase or decrease the incidence, latency, and/or severity of spontaneous disease in test species

14. Influence of Test Article Treatment on Natural Disease • Increased incidence, latency, and/or severity of spontaneous disease in test species • Can be related to adverse effects of the test article and/or the treatment itself • Example: Rat chronic progressive nephropathy (CPN) • Age-related degenerative renal disease characterized by glomerulosclerosis, various stages of tubular degeneration, dilation, and regeneration, protein casts, and interstitial inflammation and fibrosis • Increased severity and decreased latency in rats treated with the gastric proton pump inhibitor omeprazole • Enhanced disease in rats treated with acetaminophen

15. Influence of Test Article Treatment on Natural Disease • Decreased incidence, latency, and/or severity of spontaneous disease in test species • Occasionally unexpected, but more often related to an intended biological response • Examples: • Decreased incidence and severity of CPN in rats treated with the angiotensin inhibitor captopril • Decreased pituitary tumor incidence in rats treated with selective estrogen receptor modulators (SERMs, e.g., tamoxifen)

16. Importance of Appropriate Experimental Design: Where Can Pathologists Help? • Route of administration consistent with exposure • Test species • Presence of appropriate receptors and metabolizing enzymes • Appropriate level of exposure and bioavailability • Sensitivity of test species to toxic effects • Age at start of study • Chemical disposition rates can vary significantly with age • Effects on immature systems have questionable relevance to adult systems

17. Importance of Appropriate Experimental Design: Where Can Pathologists Help? • Developmental and reproductive toxicology (DART) studies • Studies that identify molecules that affect male and female fertility, early embryonic development, prenatal and postnatal development, and maternal function • In vivo endocrine assessment can be crucial • Recording and correlating female cyclicity with circulating hormones may be useful in DART studies in the rodent and nonhuman primate.

18. Primary Test Article Effects • Enzyme induction and inhibition • Organ specificity for toxicity • Site-specific and nonspecific cellular interactions • Dose dependency and response

19. Primary Test Article Effects: Enzyme Induction and Inhibition • Enzyme systems responsible for metabolism and detoxication • Cytochrome P450 monooxygenase • Nonspecific and specific induction responses can alter normal organ homeostasis, detoxication, and/or increase toxic metabolites • Exposure may also inhibit enzyme activity or form an inactivating complex

20. Primary Test Article Effects: Organ Specificity for Toxicity • Specific cellular and intracellular site and specificity of binding • Interactions with similar subcellular sites in several organs may not result in a similar degree of effect or toxicity • Organ specificity is usually caused by differences in metabolism or other tissue responses (e.g., cell proliferation)

21. Primary Test Article Effects: Site-Specific and Nonspecific Cellular Interactions • Site-specific cellular interactions depend on binding affinity • Toxicants can bind with a receptor to either promote (agonist) or block (antagonist) the effect produced by the normal endogenous compound • Competing molecules can alter normal enzyme activity by acting as a substrate, forming a stable substrate-enzyme complex that dissociates slowly, or inactivating the enzyme directly • Nonspecific cellular interactions • Reactive molecules that form free radicals and/or bind to tissue macromolecules to form adducts

22. Saturation of detoxication Linear dose response Saturation of metabolism Threshold of observable dose-level effect Dose Response Basics Dose Effect

23. Primary Test Article Effects: Dose Dependency and Response • Variability of toxic effects is dependent on the dose at a target site in addition to the binding affinity • Molecules that interact with more than one site may show different affinity for each organ • As the dose is increased, the bioavailable molecule can interact with an increasing number of different sites beginning with those of greatest affinity • The number of toxic responses may increase as the number of sites which interact with the molecule increases

24. Secondary Test Article Effects • Sequelae manifest differently depending on direct organ system injury • Differences in enzyme induction and reduction, e.g., • Liver – induced P450 enzyme activity can alter peripheral metabolism of thyroid hormone and result in thyroid follicular cell hyperplasia. • Reduced functional mass, e.g., • Kidney – renal insufficiency reduces intestinal calcium absorption, resulting in decreased serum calcium, increased resorption of bone calcium, and secondary hyperparathyroidism.

25. Secondary Test Article Effects • Relevance of the mechanism of secondary toxic effects in test species to humans must be established • Differences in the importance and relevance of specific enzyme induction or inhibition • Stress response in experimental conditions • A veterinary pathologist can provide practical insight on the importance of treatment and test article-related findings and the potential impact or relevance to the human clinical setting

26. Interpretive Responsibility of the Pathologist • Focus on toxicologically-important and clinically-relevant findings • Attribute relationship to test article • Distinguish primary and secondary effects • Provide perspective on similar lesions induced by other compounds or natural occurrence • Present evidence concerning reversibility • Propose the most plausible pathogenesis for toxic changes related to the test article

27. Refining the Risk Assessment Through Mechanistic Understanding • Establish the cellular and subcellular organelle target of toxic injury • Conduct time and dose-response studies using doses that manifest acute injury • Define primary or secondary effects and reversible, irreversible, or progressive processes • Investigate biomarkers for exposure and toxicity • Predict potential functional and long-term consequence after short-term toxic responses

28. Selected References • Greaves, Peter. Histopathology of Preclinical Toxicity Studies: Interpretation and Relevance in Drug Safety Evaluation. New York: Elsevier Science B.V., 2000. • Hard, Gordon C. and Khan,Kanwar N. (2004). A contemporary overview of chronic progressive nephropathy in the laboratory rat, and its significance for human risk assessment. Toxicologic Pathology. Mar-Apr;32(2):171-80.

29. Selected References • Haschek, Wanda M., and Rousseaux, Colin G. Fundamentals of Toxicologic Pathology. San Diego: Academic Press, 1998. • Kerns W, Schwartz L, Blanchard K, Burchiel S, Essayan D, Fung E, Johnson R, Lawton M, Louden C, MacGregor J, Miller F, Nagarkatti P, Robertson D, Snyder P, Thomas H, Wagner B, Ward A, Zhang J; Expert Working Group on Drug-Induced Vascular Injury. (2005). Drug-induced vascular injury--a quest for biomarkers. Toxicol Appl Pharmacol. Feb 15;203(1):62-87.

30. American College of Toxicology (ACT) 26th Annual Meeting:Williamsburg, VA, November 6-9, 2005 • Current Topics in Toxicologic Pathology • An STP-Sponsored Symposium, ACT annual meeting • Monday, Nov 7, 2005, 1:30 – 5:00 pm • Toxic effects on the developing nervous, endocrine, and immune systems, and the challenges of toxicology testing in juvenile animals • Introduction; Co-Chairs Dr. Kathleen Funk and Dr. Kevin McDorman • Developmental Neurotoxicity Studies - Neuropathology Procedures and Pitfalls; Dr. Robert H. Garman • Endocrine Disruptor Effects in Fish; Dr. Jeff Wolf • Developmental Immunotoxicology; Dr. Michael Holsapple • Factors Associated with Juvenile Animal Toxicity and Pathology; Dr. John Seely