Susceptibility Factors in Idiosyncratic Drug-Induced Liver Injury

1. U.S. Department of Health and Human Services National Institutes of Health National Heart, Lung, and Blood Institute Susceptibility Factors in Idiosyncratic Drug-Induced Liver Injury Steven Yee Molecular and Cellular Toxicology Section National Institutes of Health yees@nhlbi.nih.gov 8 June 2004

2. Drug-Induced Liver Injury • Occurs in small fraction of individuals • Difficult to predict • Major clinical problem; often life-threatening • Leading cause of acute liver failure • Reason drugs removed from clinical development and widespread use

3. Examples of Drugs Withdrawn Due to Liver Disease Iproniazid 1956 Ibufenac (in Europe only) 1975 Ticrynafen 1979 Benoxaprofen 1982 Perhexiline (in France) 1985 Dilevalol (in Portugal, Ireland) 1990 Bromfenac 1998 Troglitazone 2000 Serzone 2004

4. Putative Mechanisms • Mechanism often involves drug metabolites • Affect critical biochemical functions • Specific immune responses • Only a few drugs demonstrate these underlying causes • Tissue susceptibility: imbalance between protoxicants and protectants • Environmental factors • Genetic polymorphism

5. Hepatotoxicants Potentiated by Exposure to Small Doses of Lipopolysaccharide (LPS) Xenobiotics CCl4 Galactosamine Ethanol T2-toxin Cadmium Halothane Lead Allyl Alcohol Aflatoxin B1 Chlorpromazine Ranitidine Source Formal et al., 1960 Galanos et al., 1979 Nolan et al., 1980 Tai and Petska, 1988 Cook et al.., 1974 Lind et al., 1984 Honchel et al., 1991 Sneed et al., 1997 Barton et al., 2000 Buchweitz et al., 2002 Luyendyk et al., 2003

6. Cellular Level Events of LPS LPS Macrophages Neutrophils Endothelial Cells Epithelial Cells TLR4 Cytokines Coagulation Factors Platelet Activating Factor Complement Activation Leukotrienes Arachidonic Acid Metabolites Prostaglandins Reactive Oxygen Species Nitric Oxide

7. Crotalaria Spectabilis (Rattlebox) Monocrotaline (MCT) CH3 CH3 C C OH OH H3C C C=O H O=C O O CH2 N Monocrotaline

8. MCT Toxicity CH3 CH3 CH3 CH3 C C C C OH OH OH OH H3C C C=O H3C C C=O Covalent Binding to Cellular Macromolecules H H O=C O=C O O CYP 3A O CH2 O CH2 Cell Death N N MCT Monocrotaline Pyrrole (MCTP)

9. MCT/LPS Treatment Protocol 0 hr 4 hr X hr 1. Plasma 2. Liver MCT or Veh (i.p.) LPS or Veh (i.v.) • Male Sprague-Dawley Rats

10. Modest Inflammation Enhances MCT Toxicity LPS 4 hours after MCT

11. LPS Lowers the Threshold for MCT Toxicity

12. Kupffer Cell Inactivation Reduces Hepatotoxicity

13. TNF- Depletion AttenuatesMCT/LPS Hepatotoxicity

14. Neutrophil Depletion Attenuates MCT/LPS Hepatotoxicity

15. Inactivation of the Coagulation System Decreases Liver Injury

16. Summary

17. Drug Liver Reactive Metabolites Protein Adducts Protective Factors Response Cellular Homeostasis Altered IL-6, IL-10, COX-2 Stress Proteins Toxicity Protection (Inhibition) Extensive Liver Injury And Death Does a loss in hepatoprotective factors result in drug-induced liver disease?

18. Model Compound • Many of the protective factors discovered through research on acetaminophen (APAP) toxicity • Acetaminophen • Clinically relevant; analgesic, antipyretic • Over 50,000 ER visits per year • 450 death per year • Suicide • Accidental ingestion • “Therapeutic misadventures” • Unlike with drug idiosyncrasy, it is well characterized • and reproducible in animals • Bioactivation to N-acetyl-p-benzoquinone imine (NAPQI)

19. O HN CH 3 Sulfation Glucuronidation Detox Detox P450 2E1 P450 1A2 OH P450 3A4 ACETAMINOPHEN O Cysteinyl N CH 3 Detox Conjugate GSH Depleted Protein Adducts LIVER O MitochondriaDysfunction INJURY NAPQI ReactiveOxygenSpecies APAP-Induced Liver Injury

20. Bourdi (2002), Hepatology, 35:289 Serum Cytokines After Administration of 300 mg APAP/kg to C57BL/6 Mice

21. Development of Hepatotoxicity in Mice Given IL-13 Neutralizing Antibody 2 Hours before 200 mg APAP/Kg

22. Development of Hepatotoxicity in IL-13 KO Mice Treated With 200 mg APAP/Kg

23. CAb/Veh CAb/APAP CV CV CV CV APAP - KO IL-13 NAb/APAP CV CV CV CV Liver Histopathology at 8 hours

24. Citrulline IL-1, IL-6, TNF-a, IFN-γ NO +Superoxide Peroxynitrite iNOS Nitrite/Nitrate L-Arginine L-NIL Aminoguanidine (AMG) Arginase L-Ornithine IL-4, IL-10, IL-13 Urea Nitric Oxide Pathway

25. Summary • Endogenous IL-13 is a hepatoprotective factor in APAP-induced liver injury • Elevated serum TNF-α concentration – causal role? • Elevated NO levels – causal role? • Deficiency in IL-13 may increase susceptibility to drug-induced liver disease

26. Idiosyncratic Drug-Induced Liver Disease • Complex “multihit” process • Liver protoxicants and protectants have a role in the overall pathogenesis • Environmental Factors • Genetic polymorphisms • Underproduction of hepatoprotective and overproduction of hepatoprotoxicant factors (i.e., imbalance) influences susceptibility to this liver disease

27. Determination of Susceptibility Factors • Identification of liver protoxicant and protectant factors results in better understanding of mechanism and in facilitating prediction of drug-induced liver disease • Inflammatory mediators • COX-2 products • Heat shock proteins • Application of new technologies • Toxicogenomics • Proteomics • Metabonomics

28. Acknowledgments Michigan State University Robert Roth Patricia Ganey Jack Harkema Chuck Barton John Buchwietz Bryan Copple Shawn Kinser Jim Luyendyk Jane Maddox Rosie Sneed National Institutes of Health Lance Pohl Michael Adams Hamid Amouzadeh Mohammed Bourdi John George Michael Holt Mary Jane Masson Kevin Welch Santana Flores Thomas Wynn, NIAID