1. 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 U.S. Department of Health and Human Services National Institutes of Health National Heart, Lung, and Blood Institute

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

3. Examples of Drugs Withdrawn Due to Liver Disease Iproniazid1956 Ibufenac (in Europe only)1975 Ticrynafen1979 Benoxaprofen1982 Perhexiline (in France)1985 Dilevalol (in Portugal, Ireland)1990 Bromfenac 1998 Troglitazone 2000 Serzone2004

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

5. Hepatotoxicants Potentiated by Exposure to Small Doses of Lipopolysaccharide (LPS) Xenobiotics CCl 4 GalactosamineEthanolT2-toxinCadmiumHalothaneLead Allyl Alcohol Aflatoxin B 1 ChlorpromazineRanitidineSource 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. LPS MacrophagesNeutrophils Endothelial Cells Epithelial Cells TLR4 CytokinesCoagulation Factors Platelet Activating FactorComplement Activation LeukotrienesArachidonic Acid Metabolites ProstaglandinsReactive Oxygen Species Nitric Oxide Cellular Level Events of LPS

7. Monocrotaline (MCT) N CH 2 O O O=C CC=O CC OH CH 3 OH H3CH3CH3CH3C H Monocrotaline Crotalaria Spectabilis (Rattlebox)

8. N CH 2 O O O=C CC=O CC OH CH 3 OH H3CH3CH3CH3C H MCT N CH 2 O O O=C CC=O CC OH CH 3 OH H3CH3CH3CH3C H MonocrotalinePyrrole(MCTP) CYP 3A Covalent Binding to CellularMacromolecules Cell Death MCT Toxicity

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

14. Neutrophil Depletion Attenuates MCT/LPS Hepatotoxicity MCT/LPS Hepatotoxicity

15. Inactivation of the Coagulation System Decreases Liver Injury

16. Summary

17. Does a loss in hepatoprotective factors result in drug-induced liver disease? Drug Liver ReactiveMetabolites ProteinAdducts CellularHomeostasisAltered (Inhibition) Response Toxicity Protection IL-6, IL-10, COX-2 Stress Proteins ProtectiveFactors Extensive Liver Injury And Death

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

19. APAP-Induced Liver Injury HNCH 3 O OH ACETAMINOPHEN NCH 3 O O NAPQI SulfationGlucuronidation Detox GSH Detox Cysteinyl Conjugate Protein Adducts Mitochondria Dysfunction Reactive Oxygen Species LIVER INJURY P450 2E1 P450 1A2 P450 3A4 Depleted

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

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. Liver Histopathology at 8 hours CVCV CV CV CV CV CV CV CAb/VehCAb/APAP IL-13 NAb/APAP APAP - KO

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

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

26. Idiosyncratic Drug-Induced Liver Disease Complex “multihit” process Complex “multihit” process Liver protoxicants and protectants have a role in the overall pathogenesis 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 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 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 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