Joint meeting of the NC3Rs and Maths in Medicine

1. Translational models ofdrug-induced liver injury Dominic Williams MRC Centre for Drug Safety Science The University of Liverpool dom@liv.ac.uk

2. Centre Strategy for Investigating ADRs Investigation of the chemical Investigation of the patient

3. Integrated Mechanistic Drug Safety: Patient SAR SMR STR DrugClass Animal Chemistry Outcomes Biomarkers Bioanalysis Man Clinical Problem Research Question Mechanism In vitro Clinical Samples

4. Integrated Mechanistic Drug Safety: Chemical SAR SMR STR In vitro Bioanalysis Drug / Compound Biological Validation Chemical Validation Chemical Studies Man Animal Clinical Validation & Application

5. Integrated Mechanistic Drug Safety Dynamic & Iterative

6. Mechanistic Classification of Adverse Drug Reactions TYPE A (augmented) predictable exaggeration of pharmacological effect dose dependent TYPE B (idiosyncratic) unpredictable apparently dose-independent less common more severe TYPE C (chemical) predictable from chemical structure eg. Paracetamol Park et al., 1998

7. Drug Disposition Physiological, Pharmacological & Toxicological Cellular accumulation DRUG Toxicity Phase I/II/III bioactivation Chemically reactive metabolites Stable metabolites bioinactivation Excretion

9. enzyme

10. transporter

11. signalling protein

12. receptor

13. random autologous protein Phase I/II/III bioactivation Necrosis Chemically reactive metabolites Stable metabolites Apoptosis bioinactivation Hypersensitivity Excretion

14. Drug Metabolism: Pharmacology Cellular accumulation DRUG RESPONSE Concentration in Plasma Phase I/II Drug Stable metabolites Disposition Metabolism Absorption Excretion Drug plasma level Pharmacological exposure Excretion

15. Drug Metabolism: Toxicology Cellular accumulation DRUG RESPONSE Concentrations in affected organs Phase I/II Drug Stable metabolites Disposition Metabolism Absorption Excretion Drug & metabolites Pharmacological & Toxicological exposure Excretion

17. informs toxicology / drug safety

19. Drug bioactivation

20. Multi-cell cross-talk

21. Cell migrationInitiation processes Adaptation vs toxicity

22. The Hepatocyte: Defence Against Chemical Stress 1st line defence DRUG METABOLISM 2nd line defence ANTIOXIDANT RESPONSE 3rd line defence APOPTOSIS Basal expression of genes co-ordinating cell defence: Phase II enzymes, antioxidant proteins Induction of genes co-ordinating cell defence: Phase II enzymes, antioxidant proteins Stress Suicide of the cell: apoptosis Chemical / metabolite NECROSIS Reactive oxygen species Transcription factor: Nrf2 GSH Increasing levels of chemical stress

23. Mechanism of Nrf2-regulated Gene Induction Chemical (metabolite) Nrf2 Chemical Nrf2 GSH repletion Nrf2 Target genes ARE GSH depletion Adduct formation Protein oxidation Cell defence proteins: Glutamate CysteineLigase Glutathione transferases NAD(P)H quinoneoxidoreductase Haemoxygenase Glucuronyltransferase Catalase Nrf2 Keap1 Proteasomal proteolysis Restore cellular redox status ADAPTATION Goldring et al., 2004; Williams et al., 2004; Randle et al., 2008; Copple et al., 2008; Reismanet al., 2009

25. Most common form DILI in US & UK

26. 400-500 deaths/yr, 70-100,000 hospital visits/yr

27. Centrilobular damage

28. Concern over chronic administration

29. Treatment with N-acetylcysteine

30. Cannot design out toxicityLee W.B. AASLD 2009

32. Most common form DILI in US & UK

33. 400-500 deaths/yr, 70-100,000 hospital visits/yr

34. Centrilobular damage

35. Concern over chronic administration

36. Treatment with N-acetylcysteine

38. 900 800 700 600 500 Nuclear Nrf2 (% control) 400 300 200 TOXICITY 100 0 0 200 400 600 800 1000 Paracetamol (mg/Kg) Mechanism of Nrf2-regulated Gene Induction Nrf2 Nrf2 GSH depletion Adduct formation Protein oxidation Nrf2 Keap1 Proteasomal proteolysis Goldring et al., 2004; Williams et al., 2004; Randle et al., 2008; Copple et al., 2008; Reismanet al., 2009

39. Individual Nrf2-dependent genes APAP: 530mg/kg – 1hour mEH mRNA/18S mEH 5 salines 5 APAP 900 HO-1 800 HO-1 mRNA/18S 700 5 salines 5 APAP 600 5 salines 5 APAP Nuclear Nrf2 (% control) GCLC GCLC mRNA/18S 500 5 salines 5 APAP 28S 18S 400 300 200 TOXICITY 100 0 0 200 400 600 800 1000 Paracetamol Hepatic nuclear translocation of Nrf2 in paracetamol-treated mice Nrf2- regulated Genes Visualize 12500 genes (mg/kg) Cell defence Goldring et al, Hepatology, 2004 Williams et al., Chem. Res. Toxicol, 2004

40. CCl3 In vivo Induction of Nrf2 by Model Hepatotoxins Covalent Binding GSH Depletion Nrf2 Induction O O C H C H H N 3 3 N Required Yes O H O B r B r B r O Required Yes O O Yes May occur CCl4 C l C l May occur Yes H N O S H N O S 2 2 2 2 O O N N C O H H Randle et al., 2008 C O H H O 2 2

43. GSH depletion 1 hourCoppleet al. Hepatology 2008

45. GSH depletion 1 hour

46. GSH resynthesis >8hCoppleet al. Hepatology 2008

47. Systems Regulation of Redox Electrophilic and/or oxidative stress Controller Tuneable actuator Plant GCL GR CAT GPx SOD GST UGT others + Other signals (AP1, NFkB) + Redox State + + Transducer + - GSH:GSSG + + - Keap1 Nrf2 + + ARE genes - + + + Zhang et al., Toxicol App Pharmacol, 2010

48. Systems Regulation of Redox Electrophilic and/or oxidative stress Controller Tuneable actuator Plant GCL GR CAT GPx SOD GST UGT others + Other signals (AP1, NFkB) + Redox State + + Transducer + - GSH:GSSG + + - Keap1 Nrf2 + + ARE genes - + + + Zhang et al., Toxicol App Pharmacol, 2010

49. Systems Regulation of Redox Electrophilic and/or oxidative stress Controller Tuneable actuator Plant GCL GR CAT GPx SOD GST UGT others + Other signals (AP1, NFkB) + Redox State + + Transducer + - GSH:GSSG + + - Keap1 Nrf2 + + ARE genes - + + + Zhang et al., Toxicol App Pharmacol, 2010

50. Systems Regulation of Redox Electrophilic and/or oxidative stress Controller Plant Tuneable actuator GCL GR CAT GPx SOD GST UGT others + Other signals (AP1, NFkB) + Redox State + TF cross talk + Transducer + - GSH:GSSG + + - Keap1 Nrf2 + + ARE genes - + + + Zhang et al., Toxicol App Pharmacol, 2010

52. 3-hydroxyanthraniliate 3,4-dioxygenase

53. Glycine N-methyltransferase

54. Aryl sulfotransferaseCovalent Binding Oxidative stress

56. Ca2+/Mg2+ ATPase

58. 3-hydroxyanthraniliate 3,4-dioxygenase

59. Glycine N-methyltransferase

61. Urateoxidase

63. Loss in activity or function and eventual cell death and lysis

64. Loss of energy production

65. Loss of cellular ion control

67. Peroxynitrite, Ca2+, MPT, DYm

68. Inactivation SOD

69. Uncoupling of oxidative phosphorylation

70. ATP depletion

71. Caspase release

73. Superoxidesgenerated

74. Increased NO synthesis

75. Increased peroxynitrite

77. Recruitment of neutrophils

78. Release of signalling molecules

79. NO, superoxide, IL-1, IL-6, TNF-α

82. Protein-reactive metabolite(s)Both animal models and in vitro systems are limited when assessing the hazard to susceptible patients: Biology of individual Occurrence, Frequency & Severity of Drug Hepatotoxicity f1 f2 + = Chemistry of drug