Nc3R\'s Meeting

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Joint meeting of the NC3Rs and Maths in Medicine

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  • Major consequence of bioactivation is in fact bioinactivation
  • Most common consequence of bioactivation is in fact bioinactivation
  • This slide shows the STRESSED situation, where the system is subject to excessive electrophilic or oxidative stress e.g. Exposure to NAPQIElectrophilic and oxidative stress are detected directly by Keap1 through its cysteine ‘sensors’This stimulates enhanced Nrf2 activation
  • This slide shows the STRESSED situation, where the system is subject to excessive electrophilic or oxidative stress e.g. Exposure to NAPQIElectrophilic and oxidative stress are detected directly by Keap1 through its cysteine ‘sensors’This stimulates enhanced Nrf2 activation
  • ....This is reinforced by signals from the plant through depletion of GSH (depressed GSH/GSSG ratio)Resulting in further Nrf2 activation – e.g. Through phosphorylation and also by directly enhancing the activity of antioxidant enzymes, e.g. By dimerisation
  • In saddition other signalling pathways may be recruited to strenghten the defence response, e.g. AP1 and NfkB, which can act either directly or indirectly to enhance ARE-mediated signalsThere is increasing evidence of cross-talk between such pathways e.g. Nrf2 and NfkBFurther supporting the value of a full SYTEMS APPROACH
  • Nc3R\'s Meeting

    1. 1. Translational models ofdrug-induced liver injury <br />Dominic Williams<br />MRC Centre for Drug Safety Science<br />The University of Liverpool<br />dom@liv.ac.uk<br />
    2. 2. Centre Strategy for Investigating ADRs<br />Investigation of the chemical<br />Investigation of the patient<br />
    3. 3. Integrated Mechanistic Drug Safety: Patient<br />SAR<br />SMR<br />STR <br />DrugClass<br />Animal<br />Chemistry<br />Outcomes<br />Biomarkers <br />Bioanalysis<br />Man<br />Clinical Problem <br />Research<br />Question <br />Mechanism <br />In vitro<br />Clinical Samples <br />
    4. 4. Integrated Mechanistic Drug Safety: Chemical<br />SAR<br />SMR<br />STR <br />In vitro<br />Bioanalysis<br />Drug / Compound <br />Biological Validation <br />Chemical Validation <br />Chemical Studies<br />Man<br />Animal<br />Clinical Validation & Application <br />
    5. 5. Integrated Mechanistic Drug Safety<br />Dynamic & Iterative<br />
    6. 6. Mechanistic Classification of Adverse Drug Reactions<br />TYPE A (augmented)<br /> predictable<br /> exaggeration of pharmacological effect<br /> dose dependent<br />TYPE B (idiosyncratic)<br /> unpredictable<br /> apparently dose-independent<br /> less common<br /> more severe<br />TYPE C (chemical)<br /> predictable from chemical structure<br />eg. Paracetamol<br />Park et al., 1998<br />
    7. 7. Drug Disposition Physiological, Pharmacological & Toxicological <br />Cellular<br />accumulation<br />DRUG<br />Toxicity<br />Phase I/II/III<br />bioactivation<br />Chemically<br />reactive<br />metabolites<br />Stable<br />metabolites<br />bioinactivation<br />Excretion<br />
    8. 8. Consequences of drug bioactivation <br />Cellular<br />accumulation<br />DRUG<br />Toxicity<br />Carcinogenicity<br />Chemical Stress<br />Modification of:<br /><ul><li> nucleic acid
    9. 9. enzyme
    10. 10. transporter
    11. 11. signalling protein
    12. 12. receptor
    13. 13. random autologous</li></ul> protein<br />Phase I/II/III<br />bioactivation<br />Necrosis<br />Chemically <br />reactive<br />metabolites<br />Stable<br />metabolites<br />Apoptosis<br />bioinactivation<br />Hypersensitivity<br />Excretion<br />
    14. 14. Drug Metabolism: Pharmacology<br />Cellular <br />accumulation<br />DRUG<br />RESPONSE<br />Concentration in<br />Plasma<br />Phase I/II<br />Drug<br />Stable<br />metabolites<br />Disposition<br />Metabolism<br />Absorption<br />Excretion<br />Drug plasma level<br />Pharmacological exposure<br />Excretion<br />
    15. 15. Drug Metabolism: Toxicology<br />Cellular <br />accumulation<br />DRUG<br />RESPONSE<br />Concentrations in<br />affected organs<br />Phase I/II<br />Drug<br />Stable<br />metabolites<br />Disposition<br />Metabolism<br />Absorption<br />Excretion<br />Drug & metabolites<br />Pharmacological &<br />Toxicological exposure<br />Excretion<br />
    16. 16. Drug Metabolism:Chemical & biological toolbox<br />Use model or paradigm drug/chemicals to perturb biological processes<br /><ul><li> elucidates biological mechanisms
    17. 17. informs toxicology / drug safety
    18. 18. assists in biomarker discovery & drug development</li></ul>Use known, protein-reactive, hepatotoxins to understand processes which may occur in susceptible patients<br />Animals provide integrated & holistic toxicological models <br /><ul><li>Pharmaco-dynamics/kinetics
    19. 19. Drug bioactivation
    20. 20. Multi-cell cross-talk
    21. 21. Cell migration</li></ul>Initiation processes <br />Adaptation vs toxicity<br />
    22. 22. The Hepatocyte: Defence Against Chemical Stress <br />1st line defence<br />DRUG METABOLISM<br />2nd line defence<br />ANTIOXIDANT <br />RESPONSE<br />3rd line defence<br />APOPTOSIS<br />Basal expression <br />of genes<br />co-ordinating<br />cell defence:<br />Phase II enzymes, antioxidant proteins<br />Induction<br />of genes<br />co-ordinating<br />cell defence:<br />Phase II enzymes, antioxidant proteins<br />Stress<br />Suicide<br />of the cell:<br />apoptosis <br />Chemical / metabolite<br />NECROSIS<br />Reactive oxygen species<br />Transcription factor:<br />Nrf2<br />GSH<br />Increasing levels of chemical stress<br />
    23. 23. Mechanism of Nrf2-regulated Gene Induction<br />Chemical<br />(metabolite)<br />Nrf2<br />Chemical<br />Nrf2<br />GSH repletion<br />Nrf2 Target genes<br />ARE<br />GSH depletion<br />Adduct formation<br />Protein oxidation<br />Cell defence proteins:<br />Glutamate CysteineLigase<br />Glutathione transferases<br />NAD(P)H quinoneoxidoreductase<br />Haemoxygenase<br />Glucuronyltransferase<br />Catalase<br />Nrf2<br />Keap1<br />Proteasomal proteolysis<br />Restore cellular redox status<br />ADAPTATION<br />Goldring et al., 2004; Williams et al., 2004; Randle et al., 2008; Copple et al., 2008; Reismanet al., 2009<br />
    24. 24. Paracetamol (APAP; acetaminophen)<br /><ul><li>Recommended dose - 4g. Toxic dose >4g
    25. 25. Most common form DILI in US & UK
    26. 26. 400-500 deaths/yr, 70-100,000 hospital visits/yr
    27. 27. Centrilobular damage
    28. 28. Concern over chronic administration
    29. 29. Treatment with N-acetylcysteine
    30. 30. Cannot design out toxicity</li></ul>Lee W.B. AASLD 2009<br />
    31. 31. Paracetamol (APAP; acetaminophen)<br /><ul><li>Recommended dose - 4g. Toxic dose >4g
    32. 32. Most common form DILI in US & UK
    33. 33. 400-500 deaths/yr, 70-100,000 hospital visits/yr
    34. 34. Centrilobular damage
    35. 35. Concern over chronic administration
    36. 36. Treatment with N-acetylcysteine
    37. 37. Cannot design out toxicity</li></li></ul><li>Mechanism of Cell Damage: a multicellular event<br />Innate immune response<br />NK/NK T cells<br />Lymphocytes<br />Neutrophils (?)*<br />Kupffer cells<br />Infiltrating Macrophages<br />GSH depletion<br />Oxidative Stress<br />Covalent Binding<br />Pro:<br />IFNg<br />FasL<br />TNF<br />HMGB-1<br />Anti:<br />IL-10<br />IL-6<br />IL-13<br />protein damage<br />-<br />-<br />-<br />+<br />+<br />+<br />Mild<br />injury<br />Inflammation<br />Severity<br />Of<br />injury<br />Yee et al., 2007; Masson et al., 2008; Holt et al., 2008; *Williams et al., 2010<br />
    38. 38. 900<br />800<br />700<br />600<br />500<br />Nuclear Nrf2 (% control)<br />400<br />300<br />200<br />TOXICITY<br />100<br />0<br />0<br />200<br />400<br />600<br />800<br />1000<br />Paracetamol (mg/Kg)<br />Mechanism of Nrf2-regulated Gene Induction<br />Nrf2<br />Nrf2<br />GSH depletion<br />Adduct formation<br />Protein oxidation<br />Nrf2<br />Keap1<br />Proteasomal proteolysis<br />Goldring et al., 2004; Williams et al., 2004; Randle et al., 2008; Copple et al., 2008; Reismanet al., 2009<br />
    39. 39. Individual Nrf2-dependent genes<br />APAP: 530mg/kg – 1hour <br />mEH mRNA/18S<br />mEH<br />5 salines 5 APAP<br />900<br />HO-1<br />800<br />HO-1 mRNA/18S<br />700<br />5 salines 5 APAP<br />600<br />5 salines 5 APAP<br />Nuclear Nrf2 (% control)<br />GCLC<br />GCLC mRNA/18S<br />500<br />5 salines 5 APAP<br />28S<br />18S<br />400<br />300<br />200<br />TOXICITY<br />100<br />0<br />0<br />200<br />400<br />600<br />800<br />1000<br />Paracetamol<br />Hepatic nuclear translocation of Nrf2 in paracetamol-treated mice<br />Nrf2-<br />regulated<br />Genes<br />Visualize<br />12500 genes<br />(mg/kg)<br />Cell defence<br />Goldring et al, Hepatology, 2004<br />Williams et al., Chem. Res. Toxicol, 2004<br />
    40. 40. CCl3<br />In vivo Induction of Nrf2 by Model Hepatotoxins<br />Covalent<br />Binding<br />GSH<br />Depletion<br />Nrf2 Induction<br />O<br />O<br />C<br />H<br />C<br />H<br />H<br />N<br />3<br />3<br />N<br />Required<br />Yes<br />O<br />H<br />O<br />B<br />r<br />B<br />r<br />B<br />r<br />O<br />Required<br />Yes<br />O<br />O<br />Yes<br />May occur<br />CCl4<br />C<br />l<br />C<br />l<br />May occur<br />Yes<br />H<br />N<br />O<br />S<br />H<br />N<br />O<br />S<br />2<br />2<br />2<br />2<br />O<br />O<br />N<br />N<br />C<br />O<br />H<br />H<br />Randle et al., 2008<br />C<br />O<br />H<br />H<br />O<br />2<br />2<br />
    41. 41. In vitro Nrf2 activation by NAPQI<br />Nrf2<br />β-Actin<br />paracetamol<br />NAPQI<br />N-acetyl p-benzoquinoneimine (NAPQI)<br />Hepatotoxic APAP metabolite<br />Mouse hepatoma cells - Hepa 1c1c7<br /><ul><li>Nrf2 translocation 1 hour</li></ul>Coppleet al. Hepatology 2008<br />
    42. 42. In vitro Nrf2 activation by NAPQI<br />paracetamol<br />NAPQI<br />N-acetyl p-benzoquinoneimine (NAPQI)<br />Hepatotoxic APAP metabolite<br />Mouse hepatoma cells - Hepa 1c1c7<br /><ul><li>Nrf2 translocation 1 hour
    43. 43. GSH depletion 1 hour</li></ul>Coppleet al. Hepatology 2008<br />
    44. 44. In vitro Nrf2 activation by NAPQI<br />paracetamol<br />NAPQI<br />N-acetyl p-benzoquinoneimine (NAPQI)<br />Hepatotoxic APAP metabolite<br />Mouse hepatoma cells - Hepa 1c1c7<br /><ul><li>Nrf2 translocation 1 hour
    45. 45. GSH depletion 1 hour
    46. 46. GSH resynthesis >8h</li></ul>Coppleet al. Hepatology 2008<br />
    47. 47. Systems Regulation of Redox<br />Electrophilic and/or oxidative stress<br />Controller<br />Tuneable actuator<br />Plant<br />GCL<br /> GR <br />CAT<br />GPx<br />SOD<br />GST<br />UGT<br />others<br />+<br />Other signals<br />(AP1, NFkB)<br />+<br />Redox State<br />+<br />+<br />Transducer<br />+<br />-<br />GSH:GSSG<br />+<br />+<br />-<br />Keap1<br />Nrf2<br />+<br />+<br />ARE<br />genes<br />-<br />+<br />+<br />+<br />Zhang et al., Toxicol App Pharmacol, 2010<br />
    48. 48. Systems Regulation of Redox<br />Electrophilic and/or oxidative stress<br />Controller<br />Tuneable actuator<br />Plant<br />GCL<br /> GR <br />CAT<br />GPx<br />SOD<br />GST<br />UGT<br />others<br />+<br />Other signals<br />(AP1, NFkB)<br />+<br />Redox State<br />+<br />+<br />Transducer<br />+<br />-<br />GSH:GSSG<br />+<br />+<br />-<br />Keap1<br />Nrf2<br />+<br />+<br />ARE<br />genes<br />-<br />+<br />+<br />+<br />Zhang et al., Toxicol App Pharmacol, 2010<br />
    49. 49. Systems Regulation of Redox<br />Electrophilic and/or oxidative stress<br />Controller<br />Tuneable actuator<br />Plant<br />GCL<br /> GR <br />CAT<br />GPx<br />SOD<br />GST<br />UGT<br />others<br />+<br />Other signals<br />(AP1, NFkB)<br />+<br />Redox State<br />+<br />+<br />Transducer<br />+<br />-<br />GSH:GSSG<br />+<br />+<br />-<br />Keap1<br />Nrf2<br />+<br />+<br />ARE<br />genes<br />-<br />+<br />+<br />+<br />Zhang et al., Toxicol App Pharmacol, 2010<br />
    50. 50. Systems Regulation of Redox<br />Electrophilic and/or oxidative stress<br />Controller<br />Plant<br />Tuneable actuator<br />GCL<br /> GR <br />CAT<br />GPx<br />SOD<br />GST<br />UGT<br />others<br />+<br />Other signals<br />(AP1, NFkB)<br />+<br />Redox State<br />+<br />TF cross talk<br />+<br />Transducer<br />+<br />-<br />GSH:GSSG<br />+<br />+<br />-<br />Keap1<br />Nrf2<br />+<br />+<br />ARE<br />genes<br />-<br />+<br />+<br />+<br />Zhang et al., Toxicol App Pharmacol, 2010<br />
    51. 51. Critical Chemical events in APAP-DILI<br />Cytosol<br />-3-HAO <br />-Carbonate dehydratase III <br />-Glutathione peroxidase<br />-Glutathione S-transferases <br />-GAPDH<br />-Thioredoxinperoxidase 2 <br />-Selenium-binding protein <br />-Amine N-methyltransferase<br />-Tropomyosin 3 <br />-Selenium-binding protein 1 <br />-Keap1 <br />-γ-Glutamylcysteinylsynthetase<br />-Protein phosphatase<br />-Proteasome<br />-Tryptophan-2,3-dioxygenase<br />-MIF tautomerase<br />-Methionineadenosyltransferase<br />-Aldehydedehydrogenase<br />-Carbonic anhydrase<br />-N-10-formyl-H4-folate dehydrogenase <br /><ul><li>Osteoblast specific factor 33
    52. 52. 3-hydroxyanthraniliate 3,4-dioxygenase
    53. 53. Glycine N-methyltransferase
    54. 54. Aryl sulfotransferase</li></ul>Covalent Binding<br />Oxidative stress<br />
    55. 55. Critical Chemical events in APAP-DILI<br />Plasma membrane<br /><ul><li>Disulphide isomerase
    56. 56. Ca2+/Mg2+ ATPase
    57. 57. Na+/K+ ATPase</li></ul>Endoplasmic reticulum<br />-Calregulin<br />-Glutamine synthase<br />-ER transmembrane protein<br />- Thioether-s-methyltansferase<br />Cytosol<br />-3-HAO <br />-Carbonate dehydratase III <br />-Glutathione peroxidase<br />-Glutathione S-transferases <br />-GAPDH<br />-Thioredoxinperoxidase 2 <br />-Selenium-binding protein <br />-Amine N-methyltransferase<br />-Tropomyosin 3 <br />-Selenium-binding protein 1 <br />-Keap1 <br />-γ-Glutamylcysteinylsynthetase<br />-Protein phosphatase<br />-Proteasome<br />-Tryptophan-2,3-dioxygenase<br />-MIF tautomerase<br />-Methionineadenosyltransferase<br />-Aldehydedehydrogenase<br />-Carbonic anhydrase<br />-N-10-formyl-H4-folate dehydrogenase <br /><ul><li>Osteoblast specific factor 33
    58. 58. 3-hydroxyanthraniliate 3,4-dioxygenase
    59. 59. Glycine N-methyltransferase
    60. 60. Aryl sulfotransferase</li></ul>Nucleus<br />- Lamin A<br />Covalent Binding<br />Oxidative stress<br />Mitochondria<br />-Aldehydedehydrogenase<br />-Carbonic anhydrase<br />-Glutathione peroxidase<br /> -N-10-formyl-H4-folate dehydrogenase<br />-ATP synthase<br />-House keeping protein<br />-Carbamoylphosphatesynthetase<br />-Glutamate Dehydrogenase<br />-Antioxidant protein 1 <br />-Inorganic pyrophosphatase<br />-3-hydroxy-3-methylglutaryl coenzyme A synthase 2 <br />-2-4-dienoyl-CoA reductase<br />Peroxisomes<br /><ul><li>Catalase
    61. 61. Urateoxidase
    62. 62. 2,4-dienoyl-CoA reductase</li></li></ul><li>Critical Biological events in APAP-DILI:<br />Intracellular<br />Covalent Binding<br /><ul><li>NAPQI binds to cysteine groups on proteins
    63. 63. Loss in activity or function and eventual cell death and lysis
    64. 64. Loss of energy production
    65. 65. Loss of cellular ion control
    66. 66. Altered membrane ATPase activity</li></ul>Covalent Binding<br />Mitochondrial dysfunction<br /><ul><li>Superoxide generation occurs with MPT
    67. 67. Peroxynitrite, Ca2+, MPT, DYm
    68. 68. Inactivation SOD
    69. 69. Uncoupling of oxidative phosphorylation
    70. 70. ATP depletion
    71. 71. Caspase release
    72. 72. Protein nitrosylation</li></ul>Oxidative stress<br />Oxidative stress<br /><ul><li>GSH depleted reduced activity of GSH peroxidase
    73. 73. Superoxidesgenerated
    74. 74. Increased NO synthesis
    75. 75. Increased peroxynitrite
    76. 76. Oxidation of lipids, proteins, DNA bases</li></li></ul><li>Critical Biological events in APAP-DILI:<br />Extracellular<br />Pro-inflammatory cytokines<br />Anti-inflammatory cytokines<br />Other mechanistically useful proteins<br />Other hepatocytes &<br />innate immune cells<br />Inflammation<br /><ul><li> Activation of Kupffer cells
    77. 77. Recruitment of neutrophils
    78. 78. Release of signalling molecules
    79. 79. NO, superoxide, IL-1, IL-6, TNF-α
    80. 80. Pro-inflammatory cytokines contribute towards toxicity</li></li></ul><li>Adaptation vs Failure to adapt<br />Critical target vs non-critical target<br />Idiosyncratic Hepatotoxicity<br />Form a hapten recognised by adaptive immune system<br />Which organ ?<br />Which cell type ?<br />Which organelle ?<br />PROTEIN<br />Which protein ?<br />Which amino acid ?<br />Which atom ?<br /><ul><li> Low direct toxicity if: </li></ul> - no binding<br /> - binding mainly to non-critical targets<br /><ul><li> Very toxic if highly selective for a criticaltarget</li></ul>Act as ‘danger’ signals which can trigger the adaptive immune system<br />Cytotoxicity<br />Release of cell contents which activate immune cells <br />
    81. 81. Summary<br />Integration of animal models with in vitro data is crucial<br />Relationship between bioactivation and adaptation is required to assess the chemical hazard of: <br /><ul><li>Parent drug
    82. 82. Protein-reactive metabolite(s)</li></ul>Both animal models and in vitro systems are limited when assessing the hazard to susceptible patients:<br />Biology of individual<br />Occurrence, Frequency<br />& Severity of<br />Drug Hepatotoxicity<br />f1<br />f2<br />+<br />=<br />Chemistry<br />of drug<br />

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