Successfully reported this slideshow.
We use your LinkedIn profile and activity data to personalize ads and to show you more relevant ads. You can change your ad preferences anytime.

Williams Az July

1,130 views

Published on

  • Be the first to comment

Williams Az July

  1. 1. Mechanistic InvestigationsinDrug Safety<br />Dominic Williams<br />
  2. 2. Mechanistic Investigations in Drug Safety <br />Introduction<br />ADRs<br />On and off target toxicity<br />Acetaminophen model of murine toxicity<br />Mechanistic markers of injury progression<br />Apoptosis (K18), necrosis (HMGB-1), inflammation (Ac-HMGB-1)<br />Fed and fasted animal models<br />Nevirapine hypersensitivity<br />Man vs animal model<br />Chemical similarities<br />
  3. 3. Adverse Drug Reactions (ADRs)<br /><ul><li>patient morbidity
  4. 4. patient mortality
  5. 5. prohibit effective drug therapy
  6. 6. drug withdrawal
  7. 7. attrition</li></li></ul><li>Lessons for the future<br />Inform mechanism and pathogenesis<br />Inform the Medicinal Chemist<br />Inform the Clinician<br />Inform the Regulator<br />Inform the Public – what is feasible<br />Develop biomarkers for integrated patient, in vitro & animal studies<br />
  8. 8. Classification of Adverse Drug Reactions<br />ON TARGET <br />Reactions that are predictable from the known primary or secondary pharmacology of the drug. <br />Often representing an exaggeration of the pharmacological effect of the drug.<br />– clear dose-dependent relationship within the individual patient.<br />OFF TARGET <br />These are not predictable from a knowledge of the basic pharmacology of the drug and can exhibit marked inter-individual susceptibility (idiosyncratic).<br />– complex dose dependence. <br />
  9. 9. Drugs withdrawn from major markets due to hepatotoxicity<br />Drug: Therapeutic area:<br />- Alpidem* Anxiolytic<br />Aspirin (children) NSAID - <br />- Bendazac* NSAID<br />BenoxaprofenNSAID -<br />- Bromfenac* NSAID<br />Chlormezanone* Anxiolytic -<br />- Dilavelol* Anti-hypertensive<br />Ebrotidine* H2 receptor antagonist -<br />- Fipexide* Stimulant<br />Nefazodone* Anti-depressant - <br />- Nimesulide NSAID<br />Nomifensine Anti-depressant - <br />- Oxyphenisatin Laxative <br />Pemoline* ADHD - <br />- Perhexilene Anti-anginal<br />Temafloxacin* Anti-infective - <br />- Tolcapone*Anti-parkinson’s<br />Tolrestat* Anti-diabetic - <br />- Troglitazone* Anti-diabetic <br />Trovafloxacin* Antibiotic - <br />Ximelagatran- Anti-coagulant<br />- Zimeldine Anti-depressant <br />* Need et al., Nat Genetics 2005<br />
  10. 10. Drug Metabolism: Pharmacology <br />Cellular <br />accumulation<br />DRUG<br />RESPONSE<br />Phase I/II<br />Stable<br />metabolites<br />Excretion<br />
  11. 11. Drug Metabolism: Pharmacology <br />Cellular <br />accumulation<br />DRUG<br />RESPONSE<br />Phase I/II<br />Stable<br />metabolites<br />Excretion<br />
  12. 12. Liver is key player in drug metabolism and toxicity<br /><ul><li> 2 blood supplies – portal (intestinal) 75%, arterial 25 %
  13. 13. High exposure to drugs and nutrients as first organ after absorption
  14. 14. Major organ for drug metabolism </li></ul>Hepatic portal vein<br />Hepatic artery<br />Biotransformation<br />To Metabolites<br />Lipophillic compounds<br />Central vein<br />Bile<br />Enterohepatic<br />recirculation<br />Urine<br />Faeces<br />
  15. 15. Multi-Lobular Arrangement of the Liver<br />Portal Triad<br />Centrilobular<br />Region with<br />highest density<br />of CYP450 <br />metabolising<br />enzymes <br />Capilliaries with fenestrations<br />
  16. 16. Toxicity<br />Variation in Drug Metabolism - Toxicity <br />Cellular <br />accumulation<br />DRUG<br />Variation in<br />Drug Metabolism<br />CYP<br />ENZYME<br />Phase I/II<br />Stable<br />metabolites<br /><ul><li> genetics
  17. 17. enzyme induction
  18. 18. enzyme inhibition
  19. 19. disease</li></ul>Excretion<br />Shah et al 1982; Davies et al 2007<br />
  20. 20. PerhexileneMaleate and CYP2D6<br />Indications - prevention of angina pectoris<br /> - prevention of ventricular systoles<br />Toxicity - peripheral neuropathy<br /> - hepatotoxicity<br />Pharmacokinetics: <br /> T1/2 Metabolic ratio (%D/%M)<br /> - ADR patients 2-6 0.3 ± 0.4<br /> + ADR patients 9-22 2.82 ± 0.4<br /> - related to CYP2D6 phenotype<br />HO<br />(Singlaset al, 1978; Shah et al, 1982; Cooper et al, 1984)<br />
  21. 21. Lipidosis Induced by Amphiphilic Cationic Drugs<br /> PHOSPHOLIPIDS<br /> PHOSPHOLIPIDS<br />Extracellular<br />Cytosol<br />Lysosome<br />pH 7.4<br />pH 7.2<br />pH 4.5<br />
  22. 22. Prediction of Variation in Drug Metabolism <br /><ul><li> Human liver banks
  23. 23. Expression systems
  24. 24. Cell lines
  25. 25. In silico techniques / models
  26. 26. Transgenic animals
  27. 27. Volunteer & Patient studies
  28. 28. Genotype
  29. 29. Phenotype</li></ul>http://www.imm.ki.se/CYPalleles/<br /><ul><li> genetics
  30. 30. enzyme induction
  31. 31. enzyme inhibition
  32. 32. disease</li></ul>Development of<br />pre-clinical screens<br />Transfer of knowledge<br />to clinical practice<br />
  33. 33. Off Target Clinical Adverse Drug Reactions <br />Drug Adverse Reaction<br />Amodiaquine Hepatotoxicity<br />Paracetamol Hepatotoxicity<br />Halothane Hepatotoxicity<br />Diclofenac Hepatotoxicity<br />Tacrine Hepatotoxicity<br />Indomethacin Hepatotoxicity<br />Valproic Acid Hepatotoxicity<br />Vesnarinone Hepatotoxicity<br />Phenacetin Hepatotoxicity<br />PhenytoinTeratogenicity / Hepatotoxicity<br />ClozapineAgranulocytosis<br />AminopyreneAgranulocytosis<br />TiclopidineAgranulocytosis<br />Sulfamethoxazole Toxic epidermal necrolysis<br />Lamotrigene Toxic epidermal necrolysis<br />Carbamazepine Hypersensitivity<br />Tienilic acid Hypersensitivity<br />FelbamateAplasticanaemia<br />RemoxiprideAplastic Anaemia<br />Reactive Metabolite<br />Quinone imine<br />Quinone Imine<br />Acyl halide<br />Quinone imine / acylglucuronide<br />Quinone methide<br />Quinone imine / chloro-indole<br />a, b unsaturated carbonyl<br />Iminium ion<br />Quinone imine<br />Free radical<br />Nitrenium ion<br />iminium<br />S-oxide<br />Hydroxylamine / nitroso<br />epoxide<br />Quinone imine / epoxide<br />S-oxide<br />atropaldehyde<br />hydroquinone<br />
  34. 34. Drug Safety Science and DILI<br />CLEARANCE<br />20 / safety pharmacology targets<br />DRUG<br />Ca2+<br />DRUG<br />+<br />METABOLITE<br />DNA<br />TARGET<br />phospholipid<br />specific proteins<br />BIOMARKERS<br />Phospholipid<br />Clearance<br />Mitochondria<br />Ca2+<br />Proteins<br />DNA<br />PHARMACOLOGICAL EFFECT<br />ADVERSE<br />EFFECT<br />CHEMICAL STRUCTURE<br />Dose<br />SAFE DRUG DESIGN<br />
  35. 35. Drug Safety Science and DILI<br />CLEARANCE<br />20 / safety pharmacology targets<br />DRUG<br />2nd effects<br />1st effects<br />3rd effects<br />Ca2+<br />DRUG<br />+<br />METABOLITE<br />DNA<br />TARGET<br />phospholipid<br />specific proteins<br />Biology of individual<br />BIOMARKERS<br />PHARMACOLOGICAL EFFECT<br />ADVERSE<br />EFFECT<br />Occurrence, Frequency<br />& Severity of<br />Drug Hepatotoxicity<br />f1<br />f2<br />+<br />=<br />Chemistry<br />of drug<br />Dose<br />CHEMICAL STRUCTURE<br />f (chemistry)<br />f (biology)<br />SPECIES and INDIVIDUAL VARIATION<br />
  36. 36. Drug Safety Science and DILI<br />Amphiphilic<br />Cation<br />2D6 genotype /<br />phenotype<br />Biology of individual<br />Rationale for<br />Safe clinical use<br />Occurrence, Frequency<br />& Severity of<br />Drug Hepatotoxicity<br />f1<br />f2<br />+<br />=<br />Chemistry<br />of drug<br />
  37. 37. ADRs: Drug-Induced Liver Injury<br />Leading cause of acute liver failure1<br /><ul><li>APAP 46% of all cases
  38. 38. 12% other drugs</li></ul>High morbidity & mortality2<br /><ul><li>20% survival without transplant</li></ul>Main reason for late stage termination or withdrawal2<br /><ul><li>1975-1999 - 548 new drugs
  39. 39. 10 received ‘black-box’ warning
  40. 40. 4 were withdrawn</li></ul>1 Lee AASLD, 2009; 2 Verma & Kaplowitz 2009<br />
  41. 41. Drug-Induced Liver Injury: issues<br />In vivo biomarkers used for non-invasive DILI assessment <br /><ul><li>Clinic & pre-clinical toxicity screening</li></ul>Why is DILI in man still:<br /><ul><li> Main reason for late drug attrition
  42. 42. Leading cause of ALF</li></ul>Animal–human concordance is 50%1,2,3<br /><ul><li> Attrition in biomarker translation </li></ul>Caveats with current biomarkers3,4<br /><ul><li> ALT in muscle & kidney
  43. 43. ALT variation - circadian / enzyme induction
  44. 44. AST in heart, muscle, kidney & erythrocytes
  45. 45. LDH not specific</li></ul>Better biomarkers are required<br />Mechanistic understanding of perturbed physiological processes<br />1Olson et al.2000; 2Greaves et al. 2004; Amacher. 2010; 4Dufour et al., 2000<br />
  46. 46. Mechanisms of Drug Induced Liver Injury<br />Hepatic Injury<br />Biological stratification<br />Drug<br /><ul><li> accumulation
  47. 47. bioactivation
  48. 48. covalent binding
  49. 49. chemical stress
  50. 50. mitochondrial dysfunction
  51. 51. apoptosis
  52. 52. hepatocyte hypertrophy
  53. 53. hepatocyte hyperplasia
  54. 54. hepatocyte integrity
  55. 55. hepatobiliary integrity
  56. 56. innate immune activation
  57. 57. hepatic function
  58. 58. fibrosis
  59. 59. Plasma / liver level
  60. 60. GSH/mercapturate
  61. 61. Protein binding
  62. 62. chemical stress
  63. 63. glutamate dehydrogenase
  64. 64. cytochrome C
  65. 65. ALT, AST, SDH, LDH, aGST
  66. 66. ALP, gGT, 5’nucleotidase
  67. 67. Bilirubin, bile acids,
  68. 68. prothrombin, metabolism / secretion</li></ul>DRUG<br />+<br />METABOLITE<br />Biology<br />
  69. 69. Mechanisms of Drug Induced Liver Injury<br />Hepatic Injury<br />Biological stratification<br />Drug<br /><ul><li> accumulation
  70. 70. bioactivation
  71. 71. covalent binding
  72. 72. chemical stress
  73. 73. mitochondrial dysfunction
  74. 74. apoptosis
  75. 75. hepatocyte hypertrophy
  76. 76. hepatocyte hyperplasia
  77. 77. hepatocyte integrity
  78. 78. hepatobiliary integrity
  79. 79. innate immune activation
  80. 80. hepatic function
  81. 81. fibrosis
  82. 82. GSH conjugate
  83. 83. Irreversible binding
  84. 84. Keratin-18 fragment
  85. 85. Oxidised HMGB-1
  86. 86. HMGB-1, full-length K-18
  87. 87. Acetylated HMGB-1</li></ul>DRUG<br />+<br />METABOLITE<br />Biology<br />
  88. 88. Acetaminophen (APAP; paracetamol)<br /><ul><li>Recommended dose - 4g. Toxic dose >4g
  89. 89. Most common form DILI in US & UK
  90. 90. Centrilobular damage
  91. 91. Concern over chronic administration
  92. 92. Limit pack size
  93. 93. Treatment with N-acetylcysteine
  94. 94. Cannot design out toxicity</li></ul>Lee W.B. AASLD 2009<br />
  95. 95. Acetaminophen (APAP; paracetamol)<br /><ul><li>Recommended dose - 4g. Toxic dose >4g
  96. 96. Most common form DILI in US & UK
  97. 97. Centrilobular damage
  98. 98. Concern over chronic administration
  99. 99. Limit pack size
  100. 100. Treatment with N-acetylcysteine
  101. 101. Cannot design out toxicity</li></li></ul><li>Mechanism of Cell Damage: a multicellular event<br />Innate immune response<br />hepatocyte<br />NK/NK T cells<br />Lymphocytes<br />Neutrophils (?)*<br />Kupffer cells<br />Infiltrating Macrophages<br />Pro:<br />IFNg<br />FasL<br />TNF<br />HMGB-1<br />Anti:<br />IL-10<br />IL-6<br />IL-13<br />GSH depletion<br />Adduct formation <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 />
  102. 102. Mechanism of Cell Damage: Mechanistic Biomarkers<br />TLR2<br />TLR4<br />RAGE<br />TLR9<br />Release of HMGB<br />-<br />1 due to necrosis<br />injury<br />Innate<br />Inflammatory<br />Many cell types<br />Necrosis<br />Target<br />Cell <br />Necrosis:<br /><ul><li>HMGB-1 – chromatin binding protein
  103. 103. released by necrotic cells
  104. 104. But NOT by apoptotic cells
  105. 105. Cytokine activity (TLR/RAGE)</li></ul>Active Secretion of HMGB<br />-<br />1<br />Activation<br />Active<br />secretion<br />Inflammatory Cell<br />Different molecular forms<br />Release of<br />Keratin<br />fragment<br />No release<br />Release of Keratin-18 due to apoptosis<br />-<br />18<br />-<br />1<br />of HMGB<br />Apoptosis:<br /><ul><li>Keratin-18– intermediate filament protein
  106. 106. Is cleared by caspases
  107. 107. Fragment released into plasma
  108. 108. Full length K18 released by necrosis</li></ul>Apoptotic Cell<br />
  109. 109. Serum marker of necrosis<br />Necrosis:<br /><ul><li>HMGB-1 – chromatin binding protein
  110. 110. released by necrotic cells
  111. 111. But NOT by apoptotic cells
  112. 112. Cytokine activity (TLR/RAGE)</li></ul>1.<br />Release of HMGB<br />-1 due to necrosis<br />injury<br />Many cell types<br />Necrosis<br />Male CD-1 mice<br />dosed APAP 5h<br />Necrosis<br />500<br />***<br />***<br />***<br />400<br />300<br />HMGB1 (ng/ml)<br />200<br />100<br />0<br />0<br />200<br />400<br />600<br />800<br />1000<br />Antoine et al., 2009<br />Acetaminophen (mg/kg)<br />
  113. 113. Serum marker of necrosis<br />Necrosis:<br /><ul><li>HMGB-1 – chromatin binding protein
  114. 114. released by necrotic cells
  115. 115. But NOT by apoptotic cells
  116. 116. Cytokine activity (TLR/RAGE)</li></ul>Release of HMGB<br />-<br />1 due to necrosis<br />injury<br />Many cell types<br />Necrosis<br />Male CD-1 mice<br />dosed APAP 5h<br />Necrosis<br />10000<br />500<br />***<br />***<br />***<br />400<br />8000<br />300<br />6000<br />**<br />HMGB1 (ng/ml)<br />**<br />ALT activity (U/l)<br />200<br />4000<br />100<br />2000<br />**<br />0<br />0<br />0<br />200<br />400<br />600<br />800<br />1000<br />Antoine et al., 2009<br />Acetaminophen (mg/kg)<br />
  117. 117. Drug-Induced Liver Injury: mechanistic biomarkers<br />Release of HMGB<br />-<br />1 due to necrosis<br />injury<br />Many cell types<br />Necrosis<br />Male CD-1 mice<br />dosed APAP 530 mg/kg<br />Antoine et al., 2009<br />
  118. 118. Mechanism of Cell Damage: MS Analysis<br />HMGB1<br />hepatocyte<br />necrotic hepatocyte<br />Male CD-1 mice<br />dosed APAP 530 mg/kg<br />40<br />***<br />30<br />***<br />**<br />HMGB1 (Fold Inc)<br />20<br />**<br />10<br />0<br />0<br />5<br />10<br />15<br />20<br />25<br />hr<br />Antoine et al., 2009<br />
  119. 119. HMGB-1 acetylation: MS Analysis<br />Lys modification in innate immune cells – inhibits nuclear localization<br />K173<br />K180<br />K177<br />acetyl-HMGB1<br />K182-185<br />K172<br />COOH<br />H2N<br />activation<br />Nuclear<br />Localization <br />sequence<br />Cytokine<br />domain<br />innate immune cell<br />activated innate<br /> immune cell<br />Antoine et al., 2009<br />
  120. 120. Drug-Induced Liver Injury: mechanistic biomarkers<br />Active Secretion of HMGB<br />-<br />1<br />Activation<br />Active<br />secretion<br />Inflammatory Cell<br />Acetylated-HMGB-1<br />Male CD-1 mice<br />dosed APAP 530 mg/kg<br />40<br />10<br />***<br />8<br />30<br />***<br />***<br />HMGB1 (Fold Inc)<br />6<br />**<br />20<br />*<br />***<br />**<br />Hyper-acetylated HMGB1 (Fold Inc)<br />4<br />*<br />10<br />*<br />2<br />0<br />0<br />0<br />5<br />10<br />15<br />20<br />25<br />hr<br />Antoine et al., 2009<br />
  121. 121. *<br />1000<br />800<br />*<br />600<br />K18 fragments (pmol/ml)<br />400<br />200<br />0<br />0<br />200<br />400<br />600<br />800<br />1000<br />1200<br />Paracetamol (mg/kg)<br />Serum Markers of Apoptosis<br />Release of Keratin-18 due to apoptosis<br />Apoptosis:<br /><ul><li>Keratin-18– intermediate filament protein
  122. 122. Is cleared by caspases
  123. 123. Fragment released into plasma
  124. 124. Full length K18 released by necrosis</li></ul>Apoptotic Cell<br />Release of<br />Keratin-18<br />fragment<br />Antoine et al., 2009<br />N=6 ±SEM. *p<0.05, **p<0.01, ***p<0.005 compared to control<br />
  125. 125. Keratin-18 : biomarker of APAP apoptosis and necrosis<br />Release of full-length Keratin-18 due to necrosis<br />Correlation* of K18 fragment (apoptosis) vs<br />full-length K18 (necrosis) <br />Necrotic hepatocyte<br />DALD/SS motif<br />Release of fragmented Keratin-18 due to apoptosis<br />Apoptotic Hepatocyte<br />*individual mice (10 h); APAP (530 mg/kg)<br />
  126. 126. Hepatic Markers of Apoptosis<br />Hepatic DNA laddering<br />Hepatic pro-caspase-3 processing<br />Hepatic active caspase-3 IHC (3hr)<br />Antoine et al., 2009<br />
  127. 127. APAP hepatotoxicity - Pathological Time Course<br />3hr<br />15hr<br />5hr<br />24hr<br />H&E<br />Active caspase-3<br />Hepatocyte proliferation<br />PCNA<br />
  128. 128. ***<br />***<br />*<br />350<br />Caspase inhibition enhances necrosis<br />300<br />Paracetamol 530mg/kg; 5hr; mouse<br />250<br />HMGB1 (ng/ml)<br />200<br />PT<br />150<br />100<br />50<br />PT<br />0<br />ZVAD.fmk<br />CV<br />CV<br />***<br />***<br />*<br />5000<br />10X<br />10X<br />4000<br />Path score 3.4 ± 0.5<br />Path score 2.2 ± 0.4<br />3000<br />Serum ALT (U/l)<br />2000<br />1000<br />Keratin-18<br />ALT<br />HMGB-1<br />0<br />*<br />*<br />1000<br />800<br />600<br />APAP<br />control<br />APAP &<br />ZVAD.fmk<br />ZVAD.fmk<br />Fragmented K18 (pmol/ml)<br />400<br />APAP<br />control<br />APAP<br />control<br />APAP &<br />ZVAD.fmk<br />ZVAD.fmk<br />APAP &<br />ZVAD.fmk<br />ZVAD.fmk<br />200<br />0<br />Antoine et al., 2009<br />
  129. 129. Acetaminophen hepatotoxicity: Glutathione levels<br />Time (hr)<br />Time (hr)<br />0 1 2 3 5<br />0 1 2 3 5<br />p32<br />APAP<br />APAP<br /><ul><li> Glutathione depletion in fed mice</li></ul> (DEM 4.7mmol/kg in corn oil; 1 hr pre-APAP)<br /><ul><li>Earlier onset of apoptosis</li></ul>40<br />100<br />p17<br />80<br />30<br />60<br />p32<br />*<br />Hepatic ATP content<br />(nmol/mg)<br />Hepatic GSH content<br />(nmol/mg)<br />APAP<br />+ DEM<br />20<br />40<br />p17<br />**<br />APAP<br />+ DEM<br />† † <br />20<br />† † <br />**<br />**<br />10<br />0<br />0<br />Saline<br />DEM<br />0<br />1<br />2<br />3<br />4<br />5<br />6<br />Time (hr)<br />}<br />Control mice<br />Mice + DEM <br />+ APAP (530 mg/kg)<br />Hepatic caspase-3<br />processing<br />Hepatic DNA<br />laddering<br />
  130. 130. Acetaminophen hepatotoxicity: Glutathione levels<br /><ul><li> Glutathione depletion in mice</li></ul> (DEM 4.7mmol/kg in corn oil; 1 hr pre-APAP)<br /><ul><li>Earlier onset of apoptosis
  131. 131. Confirmed with biomarkers</li></ul>}<br />Control mice<br />Mice + DEM <br />+ APAP (530 mg/kg)<br />Apoptosis<br />Necrosis<br />1000<br />500<br />*<br />†<br />*<br />**<br />***<br />400<br />800<br />†<br />300<br />*<br />600<br />HMGB1 content (ng/ml)<br />Fragmented K18 (pmol/ml)<br />*<br />*<br />200<br />***<br />† <br />400<br />*<br />*<br />100<br />200<br />0<br />0<br />0<br />1<br />2<br />3<br />4<br />5<br />6<br />0<br />1<br />2<br />3<br />4<br />5<br />6<br />Time (hr)<br />Time (hr)<br />Antoine et al., in prep<br />
  132. 132. Acetaminophen hepatotoxicity: energy dependence<br /><ul><li> Apoptosis is a survival mechanism.
  133. 133. Apoptosis is an ATP-dependent process.
  134. 134. Does fasting mice alter the mechanism of cell death ?</li></ul>H & E<br />}<br />FED mice<br />24h Fasted mice <br />+ APAP (530 mg/kg)<br />40<br />Active<br />Caspase 3<br />*<br />30<br />Hepatic ATP content (nmol/mg)<br />20<br />**<br />10<br />**<br />**<br />PAS<br />reaction<br />0<br />0<br />1<br />2<br />3<br />4<br />5<br />6<br />Time (hr)<br />Antoine et al., in prep<br />
  135. 135. Biomarkers further inform hepatic pathology <br />5000<br />4500<br />*<br />4000<br />3500<br />**<br />3000<br />Serum ALT (U/L)<br />2500<br />2000<br />1500<br />1000<br />500<br />0<br />*<br />Control <br />Fed<br />APAP <br />Fed<br />Control <br />Fasted<br />APAP <br />Fasted<br />**<br />**<br />400<br />***<br />[14C] Irreversible binding <br />(nmol/mg) 5h post-dose:<br />Fed Fasted<br />1.87 ± 0.45 1.95 ± 0.44<br />300<br />***<br />HMGB1 content (ng/ml)<br />200<br />100<br />0<br />Control <br />Fed<br />APAP <br />Fed<br />Control <br />Fast<br />APAP <br />Fast<br />Antoine et al., in prep<br />
  136. 136. Acetaminophen hepatotoxicity: energy dependence<br /><ul><li> Apoptosis is a survival mechanism.
  137. 137. Apoptosis is an ATP-dependent process.
  138. 138. Does fasting mice alter the mechanism of cell death:
  139. 139. ATP & GSH levels</li></ul>*<br />*<br />**<br />*<br />Hepatic caspase-3<br />processing<br />Hepatic DNA<br />laddering<br />Antoine et al., in prep<br />
  140. 140. Acetaminophen hepatotoxicity: energy dependence<br />1000<br /><ul><li> Glucose supplementation in fasted mice</li></ul> (1000mg/kg glucose; 100mg/kg glycine; 1hr pre-APAP)<br /><ul><li>Apoptotic mechanism returns</li></ul> Basal ATP Levels<br /> (nmol/mg)<br />Fed 35.5 ± 4.0<br />Fasted 17.1 ± 5.3*<br />Fasted 24.4 ± 3.7*<br />+ Glucose/glycine<br />800<br />*<br />*<br />*<br />*<br />600<br />Fragmented K18 (pmol/ml)<br />APAP + Glucose /Glycine<br />400<br />Glucose / Glycine<br />Control<br />APAP<br /><ul><li>Necrosis is reduced:</li></ul>200<br />APAP<br />Control<br />p32<br />Glucose / Glycine<br />APAP + <br />Glucose /Glycine<br />p17<br />Hepatic caspase-3<br />processing<br />Hepatic DNA<br />laddering<br />4000<br />**<br />**<br />*<br />*<br />**<br />**<br />APAP + Glucose /Glycine<br />3000<br />Glucose / Glycine<br />Control<br />APAP<br />Serum ALT (U/l)<br />2000<br />1000<br />0<br />APAP<br />Control<br />Glucose / Glycine<br />APAP + <br />Glucose /Glycine<br />
  141. 141. Does fasting mice alter the mechanism of cell death ?<br />Fasted mouse:<br />APAP (530 mg/kg; 24h)<br />Fed mouse:<br />APAP (530mg/kg; 24h)<br />Hepatic Regeneration<br />Mitosis<br />No inflammation<br />Serum acetylated HMGB-1<br />1.7 ±0.3 fold increase vs control<br />Hepatic Damage<br />Inflammation<br />Neutrophil infiltration<br />Serum acetylated HMGB-1<br />14.9 ± 4.2 fold increase vs control<br />Antoine et al., in prep<br />
  142. 142. Mechanism of HMGB1 Release<br />Potential release<br />of oxidized HMGB-1<br />bound to DNA<br />+<br />Caspase<br />inhibition<br />Secondary<br />Necrotic fibroblast<br />Apoptotic fibroblast<br />Tolerance<br />Inflammation<br />Release of<br />non-oxidized<br />HMGB-1<br />DC Activation<br />Necrotic Cell<br />
  143. 143. Functional consequences of HMGB1 oxidation<br />REDUCED HMGB1<br />TLR2<br />TLR4<br />RAGE<br />NFkB activation<br />pro-inflammatory cytokine production<br />OXIDIZED HMGB1<br />NoNFkB activation<br />No pro-inflammatory cytokine production<br />Caspase-initiated oxidation by ROS<br />Released during apoptosis<br />
  144. 144. Circulating HMGB1 oxidation status : fed vs fasted mouse<br />FED<br />APAP<br />Sulfonic acid<br />(immune tolerance)<br />C106<br />COOH<br />H2N<br />TLR / RAGE<br />binding domain<br />FASTED<br />APAP<br />Thiol<br />(immune activation)<br />Antoine et al., in prep<br />
  145. 145. Caspase dependent oxidation of HMGB1<br />Validated with ZVAD.fmk<br />C106<br />COOH<br />H2N<br />TLR / RAGE<br />binding domain<br />Antoine et al., in prep<br />
  146. 146. HMGB1:induction of inflammation in fasted mice<br />Fasted mouse dosed APAP<br />Hepatic damage with inflammation<br />Fasted mouse dosed APAP<br />+ HMGB1 neutralizing antibody<br />Hepatic damage without inflammation<br />Antoine et al., in prep<br />
  147. 147. Mechanism of Cell Damage: a multicellular event<br />Factors: Determine: Serum Markers:<br /><ul><li> GSH status Injury onset
  148. 148. ATP Level Apoptosis
  149. 149. Apoptosis Hepatic regeneration K18 fragment</li></ul> Oxidised HMGB1<br /><ul><li> Necrosis Hepatic degeneration Full length K18</li></ul> Reduced HMGB1<br /><ul><li> Low ATP</li></ul>& Necrosis Inflammation Acetyl-HMGB1<br />Mild<br />injury<br />Innate immune response<br />Severity of injury<br />Antoine et al., 2009; Yee et al., 2007; Masson et al., 2008; Holt et al., 2008<br />
  150. 150. Reactive drug metabolites<br />Bioactivation<br />Acetaminophen<br />Nevirapine<br />
  151. 151. Nevirapine Hypersensitivity<br />Cheap and therefore widely used in Africa<br />Rash (16%)1 , SJS (1%)2, DILI (10%)3<br />1000 patient cohort study in Malawi<br />Systematic investigation of mechanisms<br />Bioactivation?<br />1.Pollard et al. Clin. Ther 1998. 20: 1071−1092. <br />2. Fagot JP et al. AIDS 2001) 15: 1843-1848.<br />3. Patel et al. J Acquir Immune DeficSyndr 2004; 35:120-125. <br />
  152. 152. Nevirapine Hypersensitivity<br />Abnormal LFTs 13 / 700 2%<br />Rash treated through 4 / 700 0.6%<br />ALT on nevirapine<br />1200<br />Patient 1<br />1000<br />Patient 2<br />Patient 3<br />800<br />Patient 4<br />ALT (U/L)<br />600<br />400<br />200<br />0<br />0<br />6<br />10<br />14<br />18<br />22<br />26<br />Weeks<br />
  153. 153. Nevirapine: Skin rash in an animal model <br />Nevirapine<br />12-OH NVP<br />Skin rash in Brown Norway rats is due to 12-OH Nevirapine pathway:<br />12-OH-NVP caused a rash at a lower dose than NVP<br />Less severe rash when CH3 hydrogens substituted by deuterium <br />Incidence of rash is increased by co-treatment with ABT<br />(ABT increases 12-OH NVP by blocking oxidation to 4-carboxy metabolite)<br />Quinone methide is potential reactive metabolite <br />NVP 12-O-sulfonate<br />NVP-12-mercapturate<br />Chen J, Mannargudi BM, Xu L, Uetrecht J (2008). Chem Res Toxicol 21:1862-1870<br />Uetrecht J (2006). Drug Metab Rev 38:745-753<br />Popovic M, Caswell JL,.Mannargudi B et al (2006). Chem Res Toxicol 19:1205-1214<br />Shenton J (2007).In: Pichler WJ (ed) Drug Hypersensitivity Karger AG, Basel, pp 115-128<br />
  154. 154. Integrated LC-MS NMR: biomarker for bioactivation<br />LC-MS/MRM<br />Human<br />Rat <br />In vitro<br />In vivo<br />mercapturate<br />detected by MRM<br />sample<br />22 g<br />Absolute quantification<br />HPLC bulk<br />Sample preparation<br />22 g<br />NMR<br />Structural<br />Confirmation<br />NVP-3-mercapturate (Major)<br />
  155. 155. Integrated LC-MS NMR: biomarker for bioactivation<br />LC-MS/MRM<br />Human<br />Rat <br />In vitro<br />In vivo<br />mercapturate<br />detected by MRM<br />sample<br />2nd mercapturate detected by MRM<br />Absolute quantification<br />22 g (major)<br />6mg (minor)<br />HPLC bulk<br />Sample preparation<br />NMR<br />Structural<br />Confirmation<br />NVP-12-mercapturate (Minor)<br />NVP-3-mercapturate (Major)<br />
  156. 156. Nevirapine: Skin rash in an animal model <br />
  157. 157. Nevirapine:<br />Common chemistry between rodent & man <br />21 Day CTRL II<br />Dehydrogenation*<br />NVP 3 & 12 Mercapturates<br />NVP 3 & 12 GSH conjugates<br />NVP Quinone Methide<br />or Epoxide<br />NVP 12-O-sulfonate<br />12-OH NVP<br />Nevirapine<br />NVP 3 & 12 Mercapturates<br />*Wenet al., 2009; Srivastava et al., 2010<br />
  158. 158. Complete Metabolic Profile of NVP in Man<br />Labels in red represent % metabolite in urine<br />Proportions represent the induced metabolic profile. <br />Riska et al. (1999a), Erickson et al. (1999), Wen et al. (2009), Srivastava et al. (2010)<br />
  159. 159. Complete Metabolic Profile of NVP in Rat<br />CYP3A1?<br />Riska et al. (1999b), Chen et al. (2008), Srivastava et al. (2010)<br />
  160. 160. 12-OH Nevirapine is a precursor of Nevirapine -12-mercapturate<br />100<br />100<br />Brown Norway Rat<br />Wistar Rat<br />NVP-3-NAC<br />(M2)<br />NVP-3-NAC<br />(M2)<br />80<br />60<br />Relative Abundance %<br />50<br />Relative Abundance %<br />NVP-12-NAC<br />(M1)<br />NVP-12-NAC<br />(M1)<br />40<br />Dosed:<br />Nevirapine<br />20<br />0<br />0<br />5<br />4<br />2<br />4<br />3<br />1<br />2<br />0<br />0<br />100<br />100<br />Dosed:<br />12-OH-Nevirapine<br />80<br />NVP-12-NAC<br />(M1)<br />NVP-12-NAC<br />(M1)<br />60<br />Relative Abundance %<br />50<br />Relative Abundance %<br />40<br />20<br />0<br />0<br />5<br />2<br />4<br />0<br />3<br />1<br />4<br />2<br />0<br />Time (min)<br />Time (min)<br />Nevirapine-12-mercapturate is formed from 12-OH-Nevirapine <br />
  161. 161. Chemical Rationale:<br />12-OH Nevirapine is responsible for the Brown Norway Rat skin rash <br />12-OH NVP<br />Nevirapine<br />Quinone methide<br />12-OH NVP<br />NVP-12-mercapturate<br />Higher incidence of skin toxicity<br />Quinone methide<br />Epoxide<br />Next Question:<br />Could the epoxide which forms NVP-3-mercapturate be responsible for liver toxicity ?<br />NVP-12-mercapturate<br />NVP-3-mercapturate<br />Lower incidence of skin toxicity<br />
  162. 162. Mechanistic Drug Safety: Integrated Approach <br />Model hepatotoxins,<br />withdrawn drugs:<br />Drugs in clinical use:<br />
  163. 163. Mechanistic Drug Safety: Integrated Approach <br /><ul><li>Integrated safety / toxicity screens are required to study the multiplebiological consequences of cell defence and cell destruction.
  164. 164. If the pharmacophore and toxicophore cannot be separated, rational risk assessment cannot be performed.
  165. 165. Integrated chemical,biological and genomic systems are required for a complete mechanistic understanding of the chemical, molecular, cellular and immunological basis of idiosyncratic drug toxicity.
  166. 166. Idiosyncratic drug toxicity cannot be predicted from the chemistry of the drug and/or its metabolite because such reactions are by definition (largely) a function of the biology of the individual.</li></ul>Biology of individual<br />Occurrence, Frequency<br />& Severity of<br />Drug Hepatotoxicity<br />f1<br />f2<br />+<br />=<br />Chemistry<br />of drug<br />
  167. 167. Acknowledgements<br />Dan Antoine<br />Hayley Webb<br />Abhi Srivastava<br />Jean Sathish<br />Anja Kipar (Vet Pathology)<br />Val Tilston<br />Roz Jenkins<br />Sophie Regan<br />Kevin Park<br />Phill Roberts<br />Luke Palmer<br />

×