Background & Current IssuesinDrug Metabolism<br />Dominic Williams<br />Dept. of Molecular & Clinical Pharmacology<br />
Overview & Learning Outcomes<br /><ul><li>Introduction to Adverse Drug Reactions
Drug Metabolism
Phase I
Aromatic hydroxylation
Epoxidation
Phase II
Glucuronidation
Glutathione conjugation
Detection of Drug Bioactivation
Reactive metabolites
Covalent binding
Prediction of Toxic Metabolites
Metabolites in Safety Testing – regulatory issues</li></li></ul><li>Adverse Drug Reactions<br /><ul><li>patient morbidity ...
4th – 6th leading cause of death in USA*
precludes otherwise effective drug therapy
drug withdrawal (4%, 1974 - 1994)~
> $4 billion 1998 – screening & toxicity testing
Drug attrition
Liver, skin, blood, cardiovascular</li></ul>*Lazarou et al., 1998<br />~Jefferys et al., 1994<br />
Lessons for the future<br />Inform mechanism and pathogenesis<br />Inform the Medicinal Chemist<br />Inform the Clinician<...
Mechanistic Classification of Adverse Drug Reactions<br />Type A or On-Target <br /><ul><li> predictable
 exaggeration of pharmacological effect
 dose dependent</li></ul>Type B or Off-Target (idiosyncratic)<br /><ul><li>not predictable from pharmacology
 apparently dose-independent
marked inter-individual susceptibility
 more severe</li></ul>TYPE C (chemical)<br /><ul><li> predictable from chemical structure
eg. Paracetamol</li></ul>Park et al., 1998<br />
Liver is key player in drug metabolism and toxicity<br /><ul><li> 2 blood supplies – portal (intestinal) 75%, arterial 25 %
 High exposure to drugs and nutrients as first organ after absorption
 Major organ for drug metabolism </li></ul>Hepatic portal vein<br />Hepatic artery<br />Biotransformation<br />To Metaboli...
Multi-Lobular Arrangement of the Liver<br />Portal Triad<br />Centrilobular<br />Region with<br />highest density<br />of ...
Reminder: Drug Metabolism<br />Phase I<br />Phase II<br />Lipophilic drug<br />Drug metabolism<br />Water soluble metaboli...
Phase I Drug Metabolism:  Cytochrome P450<br /><ul><li>Multigene family of haemoproteinmonooxygenases
Membrane bound in smooth endoplasmic reticulum
Conducts majority of phase I drug oxidation reactions
Broad and overlapping substrate specificities
Polymorphic in human population</li></li></ul><li>Cytochrome P450 oxidationsAromatic hydroxylation<br />Example<br />HO<br...
Cytochrome P450 oxidationsEpoxidation<br />Carbamazepine: anticonvulsant for epilepsy   <br />Carbamazepine epoxide<br />[...
Cytochrome P450 oxidationsEpoxidation<br />Benzo[a]pyrene:	carcinogen found in cigarette smoke<br />Benzo[a]pyrene-4,5-epo...
Epoxide hydrolysis<br />HO<br />OH<br />H2O<br />P450<br />mEH<br />example<br />epoxide<br />dihydrodiol<br />mEH<br />P4...
Epoxide hydrolase (mEH)- microsomal enzyme</li></li></ul><li>Phase II Biotransformations:  chemical change<br />Major chan...
Coordinated Phase I and Phase II metabolism<br />sulphate<br />and <br />glucuronide<br />
Glucuronidation<br /><ul><li>Glucuronides are water soluble and excreted more easily in bile or urine.
Molecular recognition by transport systems in various tissues
kidney
Liver
Glucuronides may be hydrolysed by b-glucuronidase in the gut resulting in Enterohepatic Recirculation.
Physiologically, glucuronidation is important for clearance of bilirubin, steroids and 5-hydroxy tryptamine.
Most glucuronides are pharmacologically inactive, although there are some exceptions e.g. Morphine
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Current Issues In Drug Metabolism

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  • Major consequence of bioactivation is in fact bioinactivation
  • Mjajorr consequence of bioactivation is in fact bioinactivation
  • Most common consequence of bioactivation is in fact bioinactivation
  • The lone pair of electrons attack the central carbon forming a bond between the oxygen and the carbon. This causes the electrons from the double bond to move back to the oxygen atom.This causes a bond to be formed between the oxygen and C forming a 5 member ring. The electrons from the O- fall back to the central carbon, resulting in an electron shift to the oxygen above it. This results in the bond breaking, and the acyl group moving around the ring.The lone pair of electrons on the OH group attach the C-OH bond, resulting in a shift of electrons back to the O atom in the ring. This then causes the ring to open and a double bond to be formed between the C=O. This bond is susceptible to attack from a the nucleophillic NH2 group of a protein, resulting in the protein binding to the carbon atom, and separating the =O bond into a single –Oh2 bond. This OH2 is a good leaving group and the +ve charge pulls electrons towards it, resulting in the loss of the group from the molecule. The electrons from NH are pulled towards the carbon, resulting in a C=N bond, and the loss of a hydrogen, which moves to the ring.The Hydrogen that moves to the ring, has a lone pair of electrons which attack the C-C bond, resulting in the formation of a double bond, with electons being pulled up from the C=N.A lone pair of electrons then is pulled towards the C on the ring, causing a C=O bond to be formed. A proton is also pulled into the structure by the double bond, resulting in the loss of the double bond, and stabilization of the adduct.NH3+ does not have a loan pair of electrons, and hence is unable to form adducts with acyl glucuronides. Usually in physiological conditions, proteins are in the state of containing NH3+ however other amino acids on the protein are able to interact with the NH3+ group, reverting it to the NH2 with the lone pair, resulting in adduct formation
  • Current Issues In Drug Metabolism

    1. 1. Background & Current IssuesinDrug Metabolism<br />Dominic Williams<br />Dept. of Molecular & Clinical Pharmacology<br />
    2. 2. Overview & Learning Outcomes<br /><ul><li>Introduction to Adverse Drug Reactions
    3. 3. Drug Metabolism
    4. 4. Phase I
    5. 5. Aromatic hydroxylation
    6. 6. Epoxidation
    7. 7. Phase II
    8. 8. Glucuronidation
    9. 9. Glutathione conjugation
    10. 10. Detection of Drug Bioactivation
    11. 11. Reactive metabolites
    12. 12. Covalent binding
    13. 13. Prediction of Toxic Metabolites
    14. 14. Metabolites in Safety Testing – regulatory issues</li></li></ul><li>Adverse Drug Reactions<br /><ul><li>patient morbidity & mortality
    15. 15. 4th – 6th leading cause of death in USA*
    16. 16. precludes otherwise effective drug therapy
    17. 17. drug withdrawal (4%, 1974 - 1994)~
    18. 18. > $4 billion 1998 – screening & toxicity testing
    19. 19. Drug attrition
    20. 20. Liver, skin, blood, cardiovascular</li></ul>*Lazarou et al., 1998<br />~Jefferys et al., 1994<br />
    21. 21. 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 />
    22. 22. Mechanistic Classification of Adverse Drug Reactions<br />Type A or On-Target <br /><ul><li> predictable
    23. 23. exaggeration of pharmacological effect
    24. 24. dose dependent</li></ul>Type B or Off-Target (idiosyncratic)<br /><ul><li>not predictable from pharmacology
    25. 25. apparently dose-independent
    26. 26. marked inter-individual susceptibility
    27. 27. more severe</li></ul>TYPE C (chemical)<br /><ul><li> predictable from chemical structure
    28. 28. eg. Paracetamol</li></ul>Park et al., 1998<br />
    29. 29. Liver is key player in drug metabolism and toxicity<br /><ul><li> 2 blood supplies – portal (intestinal) 75%, arterial 25 %
    30. 30. High exposure to drugs and nutrients as first organ after absorption
    31. 31. 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 />
    32. 32. 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 />
    33. 33. Reminder: Drug Metabolism<br />Phase I<br />Phase II<br />Lipophilic drug<br />Drug metabolism<br />Water soluble metabolite<br />Excretion<br />Urine<br />Bile<br /> CHEMICAL REACTIVITY<br />CONJUGATION WITH POLAR GROUP<br />Glucuronidation<br />Sulphation<br />Glutathione conjugation<br />etc.<br />Oxidation<br />Reduction<br />Hydrolysis<br />Cytochrome P450<br />Transferases<br />
    34. 34. Phase I Drug Metabolism: Cytochrome P450<br /><ul><li>Multigene family of haemoproteinmonooxygenases
    35. 35. Membrane bound in smooth endoplasmic reticulum
    36. 36. Conducts majority of phase I drug oxidation reactions
    37. 37. Broad and overlapping substrate specificities
    38. 38. Polymorphic in human population</li></li></ul><li>Cytochrome P450 oxidationsAromatic hydroxylation<br />Example<br />HO<br />[O]<br />gentisic acid<br />Salicylic acid: treatment for psoriasis; analgesic<br />[O]<br />
    39. 39. Cytochrome P450 oxidationsEpoxidation<br />Carbamazepine: anticonvulsant for epilepsy <br />Carbamazepine epoxide<br />[O]<br />Example<br />
    40. 40. Cytochrome P450 oxidationsEpoxidation<br />Benzo[a]pyrene: carcinogen found in cigarette smoke<br />Benzo[a]pyrene-4,5-epoxide<br />[O]<br />Example<br />
    41. 41. Epoxide hydrolysis<br />HO<br />OH<br />H2O<br />P450<br />mEH<br />example<br />epoxide<br />dihydrodiol<br />mEH<br />P450<br />carbamazepine dihydrodiol<br />carbamazepine epoxide<br /><ul><li>Addition of water to epoxide
    42. 42. Epoxide hydrolase (mEH)- microsomal enzyme</li></li></ul><li>Phase II Biotransformations: chemical change<br />Major change in charge & molecular weight…<br />Glucuronidation - 176<br />Sulphonation - 80<br />Glutathione Conjugation - 305<br />Species differences in route of elimination<br />Molecular weight thresholds in biliary clearance:<br />Rat 300-400<br />Man 500-600<br />
    43. 43. Coordinated Phase I and Phase II metabolism<br />sulphate<br />and <br />glucuronide<br />
    44. 44. Glucuronidation<br /><ul><li>Glucuronides are water soluble and excreted more easily in bile or urine.
    45. 45. Molecular recognition by transport systems in various tissues
    46. 46. kidney
    47. 47. Liver
    48. 48. Glucuronides may be hydrolysed by b-glucuronidase in the gut resulting in Enterohepatic Recirculation.
    49. 49. Physiologically, glucuronidation is important for clearance of bilirubin, steroids and 5-hydroxy tryptamine.
    50. 50. Most glucuronides are pharmacologically inactive, although there are some exceptions e.g. Morphine
    51. 51. Morphine 6-glucuronide 3 X potency of morphine.</li></li></ul><li>Glucuronidation<br />alcohols<br />phenols<br />b<br />HO-R<br />R<br />+<br />UDPGT<br />UDP<br />a<br />ether glucuronide<br />carboxylic acids<br />b<br />HOOC-R<br />R<br />+<br />UDPGT<br />UDP<br />a<br />ester glucuronide<br />O-Glucuronidation<br />
    52. 52. Glucuronidation<br />UDPGT<br />glucuronide<br />Salicylphenolic (ether) glucuronide<br />UDPGT<br />glucuronide<br />Salicylacyl (ester) glucuronide<br />O-Glucuronidation: - phenols<br /> - carboxylic acids<br />SALICYLIC ACID<br />+ UDPGA<br />
    53. 53. Acyl Glucuronides<br /><ul><li> One type of biogenic reactive ester </li></ul>(cfthioesters and acyl adenylates, bilirubin acyl glucuronide)<br /><ul><li> Can be major metabolite of drugs containing carboxylic acid moiety
    54. 54. Electrophilicity recognised for nearly 30 years
    55. 55. Categorised as reactive metabolites, but reactivity varies greatly</li></li></ul><li>Acyl Glucuronides: the concern<br />Drug withdrawals:<br /><ul><li> also: aclofenac, bendazac, zomepirac, bromfenac, fenclofenac, indoprofen, suprofen…etc
    56. 56. 25% of withdrawn drugs are carboxylic acids
    57. 57. Adverse drug reactions: Hepatotoxicity, hypersensitivity & immune cytopenias</li></li></ul><li>Acyl glucuronides: reactive speciesthat can bind covalently to proteins<br /><ul><li>Transacylation</li></ul>Direct reaction with nucleophiles leads to displacement of the acyl residue<br /><ul><li>Glycation</li></ul> Acyl migration yields isomeric acyl glucuronides followed by the formation a Schiff base with the amino group of a protein and further rearrangement to a stable amino-keto product.<br />
    58. 58. AcylGlucuronides and Drug Toxicity<br />What is the evidence?<br /><ul><li>Biochemistry of drug metabolism
    59. 59. Chemistry of covalent binding
    60. 60. Specific targets
    61. 61. Hepatocytes: metabolism & toxicity
    62. 62. Animal models; metabolism and toxicity
    63. 63. Clinical evidence
    64. 64. covalent binding
    65. 65. immunological
    66. 66. genetic (UGT2B7*2)</li></li></ul><li>Formation of glutathione (GSH) conjugates<br /><ul><li> Glutathione conjugation is part of Phase II metabolism
    67. 67. Electrophiles can either bind directly or enzymatically (GST)
    68. 68. The thiol group is the active binding group
    69. 69. Binding to GSH detoxifies the electrophile
    70. 70. Increases hydrophilicity, allowing excretion
    71. 71. ~10mM GSH in the liver
    72. 72. Reactive metabolites bind to protein via cysteine residues (-SH)</li></ul>glycine cysteine g-glutamyl<br />Williams & Naisbitt, CurrOpin Drug Disc Develop 2002, 5(1): 104<br />
    73. 73. Formation of glutathione (GSH) conjugates<br />Quinone imine<br />epoxide<br /><ul><li> Electrophilic metabolites
    74. 74. epoxides
    75. 75. quinone imines
    76. 76. Glutathione S-transferase
    77. 77. cytosolic enzyme
    78. 78. no high energy donor as substrates are chemically reactive
    79. 79. Products
    80. 80. excreted in urine, bile
    81. 81. undergo further metabolism to Mercapturic acids</li></ul>Williams, Bioanalysis (2010) 2(4), 693–697<br />
    82. 82. 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 />
    83. 83. Drug Metabolism: Toxicology<br />Cellular <br />accumulation<br />DRUG<br />RESPONSE<br />Concentrations in<br />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 />
    84. 84. 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 />
    85. 85. Hepatotoxin Accumulation<br />Energy Depletion<br />ROS Generation<br />Apoptosis<br />Steatosis<br />Mt-DNA<br /> OXPHOS<br /> Fatty acid synthesis<br />HepatocellularTargets<br />Toxic Consequences<br />Toxicity Mechanisms Independent of Reactive Metabolites<br />Perhexiline<br />Fialuridine<br />Aplovirac<br />Tacrine<br />Valproic Acid<br />Amiodarone<br />Troglitazone<br />Ritonavir<br />Rifampin<br />NRTIs egStavudine<br />Oxidative Stress<br />Transporters<br />Protein Oxidation<br />Fatty Acids<br />Apoptosis<br />Metabolizing Enzymes<br />Necrosis<br />Steatosis<br />Mitochondria<br />Accumulation<br /><ul><li>Physicochemical
    86. 86. Biochemical
    87. 87. Transport</li></li></ul><li>Mechanisms of Drug Induced Liver Injury<br />CLEARANCE<br />DRUG<br /><ul><li> accumulation
    88. 88. bioactivation
    89. 89. covalent binding
    90. 90. chemical stress
    91. 91. mitochondrial dysfunction
    92. 92. apoptosis
    93. 93. necrosis
    94. 94. hepatocyte hypertrophy
    95. 95. hepatocyte hyperplasia
    96. 96. fibrosis
    97. 97. cholestasis</li></ul>DRUG<br />+<br />METABOLITE<br />
    98. 98. 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 />
    99. 99. Consequences of bioactivation - I <br />Cellular<br />accumulation<br />DRUG<br />Toxicity<br />Phase I/II/III<br />bioactivation<br /><ul><li>heme complex
    100. 100. protein alkylation</li></ul>Chemically<br />reactive<br />metabolites<br />Inhibition<br />Of<br />P450s<br />bioinactivation<br />Excretion<br />
    101. 101. Consequences of bioactivation - II <br />Cellular<br />accumulation<br />DRUG<br />Toxicity<br />Carcinogenicity<br />Chemical Stress<br />Modification of:<br /><ul><li> nucleic acid
    102. 102. enzyme
    103. 103. transporter
    104. 104. signalling protein
    105. 105. receptor
    106. 106. 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 />
    107. 107. Consequences of bioactivation – structural alerts <br />Cellular<br />accumulation<br />DRUG<br />Toxicity ??<br /><ul><li>furan
    108. 108. thiophene
    109. 109. aliphatic amine
    110. 110. aromatic amine
    111. 111. epoxide
    112. 112. quinone
    113. 113. quinoneimine
    114. 114. carbocation
    115. 115. acyl halide
    116. 116. hydroxylamine
    117. 117. allylic alcohol
    118. 118. acylglucuronide</li></ul>bioactivation<br />Phase I/II/III<br />Chemically<br /> reactive<br />metabolites<br />Stable<br />metabolites<br />bioinactivation<br />Excretion<br />PHARMACOLOGICAL EFFECT<br />ADVERSE<br />EFFECT<br />CHEMICAL STRUCTURE<br />
    119. 119. Metabolic basis of Bioactivation and Toxicity<br />P450 null mice<br />Hepatotoxicity <br />Hepatotoxicity<br />Myelotoxicity<br />Nephrotoxicity<br />CNS toxicity<br />Hepatotoxicity & Nephrotoxicity<br />Hepatocarcinogenesis<br />Multi-organ hyperplasia and tumours<br /> Paracetamol<br /> Carbon tetrachloride<br /> Benzene<br /> Cisplatin<br /> Acrylonitrile<br /> Chloroform<br /> 4-Aminobiphenol<br /> DMBA<br />Lee et al., 1996; Zaher et al., 1998: Wong et al., 1998; Valentine et al., 1996; Liu et al., 2003; Wang, et al., 2002; Constan et al., 1999; Kimira et al., 1999 ;Buters et al., 1999<br />
    120. 120. 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 />FelbamateAplastic anaemia<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 />
    121. 121. Rational risk assessment for new inhaled anaesthetics<br />Isoflurane<br />Enflurane<br />Desflurane<br />2%<br />0.2%<br />0.01%<br />20%<br />CYP450 2E1<br />?<br />Halothane<br />Reactive<br />Metabolites<br />Sevoflurane<br />Immune-mediated<br />hepatotoxicity<br />Covalent Binding<br />to macromolecules<br />Potential to cause immune-mediated hepatotoxicity in man correlates with extent of metabolic bioactivation and liver protein adduct formation<br /> - which can be quantified experimentally (in vivo/ in vitro) <br />rational risk assessment for new inhaled anaesthetics<br />
    122. 122. Prediction of Toxic Metabolites<br />Chemically Reactive Metabolite <br />Screens<br /><ul><li>human liver banks
    123. 123. expression systems
    124. 124. cell lines
    125. 125. In silico techniques
    126. 126. Freshly isolated cells
    127. 127. Animal models
    128. 128. transgenic animals
    129. 129. volunteers - phenotype and genotype
    130. 130. patients</li></li></ul><li> Prediction of Toxic Metabolites<br /><ul><li>Screens for metabolic reactivity
    131. 131. Glutathione conjugation
    132. 132. Covalent binding
    133. 133. Hazard assessment
    134. 134. Biology
    135. 135. Provided retrospective mechanistic insight
    136. 136. Use for predictivity has been challenged
    137. 137. Many non-hepatotoxins undergo covalent binding
    138. 138. Distinguish critical vs non-critical proteins</li></ul>Park et al., 2005, Obach et al., 2008, Bauman et al., 2009<br />
    139. 139. Screens for metabolic reactivity – GSH conjugation<br />-129<br />Neutral loss of 129mu = g-glutamyl group<br />Regardless of structure of intermediate<br />Paracetamol<br />Clozapine<br />
    140. 140. Nucleophile Traps for Electrophilic Intermediates<br />Recent: radiolabelled GSH – ease of detection<br />g-glutamylcysteinelysine – simultaneous hard and soft electrophiles<br />
    141. 141. Prediction of Toxic Metabolites<br /><ul><li>Screens for metabolic reactivity
    142. 142. Glutathione conjugation
    143. 143. Covalent binding
    144. 144. Hazard assessment
    145. 145. Biology
    146. 146. Provided retrospective mechanistic insight
    147. 147. Use for predictivity has been challenged
    148. 148. Many non-hepatotoxins undergo covalent binding
    149. 149. Distinguish critical vs non-critical proteins</li></ul>Park et al., 2005, Obach et al., 2008, Bauman et al., 2009<br />
    150. 150. How much covalent binding is acceptable ?<br /><ul><li>Model hepatotoxins show 1 – 1.4 nmol equivalent bound / mg protein
    151. 151. 20 fold reduction is a CONSERVATIVE TARGET UPPER LIMIT
    152. 152. 50 pmol drug equivalent / mg total liver protein
    153. 153. Equates to 10 x background level binding
    154. 154. Consideration of the risk : benefit ratio
    155. 155. Provided retrospective mechanistic insight
    156. 156. Hazard assessment
    157. 157. Many false positives
    158. 158. non-hepatotoxins undergo covalent binding
    159. 159. critical vs non-critical proteins</li></ul>Evans et al., 2004<br />Obach et al ., 2008<br />Bauman et al., 2009<br />Nakayama et al., 2009<br />
    160. 160. Critical protein target vs non-critical target:<br />General situation<br />Idiosyncratic Toxicity<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 toxicity if: </li></ul> - no binding<br /> - binding mainly to non-critical targets<br /><ul><li> Toxic: -if highly selective for a criticaltarget</li></ul> -high non-selective binding<br />Act as ‘danger’ signals which can trigger the adaptive immune system<br />Cytotoxicity<br />Release of cell contents which activate immune cells <br />Likely to be screened out in non-clinical tests<br />
    161. 161. <ul><li> Extensive dialogue after Industrial ‘best practice’ position paper
    162. 162. Concerned with non-clinical toxicity testing of drug metabolites
    163. 163. Metabolites
    164. 164. human-only
    165. 165. disproportionately higher in man vs animal </li></ul>‘Human metabolites that can raise a safety concern are those formed at >10% of parent drug systemic exposure at steady state’<br /><ul><li> Consistent with FDA and EPA guidelines
    166. 166. Chemically reactive metabolites can be difficult to measure
    167. 167. ‘if they form chemically stable product – no further testing’
    168. 168. ‘if the conjugate forms a toxic compound such as acyl glucuronide, additional safety testing may be needed’</li></ul>Baillie et al., 2002 TAAP<br />
    169. 169. MIST Problems<br /><ul><li> Relative rather than absolute thresholds</li></ul>eg. a 10% metabolite from a drug given at 1mg/day may have less toxicological significance than a 5% metabolite from a drug given at 1g/day<br /><ul><li> 2010 FDA guidelines were combined with ICH M3(R2) ‘Non-clinical Safety Studies for the conduct of Human Clinical Trials and marketing authorization for pharmaceuticals’ </li></ul>Expert Opin. Drug Metab. Toxicol. (2010) 6(12):1539-1549<br />
    170. 170. Summary<br /><ul><li>The chemistry and biochemistry of reactive metabolites provides an essential platform to investigate the multiplebiological consequences of cell defence and cell destruction.
    171. 171. Acyl glucuronides represent a clear chemical hazard because of their propensity to covalently modify macromolecules which has been demonstrated both in vitro and in vivo.
    172. 172. Reactive metabolite screens can be used to detect the chemical hazard associated with bioactivation and are a useful tool in drug design, discovery and development.</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 />
    173. 173. Extra Slides<br />
    174. 174. Chemistry of acyl glucuronide binding to protein<br />Acyl migration<br />Rearrangement<br />
    175. 175. Irreversible plasma protein binding of tolmetin in humans<br />Pharmacokinetics and irreversible plasma binding of tolmetin studied in six healthy volunteers after a single dose<br />Irreversible binding of tolmetin to plasma proteins occurred in all volunteers<br />Correlation between binding and glucuronylation<br />Tolmetin<br />Tolmetin glucuronide<br />Hyneck et al, ClinPharmTher1988<br />
    176. 176. Mrp2-driven up-concentration of the reactive diclofenacacylglucuronide in bile canaliculi favours covalent binding to the canalicular protein target, DPP IV<br />Plasma<br />Hepatocyte<br />Diclofenac<br />Acyl glucuronide<br />Acylation of target proteins<br />Mrp2<br />SH<br />HS<br />HS<br />Bile<br />(pH ~ 8)<br />DPP IV<br />

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