Current Issues In Drug Metabolism

  • 1,632 views
Uploaded on

 

  • Full Name Full Name Comment goes here.
    Are you sure you want to
    Your message goes here
    Be the first to comment
No Downloads

Views

Total Views
1,632
On Slideshare
0
From Embeds
0
Number of Embeds
1

Actions

Shares
Downloads
0
Comments
0
Likes
2

Embeds 0

No embeds

Report content

Flagged as inappropriate Flag as inappropriate
Flag as inappropriate

Select your reason for flagging this presentation as inappropriate.

Cancel
    No notes for slide
  • 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

Transcript

  • 1. Background & Current IssuesinDrug Metabolism
    Dominic Williams
    Dept. of Molecular & Clinical Pharmacology
  • 2. Overview & Learning Outcomes
    • Introduction to Adverse Drug Reactions
    • 3. Drug Metabolism
    • 4. Phase I
    • 5. Aromatic hydroxylation
    • 6. Epoxidation
    • 7. Phase II
    • 8. Glucuronidation
    • 9. Glutathione conjugation
    • 10. Detection of Drug Bioactivation
    • 11. Reactive metabolites
    • 12. Covalent binding
    • 13. Prediction of Toxic Metabolites
    • 14. Metabolites in Safety Testing – regulatory issues
  • Adverse Drug Reactions
    • patient morbidity & mortality
    • 15. 4th – 6th leading cause of death in USA*
    • 16. precludes otherwise effective drug therapy
    • 17. drug withdrawal (4%, 1974 - 1994)~
    • 18. > $4 billion 1998 – screening & toxicity testing
    • 19. Drug attrition
    • 20. Liver, skin, blood, cardiovascular
    *Lazarou et al., 1998
    ~Jefferys et al., 1994
  • 21. Lessons for the future
    Inform mechanism and pathogenesis
    Inform the Medicinal Chemist
    Inform the Clinician
    Inform the Regulator
    Inform the Public – what is feasible
    Develop biomarkers for integrated patient, in vitro & animal studies
  • 22. Mechanistic Classification of Adverse Drug Reactions
    Type A or On-Target
    • predictable
    • 23. exaggeration of pharmacological effect
    • 24. dose dependent
    Type B or Off-Target (idiosyncratic)
    • not predictable from pharmacology
    • 25. apparently dose-independent
    • 26. marked inter-individual susceptibility
    • 27. more severe
    TYPE C (chemical)
    • predictable from chemical structure
    • 28. eg. Paracetamol
    Park et al., 1998
  • 29. Liver is key player in drug metabolism and toxicity
    • 2 blood supplies – portal (intestinal) 75%, arterial 25 %
    • 30. High exposure to drugs and nutrients as first organ after absorption
    • 31. Major organ for drug metabolism
    Hepatic portal vein
    Hepatic artery
    Biotransformation
    To Metabolites
    Lipophillic compounds
    Central vein
    Bile
    Enterohepatic
    recirculation
    Urine
    Faeces
  • 32. Multi-Lobular Arrangement of the Liver
    Portal Triad
    Centrilobular
    Region with
    highest density
    of CYP450
    metabolising
    enzymes
    Capilliaries with fenestrations
  • 33. Reminder: Drug Metabolism
    Phase I
    Phase II
    Lipophilic drug
    Drug metabolism
    Water soluble metabolite
    Excretion
    Urine
    Bile
     CHEMICAL REACTIVITY
    CONJUGATION WITH POLAR GROUP
    Glucuronidation
    Sulphation
    Glutathione conjugation
    etc.
    Oxidation
    Reduction
    Hydrolysis
    Cytochrome P450
    Transferases
  • 34. Phase I Drug Metabolism: Cytochrome P450
    • Multigene family of haemoproteinmonooxygenases
    • 35. Membrane bound in smooth endoplasmic reticulum
    • 36. Conducts majority of phase I drug oxidation reactions
    • 37. Broad and overlapping substrate specificities
    • 38. Polymorphic in human population
  • Cytochrome P450 oxidationsAromatic hydroxylation
    Example
    HO
    [O]
    gentisic acid
    Salicylic acid: treatment for psoriasis; analgesic
    [O]
  • 39. Cytochrome P450 oxidationsEpoxidation
    Carbamazepine: anticonvulsant for epilepsy
    Carbamazepine epoxide
    [O]
    Example
  • 40. Cytochrome P450 oxidationsEpoxidation
    Benzo[a]pyrene: carcinogen found in cigarette smoke
    Benzo[a]pyrene-4,5-epoxide
    [O]
    Example
  • 41. Epoxide hydrolysis
    HO
    OH
    H2O
    P450
    mEH
    example
    epoxide
    dihydrodiol
    mEH
    P450
    carbamazepine dihydrodiol
    carbamazepine epoxide
    • Addition of water to epoxide
    • 42. Epoxide hydrolase (mEH)- microsomal enzyme
  • Phase II Biotransformations: chemical change
    Major change in charge & molecular weight…
    Glucuronidation - 176
    Sulphonation - 80
    Glutathione Conjugation - 305
    Species differences in route of elimination
    Molecular weight thresholds in biliary clearance:
    Rat 300-400
    Man 500-600
  • 43. Coordinated Phase I and Phase II metabolism
    sulphate
    and
    glucuronide
  • 44. Glucuronidation
    • Glucuronides are water soluble and excreted more easily in bile or urine.
    • 45. Molecular recognition by transport systems in various tissues
    • 46. kidney
    • 47. Liver
    • 48. Glucuronides may be hydrolysed by b-glucuronidase in the gut resulting in Enterohepatic Recirculation.
    • 49. Physiologically, glucuronidation is important for clearance of bilirubin, steroids and 5-hydroxy tryptamine.
    • 50. Most glucuronides are pharmacologically inactive, although there are some exceptions e.g. Morphine
    • 51. Morphine 6-glucuronide 3 X potency of morphine.
  • Glucuronidation
    alcohols
    phenols
    b
    HO-R
    R
    +
    UDPGT
    UDP
    a
    ether glucuronide
    carboxylic acids
    b
    HOOC-R
    R
    +
    UDPGT
    UDP
    a
    ester glucuronide
    O-Glucuronidation
  • 52. Glucuronidation
    UDPGT
    glucuronide
    Salicylphenolic (ether) glucuronide
    UDPGT
    glucuronide
    Salicylacyl (ester) glucuronide
    O-Glucuronidation: - phenols
    - carboxylic acids
    SALICYLIC ACID
    + UDPGA
  • 53. Acyl Glucuronides
    • One type of biogenic reactive ester
    (cfthioesters and acyl adenylates, bilirubin acyl glucuronide)
    • Can be major metabolite of drugs containing carboxylic acid moiety
    • 54. Electrophilicity recognised for nearly 30 years
    • 55. Categorised as reactive metabolites, but reactivity varies greatly
  • Acyl Glucuronides: the concern
    Drug withdrawals:
    • also: aclofenac, bendazac, zomepirac, bromfenac, fenclofenac, indoprofen, suprofen…etc
    • 56. 25% of withdrawn drugs are carboxylic acids
    • 57. Adverse drug reactions: Hepatotoxicity, hypersensitivity & immune cytopenias
  • Acyl glucuronides: reactive speciesthat can bind covalently to proteins
    • Transacylation
    Direct reaction with nucleophiles leads to displacement of the acyl residue
    • Glycation
    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.
  • 58. AcylGlucuronides and Drug Toxicity
    What is the evidence?
    • Biochemistry of drug metabolism
    • 59. Chemistry of covalent binding
    • 60. Specific targets
    • 61. Hepatocytes: metabolism & toxicity
    • 62. Animal models; metabolism and toxicity
    • 63. Clinical evidence
    • 64. covalent binding
    • 65. immunological
    • 66. genetic (UGT2B7*2)
  • Formation of glutathione (GSH) conjugates
    • Glutathione conjugation is part of Phase II metabolism
    • 67. Electrophiles can either bind directly or enzymatically (GST)
    • 68. The thiol group is the active binding group
    • 69. Binding to GSH detoxifies the electrophile
    • 70. Increases hydrophilicity, allowing excretion
    • 71. ~10mM GSH in the liver
    • 72. Reactive metabolites bind to protein via cysteine residues (-SH)
    glycine cysteine g-glutamyl
    Williams & Naisbitt, CurrOpin Drug Disc Develop 2002, 5(1): 104
  • 73. Formation of glutathione (GSH) conjugates
    Quinone imine
    epoxide
    • Electrophilic metabolites
    • 74. epoxides
    • 75. quinone imines
    • 76. Glutathione S-transferase
    • 77. cytosolic enzyme
    • 78. no high energy donor as substrates are chemically reactive
    • 79. Products
    • 80. excreted in urine, bile
    • 81. undergo further metabolism to Mercapturic acids
    Williams, Bioanalysis (2010) 2(4), 693–697
  • 82. 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
  • 83. Drug Metabolism: Toxicology
    Cellular
    accumulation
    DRUG
    RESPONSE
    Concentrations in
    organs
    Phase I/II
    Drug
    Stable
    metabolites
    Disposition
    Metabolism
    Absorption
    Excretion
    Drug & metabolites
    Pharmacological &
    Toxicological exposure
    Excretion
  • 84. Drug Safety Science and DILI
    CLEARANCE
    20 / safety pharmacology targets
    DRUG
    2nd effects
    1st effects
    3rd effects
    Ca2+
    DRUG
    +
    METABOLITE
    DNA
    TARGET
    phospholipid
    specific proteins
    Biology of individual
    BIOMARKERS
    PHARMACOLOGICAL EFFECT
    ADVERSE
    EFFECT
    Occurrence, Frequency
    & Severity of
    Drug Hepatotoxicity
    f1
    f2
    +
    =
    Chemistry
    of drug
    Dose
    CHEMICAL STRUCTURE
    f (chemistry)
    f (biology)
    SPECIES and INDIVIDUAL VARIATION
  • 85. Hepatotoxin Accumulation
    Energy Depletion
    ROS Generation
    Apoptosis
    Steatosis
    Mt-DNA
    OXPHOS
    Fatty acid synthesis
    HepatocellularTargets
    Toxic Consequences
    Toxicity Mechanisms Independent of Reactive Metabolites
    Perhexiline
    Fialuridine
    Aplovirac
    Tacrine
    Valproic Acid
    Amiodarone
    Troglitazone
    Ritonavir
    Rifampin
    NRTIs egStavudine
    Oxidative Stress
    Transporters
    Protein Oxidation
    Fatty Acids
    Apoptosis
    Metabolizing Enzymes
    Necrosis
    Steatosis
    Mitochondria
    Accumulation
  • Mechanisms of Drug Induced Liver Injury
    CLEARANCE
    DRUG
    DRUG
    +
    METABOLITE
  • 98. Drug Disposition Physiological, Pharmacological & Toxicological
    Cellular
    accumulation
    DRUG
    Toxicity
    Phase I/II/III
    bioactivation
    Chemically
    reactive
    metabolites
    Stable
    metabolites
    bioinactivation
    Excretion
  • 99. Consequences of bioactivation - I
    Cellular
    accumulation
    DRUG
    Toxicity
    Phase I/II/III
    bioactivation
    • heme complex
    • 100. protein alkylation
    Chemically
    reactive
    metabolites
    Inhibition
    Of
    P450s
    bioinactivation
    Excretion
  • 101. Consequences of bioactivation - II
    Cellular
    accumulation
    DRUG
    Toxicity
    Carcinogenicity
    Chemical Stress
    Modification of:
    protein
    Phase I/II/III
    bioactivation
    Necrosis
    Chemically
    reactive
    metabolites
    Stable
    metabolites
    Apoptosis
    bioinactivation
    Hypersensitivity
    Excretion
  • 107. Consequences of bioactivation – structural alerts
    Cellular
    accumulation
    DRUG
    Toxicity ??
    bioactivation
    Phase I/II/III
    Chemically
    reactive
    metabolites
    Stable
    metabolites
    bioinactivation
    Excretion
    PHARMACOLOGICAL EFFECT
    ADVERSE
    EFFECT
    CHEMICAL STRUCTURE
  • 119. Metabolic basis of Bioactivation and Toxicity
    P450 null mice
    Hepatotoxicity
    Hepatotoxicity
    Myelotoxicity
    Nephrotoxicity
    CNS toxicity
    Hepatotoxicity & Nephrotoxicity
    Hepatocarcinogenesis
    Multi-organ hyperplasia and tumours
    Paracetamol
    Carbon tetrachloride
    Benzene
    Cisplatin
    Acrylonitrile
    Chloroform
    4-Aminobiphenol
    DMBA
    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
  • 120. Off Target Clinical Adverse Drug Reactions
    Drug Adverse Reaction
    Amodiaquine Hepatotoxicity
    Paracetamol Hepatotoxicity
    Halothane Hepatotoxicity
    Diclofenac Hepatotoxicity
    Tacrine Hepatotoxicity
    Indomethacin Hepatotoxicity
    Valproic Acid Hepatotoxicity
    Vesnarinone Hepatotoxicity
    Phenacetin Hepatotoxicity
    PhenytoinTeratogenicity/ Hepatotoxicity
    ClozapineAgranulocytosis
    AminopyreneAgranulocytosis
    TiclopidineAgranulocytosis
    Sulfamethoxazole Toxic epidermal necrolysis
    Lamotrigene Toxic epidermal necrolysis
    Carbamazepine Hypersensitivity
    Tienilic acid Hypersensitivity
    FelbamateAplastic anaemia
    RemoxiprideAplastic Anaemia
    Reactive Metabolite
    Quinone imine
    Quinone Imine
    Acyl halide
    Quinone imine / acylglucuronide
    Quinone methide
    Quinone imine / chloro-indole
    a, b unsaturated carbonyl
    Iminium ion
    Quinone imine
    Free radical
    Nitrenium ion
    iminium
    S-oxide
    Hydroxylamine / nitroso
    epoxide
    Quinone imine / epoxide
    S-oxide
    atropaldehyde
    hydroquinone
  • 121. Rational risk assessment for new inhaled anaesthetics
    Isoflurane
    Enflurane
    Desflurane
    2%
    0.2%
    0.01%
    20%
    CYP450 2E1
    ?
    Halothane
    Reactive
    Metabolites
    Sevoflurane
    Immune-mediated
    hepatotoxicity
    Covalent Binding
    to macromolecules
    Potential to cause immune-mediated hepatotoxicity in man correlates with extent of metabolic bioactivation and liver protein adduct formation
    - which can be quantified experimentally (in vivo/ in vitro)
    rational risk assessment for new inhaled anaesthetics
  • 122. Prediction of Toxic Metabolites
    Chemically Reactive Metabolite
    Screens
  • Prediction of Toxic Metabolites
    • Screens for metabolic reactivity
    • 131. Glutathione conjugation
    • 132. Covalent binding
    • 133. Hazard assessment
    • 134. Biology
    • 135. Provided retrospective mechanistic insight
    • 136. Use for predictivity has been challenged
    • 137. Many non-hepatotoxins undergo covalent binding
    • 138. Distinguish critical vs non-critical proteins
    Park et al., 2005, Obach et al., 2008, Bauman et al., 2009
  • 139. Screens for metabolic reactivity – GSH conjugation
    -129
    Neutral loss of 129mu = g-glutamyl group
    Regardless of structure of intermediate
    Paracetamol
    Clozapine
  • 140. Nucleophile Traps for Electrophilic Intermediates
    Recent: radiolabelled GSH – ease of detection
    g-glutamylcysteinelysine – simultaneous hard and soft electrophiles
  • 141. Prediction of Toxic Metabolites
    • Screens for metabolic reactivity
    • 142. Glutathione conjugation
    • 143. Covalent binding
    • 144. Hazard assessment
    • 145. Biology
    • 146. Provided retrospective mechanistic insight
    • 147. Use for predictivity has been challenged
    • 148. Many non-hepatotoxins undergo covalent binding
    • 149. Distinguish critical vs non-critical proteins
    Park et al., 2005, Obach et al., 2008, Bauman et al., 2009
  • 150. How much covalent binding is acceptable ?
    • Model hepatotoxins show 1 – 1.4 nmol equivalent bound / mg protein
    • 151. 20 fold reduction is a CONSERVATIVE TARGET UPPER LIMIT
    • 152. 50 pmol drug equivalent / mg total liver protein
    • 153. Equates to 10 x background level binding
    • 154. Consideration of the risk : benefit ratio
    • 155. Provided retrospective mechanistic insight
    • 156. Hazard assessment
    • 157. Many false positives
    • 158. non-hepatotoxins undergo covalent binding
    • 159. critical vs non-critical proteins
    Evans et al., 2004
    Obach et al ., 2008
    Bauman et al., 2009
    Nakayama et al., 2009
  • 160. Critical protein target vs non-critical target:
    General situation
    Idiosyncratic Toxicity
    Form a hapten recognised by adaptive immune system
    Which organ ?
    Which cell type ?
    Which organelle ?
    PROTEIN
    Which protein ?
    Which amino acid ?
    Which atom ?
    • Low toxicity if:
    - no binding
    - binding mainly to non-critical targets
    • Toxic: -if highly selective for a criticaltarget
    -high non-selective binding
    Act as ‘danger’ signals which can trigger the adaptive immune system
    Cytotoxicity
    Release of cell contents which activate immune cells
    Likely to be screened out in non-clinical tests
  • 161.
    • Extensive dialogue after Industrial ‘best practice’ position paper
    • 162. Concerned with non-clinical toxicity testing of drug metabolites
    • 163. Metabolites
    • 164. human-only
    • 165. disproportionately higher in man vs animal
    ‘Human metabolites that can raise a safety concern are those formed at >10% of parent drug systemic exposure at steady state’
    • Consistent with FDA and EPA guidelines
    • 166. Chemically reactive metabolites can be difficult to measure
    • 167. ‘if they form chemically stable product – no further testing’
    • 168. ‘if the conjugate forms a toxic compound such as acyl glucuronide, additional safety testing may be needed’
    Baillie et al., 2002 TAAP
  • 169. MIST Problems
    • Relative rather than absolute thresholds
    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
    • 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’
    Expert Opin. Drug Metab. Toxicol. (2010) 6(12):1539-1549
  • 170. Summary
    • The chemistry and biochemistry of reactive metabolites provides an essential platform to investigate the multiplebiological consequences of cell defence and cell destruction.
    • 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. 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.
    Biology of individual
    Occurrence, Frequency
    & Severity of
    Drug Hepatotoxicity
    f1
    f2
    +
    =
    Chemistry
    of drug
  • 173. Extra Slides
  • 174. Chemistry of acyl glucuronide binding to protein
    Acyl migration
    Rearrangement
  • 175. Irreversible plasma protein binding of tolmetin in humans
    Pharmacokinetics and irreversible plasma binding of tolmetin studied in six healthy volunteers after a single dose
    Irreversible binding of tolmetin to plasma proteins occurred in all volunteers
    Correlation between binding and glucuronylation
    Tolmetin
    Tolmetin glucuronide
    Hyneck et al, ClinPharmTher1988
  • 176. Mrp2-driven up-concentration of the reactive diclofenacacylglucuronide in bile canaliculi favours covalent binding to the canalicular protein target, DPP IV
    Plasma
    Hepatocyte
    Diclofenac
    Acyl glucuronide
    Acylation of target proteins
    Mrp2
    SH
    HS
    HS
    Bile
    (pH ~ 8)
    DPP IV