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  1. Biotransformation of Xenobiotics Barbara M. Davit, PhD, DABT Division of Bioequivalence, Office of Generic Drugs, CDER, FDA Introduction to the Theory and Methods in Toxicology Sept. 17, 2001

  2. Overview • Major Phase I and Phase II enzymes • Reaction mechanisms, substrates • Enzyme inhibitors and inducers • Genetic polymorphism • Detoxification • Metabolic activation • FDA guidances related tobiotransformation

  3. Introduction • Purpose • Converts lipophilic to hydrophilic compounds • Facilitates excretion • Consequences • Changes in PK characteristics • Detoxification • Metabolic activation

  4. Comparing Phase I & Phase II

  5. First Pass Effect • Biotransformation by liver or gut enzymes before compound reaches systemic circulation • Results in lower systemic bioavailbility of parent compound • Examples: propafenone, isoniazid, propanolol

  6. Phase I: Hydrolysis • Carboxyesterases & peptidases • hydrolysis of esters • eg: valacyclovir, midodrine • hydrolysis of peptide bonds • e.g.: insulin (peptide) • Epoxide hydrolase • H2O added to expoxides • eg: carbamazepine

  7. Phase I: Reductions • Azo reduction • N=N to 2 -NH2 groups • eg: prontosil to sulfanilamide • Nitro reduction • N=O to one -NH2 group • eg: 2,6-dinitrotoluene activation • N-glucuronide conjugate hydrolyzed by gut microflora • Hepatotoxic compound reabsorbed

  8. Reductions • Carbonyl reduction • Alcohol dehydrogenase (ADH) • Chloral hydrate is reduced to trichlorothanol • Disulfide reduction • First step in disulfiram metabolism • Sulfoxide reduction • NSAID prodrug Sulindac converted to active sulfide moiety

  9. Reductions • Quinone reduction • Cytosolic flavoprotein NAD(P)H quinone oxidoreductase • two-electron reduction, no oxidative stress • high in tumor cells; activates diaziquone to more potent form • Flavoprotein P450-reductase • one-electron reduction, produces superoxide ions • metabolic activation of paraquat, doxorubicin

  10. Reductions • Dehalogenation • Reductive (H replaces X) • Enhances CCl4 toxicity by forming free radicals • Oxidative (X and H replaced with =O) • Causes halothane hepatitis via reactive acylhalide intermediates • Dehydrodechlorination (2 X’s removed, form C=C) • DDT to DDE

  11. Phase I: Oxidation-Reduction • Alcohol dehydrogenase • Alcohols to aldehydes • Genetic polymorphism; Asians metabolize alcohol rapidly • Inhibited by ranitidine, cimetidine, aspirin • Aldehyde dehydrogenase • Aldehydes to carboxylic acids • Inhibited by disulfiram

  12. Phase I: Monooxygenases • Monoamine oxidase • Primaquine, haloperidol, tryptophan are substrates • Activates 1-methyl-4-phenyl-1,2,5,6-tetrahydropyridine (MPTP) to neurotoxic toxic metabolite in nerve tissue, resulting in Parkinsonian-like symptoms

  13. Monooxygenases • Peroxidases couple oxidation to reduction of H2O2 & lipid hydroperoxidase • Prostaglandin H synthetase (prostaglandin metabolism) • Causes nephrotoxicity by activating aflatoxin B1, acetaminophen to DNA-binding compounds • Lactoperoxidase (mammary gland) • Myleoperoxidase (bone marrow) • Causes bone marrow suppression by activating benzene to DNA-reactive compound

  14. Monooxygenases • Flavin-containing mono-oxygenases • Generally results in detoxification • Microsomal enzymes • Substrates: nicotine, cimetidine, chlopromazine, imipramine • Repressed rather than induced by phenobarbital, 3-methylcholanthrene

  15. Phase I: Cytochrome P450 • Microsomal enzyme ranking first among Phase I enzymes with respect to catalytic versatility • Heme-containing proteins • Complex formed between Fe2+ and CO absorbs light maximally at 450 (447-452) nm • Overall reaction proceeds by catalytic cycle: RH+O2+H++NADPH ROH+H2O+NADP+

  16. Cytochrome P450 catalytic cycle

  17. Cytochrome P450 reactions • Hydroxylation of aliphatic or aromatic carbon • (S)-mephenytoin to 4’-hydroxy-(S)-mephenytoin (CYP2C19) • Testosterone to 6-hydroxytestosterone (CYP3A4)

  18. Cytochrome P450 reactions • Expoxidation of double bonds • Carbamazepine to 10,11-epoxide • Heteroatom oxygenation, N-hydroxylation • Amines to hydroxylamines • Omeprazole to sulfone (CYP3A4)

  19. Cytochrome P450 reactions • Heteroatom dealkylation • O-dealkylation (e.g., dextromethorphan to dextrophan by CYP2D6) • N-demethylation of caffeine to: theobromine (CYP2E1) paraxanthine (CYP1A2) theophylline (CYP2E1)

  20. Cytochrome P450 reactions • Oxidative group transfer • N, S, X replaced with O • Parathion to paroxon (S by O) • Activation of halothane to trifluoroacetylchloride (immune hepatitis)

  21. Cytochrome P450 reactions • Cleavage of esters • Cleavage of functional group, with O incorporated into leaving group • Loratadine to Desacetylated loratadine (CYP3A4, 2D6)

  22. Cytochrome P450 reactions • Dehydrogenation • Abstraction of 2 H’s with formation of C=C • Activation of Acetaminophen to hepatotoxic metabolite N-acetylbenzoquinoneimine

  23. Cytochrome P450 expression • Gene family, subfamily names based on amino acid sequences • At least 15 P450 enzymes identified in human liver microsomes

  24. Cytochrome P450 expression • Variation in levels, activity due to: • Genetic polymorphism • Environmental factors: inducers, inhibitors, disease • Multiple P450’s can catalyze same reaction (lowest Km is predominant) • A single P450 can catalyze multiple pathways

  25. Major P450 Enzymes in Humans

  26. Major P450 Enzymes in Humans

  27. Major P450 Enzymes in Humans

  28. Major P450 Enzymes in Humans

  29. Major P450 Enzymes in Humans

  30. Major P450 Enzymes in Humans

  31. Major P450 Enzymes in Humans

  32. Major P450 Enzymes in Humans

  33. Metabolic activation by P450 • Formation of toxic species • Dechlorination of chloroform to phosgene • Dehydrogenation and subsequent epoxidation of urethane (CYP2E1) • Formation of pharmacologically active species • Cyclophosphamide to electrophilic aziridinum species (CYP3A4, CYP2B6)

  34. Inhibition of P450 • Drug-drug interactions due to reduced rate of biotransformation • Competitive • S and I compete for active site • e.g., rifabutin & ritonavir; dextromethorphan & quinidine • Mechanism-based • Irreversible; covalent binding to active site

  35. Induction and P450 • Increased rate of biotransformation due to new protein synthesis • Must give inducers for several days for effect • Drug-drug interactions • Possible subtherapeutic plasma concentrations • eg, co-administration of rifampin and oral contraceptives is contraindicated • Some drugs induce, inhibit same enzyme (isoniazid, ethanol (2E1), ritonavir (3A4)

  36. Phase II: Glucuronidation • Major Phase II pathway in mammals • UDP-glucuronyltransferase forms O-, N-, S-, C- glucuronides; six forms in human liver • Cofactor is UDP-glucuronic acid • Inducers: phenobarbital, indoles, 3-methylcholanthrene, cigarette smoking • Substrates include dextrophan, methadone, morphine, p-nitrophenol, valproic acid, NSAIDS, bilirubin, steroid hormones

  37. Glucuronidation & genetic polymorphism • Crigler-Nijar syndrome (severe): inactive enzyme; severe hyperbilirubinemia; inducers have no effect • Gilbert’s syndrome (mild): reduced enzyme activity; mild hyperbilirubinemia; phenobarbital increases rate of bilirubin glucuronidation to normal • Patients can glucuronidate p-nitrophenol, morphine, chloroamphenicol

  38. Glucuronidation & -glucuronidase • Conjugates excreted in bile or urine (MW) • -glucuronidase from gut microflora cleaves glucuronic acid • Aglycone can be reabsorbed & undergo enterohepatic recycling

  39. Glucuronidation and -glucuronidase • Metabolic activation of 2.6-dinitrotoluene) by -glucuronidase • -glucuronidase removes glucuronic acid from N-glucuronide • nitro group reduced by microbial N-reductase • resulting hepatocarcinogen is reabsorbed

  40. Phase II: Sulfation • Sulfotransferases are widely-distributed enzymes • Cofactor is 3’-phosphoadenosine-5’-phosphosulfate (PAPS) • Produce highly water-soluble sulfate esters, eliminated in urine, bile • Xenobiotics & endogenous compounds are sulfated (phenols, catechols, amines, hydroxylamines)

  41. Sulfation • Sulfation is a high affinity, low capacity pathway • Glucuronidation is low affinity, high capacity • Capacity limited by low PAPS levels • Acetaminophen undergoes both sulfation and glucuronidation • At low doses sulfation predominates • At high doses, glucuronidation predominates

  42. Sulfation • Four sulfotransferases in human liver cytosol • Aryl sulfatases in gut microflora remove sulfate groups; enterohepatic recycling • Usually decreases pharmacologic, toxic activity • Activation to carcinogen if conjugate is chemically unstable • Sulfates of hydroxylamines are unstable (2-AAF)

  43. Phase II: Methylation • Common, minor pathway which generally decreases water solubility • Methyltransferases • Cofactor: S-adenosylmethionine (SAM) • -CH3 transfer to O, N, S, C • Substrates include phenols, catechols, amines, heavy metals (Hg, As, Se)

  44. Methylation & genetic polymorphism • Several types of methyltransferases in human tissues • Phenol O-methyltransferase, Catechol O-methyltransferase, N-methyltransferase, S-methyltransferase • Genetic polymorphism in thiopurine metabolism • high activity allele, increased toxicity • low activity allele, decreased efficacy

  45. Phase II: Acetylation • Major route of biotransformation for aromatic amines, hydrazines • Generally decreases water solubility • N-acetyltransferase (NAT) • Cofactor is AcetylCoenzyme A • Humans express two forms • Substrates include sulfanilamide, isoniazid, dapsone

  46. Acetylation & genetic polymorphism • Rapid and slow acetylators • Various mutations result in decreased enzyme activity or stability • Incidence of slow acetylators • 70% in Middle Eastern populations; 50% in Caucasians; 25% in Asians • Drug toxicities in slow acetylators • nerve damage from dapsone; bladder cancer in cigarette smokers due to increased levels of hydroxylamines

  47. Phase II:Amino Acid Conjugation • Alternative to glucuronidation • Two principle pathways • -COOH group of substrate conjugated with -NH2 of glycine, serine, glutamine, requiring CoA activation • e.g: conjugation of benzoic acid with glycine to form hippuric acid • Aromatic -NH2 or NHOH conjugated with -COOH of serine, proline, requiring ATP activation

  48. Amino Acid Conjugation • Substrates: bile acids, NSAIDs • Species specificity in amino acid acceptors • mammals: glycine (benzoic acid) • birds: ornithine (benzoic acid) • dogs, cats, taurine (bile acids) • nonhuman primates: glutamine • Metabolic activation • Serine or proline N-esters of hydroxylamines are unstable & degrade to reactive electrophiles

  49. Phase II:Glutathione Conjugation • Enormous array of substrates • Glutathione-S-transferase catalyzes conjugation with glutathione • Glutathione is tripeptide of glycine, cysteine, glutamic acid • Formed by -glutamylcysteine synthetase, glutathione synthetase • Buthione-S-sulfoxine is inhibitor

  50. Glutathione Conjugation • Two types of reactions with glutathione • Displacement of halogen, sulfate, sulfonate, phospho, nitro group • Glutathione added to activated double bond or strained ring system • Glutathione substrates • Hydrophobic, containing electrophilic atom • Can react with glutathione nonenzymatically