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Biotransformation of Xenobiotics
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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. www.dvmdocs.webs.com 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. www.dvmdocs.webs.com Introduction • Purpose • Converts lipophilic to hydrophilic compounds • Facilitates excretion • Consequences • Changes in PK characteristics • Detoxification • Metabolic activation

  4. www.dvmdocs.webs.com Comparing Phase I & Phase II

  5. www.dvmdocs.webs.com 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. www.dvmdocs.webs.com 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. www.dvmdocs.webs.com 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. www.dvmdocs.webs.com 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. www.dvmdocs.webs.com 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. www.dvmdocs.webs.com 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. www.dvmdocs.webs.com 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. www.dvmdocs.webs.com 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. www.dvmdocs.webs.com 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. www.dvmdocs.webs.com 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. www.dvmdocs.webs.com 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. www.dvmdocs.webs.com Cytochrome P450 catalytic cycle

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

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

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

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

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

  24. www.dvmdocs.webs.com 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. www.dvmdocs.webs.com Major P450 Enzymes in Humans

  26. www.dvmdocs.webs.com Major P450 Enzymes in Humans

  27. www.dvmdocs.webs.com Major P450 Enzymes in Humans

  28. www.dvmdocs.webs.com Major P450 Enzymes in Humans

  29. www.dvmdocs.webs.com Major P450 Enzymes in Humans

  30. www.dvmdocs.webs.com Major P450 Enzymes in Humans

  31. www.dvmdocs.webs.com Major P450 Enzymes in Humans

  32. www.dvmdocs.webs.com Major P450 Enzymes in Humans

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

  39. www.dvmdocs.webs.com 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. www.dvmdocs.webs.com 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. www.dvmdocs.webs.com 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. www.dvmdocs.webs.com 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. www.dvmdocs.webs.com 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. www.dvmdocs.webs.com 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. www.dvmdocs.webs.com 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. www.dvmdocs.webs.com 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. www.dvmdocs.webs.com 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. www.dvmdocs.webs.com 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. www.dvmdocs.webs.com 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. www.dvmdocs.webs.com 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