Biotransformation of xenobiotics

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Biotransformation of xenobiotics

  1. 1. Biotransformation of Xenobiotics
  2. 2. Biotransformation of Xenobiotics... Overview • Major Phase I and Phase II enzymes • Reaction mechanisms, substrates • Enzyme inhibitors and inducers • Genetic polymorphism • Detoxification • Metabolic activation • FDA guidances related to biotransformation 2
  3. 3. Biotransformation of Xenobiotics... Introduction • Purpose – Converts lipophilic to hydrophilic compounds – Facilitates excretion • Consequences – Changes in PK characteristics – Detoxification – Metabolic activation 3
  4. 4. Biotransformation of Xenobiotics... Comparing Phase I & Phase II Enzym e Phase I Phase I I Types of reactions Hydrolysis Conjugations Oxidation Reduction Increase in Small Large hydrophilicity General mechanism Exposes functional Polar compound added group to functional group Consquences May result in Facilitates excretion metabolic activation 4
  5. 5. Biotransformation of Xenobiotics... 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 5
  6. 6. Biotransformation of Xenobiotics... 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 6
  7. 7. Biotransformation of Xenobiotics... 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 7
  8. 8. Biotransformation of Xenobiotics... 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 8
  9. 9. Biotransformation of Xenobiotics... 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 9
  10. 10. Biotransformation of Xenobiotics... 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 10
  11. 11. Biotransformation of Xenobiotics... 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 11
  12. 12. Biotransformation of Xenobiotics... 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 12
  13. 13. Biotransformation of Xenobiotics... 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 13
  14. 14. Biotransformation of Xenobiotics... Monooxygenases • Flavin-containing mono-oxygenases – Generally results in detoxification – Microsomal enzymes – Substrates: nicotine, cimetidine, chlopromazine, imipramine – Repressed rather than induced by phenobarbital, 3-methylcholanthrene 14
  15. 15. Biotransformation of Xenobiotics... 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+ 15
  16. 16. Biotransformation of Xenobiotics... Cytochrome P450 catalytic 16
  17. 17. Biotransformation of Xenobiotics... Cytochrome P450 reactions • Hydroxylation of aliphatic or aromatic carbon – (S)-mephenytoin to 4’-hydroxy-(S)- mephenytoin (CYP2C19) – Testosterone to 6-hydroxytestosterone (CYP3A4) 17
  18. 18. Biotransformation of Xenobiotics... Cytochrome P450 reactions • Expoxidation of double bonds – Carbamazepine to 10,11-epoxide • Heteroatom oxygenation, N-hydroxylation – Amines to hydroxylamines – Omeprazole to sulfone (CYP3A4) 18
  19. 19. Biotransformation of Xenobiotics... 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) 19
  20. 20. Biotransformation of Xenobiotics... 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) 20
  21. 21. Biotransformation of Xenobiotics... Cytochrome P450 reactions • Cleavage of esters – Cleavage of functional group, with O incorporated into leaving group – Loratadine to Desacetylated loratadine (CYP3A4, 2D6) 21
  22. 22. Biotransformation of Xenobiotics... Cytochrome P450 reactions • Dehydrogenation – Abstraction of 2 H’s with formation of C=C – Activation of Acetaminophen to hepatotoxic metabolite N-acetylbenzoquinoneimine 22
  23. 23. Biotransformation of Xenobiotics... Cytochrome P450 expression • Gene family, subfamily names based on amino acid sequences • At least 15 P450 enzymes identified in human liver microsomes 23
  24. 24. Biotransformation of Xenobiotics... 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 24
  25. 25. Biotransformation of Xenobiotics... Major P450 Enzymes in Humans CYP1A1/ 2 Expressed Substrates Inducers Inhibitors in: Liver Caffeine Cigarrette Furafylline Lung Theophylline smoke; (mechanism- Skin Cruciferous based); GI veggies; ∀-naphtho- Placenta Charcoal- flavone broiled meat (reversible) 25
  26. 26. Biotransformation of Xenobiotics... Major P450 Enzymes in Humans CYP2B6 Expressed Substrates Inducers Inhibitors in: Liver Diazepam ??? Orphenadrine Phenanthrene (mechanism- based) 26
  27. 27. Biotransformation of Xenobiotics... Major P450 Enzymes in Humans CYP2C19 Genetic polymorphism Substrates Inducers Inhibitors Poor metabolizers have defective Phenytoin Rifampin Sulfafenazole CYP2C9 Piroxicam Tolbutamide Warfarin 27
  28. 28. Biotransformation of Xenobiotics... Major P450 Enzymes in Humans CYP2C19 Genetic polymorphism Substrates Inducers Inhibitors  Rapid and slow S-mephenytoin Rifampin Tranylcypromine metabolizers of S- (4’-hydroxylation mephenytoin is catalyzed by  N-demethylation CYP2C19) pathway of S- mephenytoin metabolism predominates in slow metabolizers 28
  29. 29. Biotransformation of Xenobiotics... Major P450 Enzymes in Humans CYP2D6 Genetic polymorphism Substrates I nducers Inhibitors  Poor metabolizers lack Propafenone None known Fluoxetine CYP2D6 Desipramine Quinidine  Debrisoquine causes marked, Propanolol prolonged hypotension in Codeine slow metabolizers Dextromethorphan  No effect on response to Fluoxetine propanolol in poor Clozapine metabolizers; alternate Captopril pathway (CYP2C19) will predominate Poor metabolizers  5-10% of Caucasians are identified by poor metabolizers urinary exrection of  < 2% of Asians, African Dextrorphan Americans are poor metabolizers 29
  30. 30. Biotransformation of Xenobiotics... Major P450 Enzymes in Humans CYP2E1 Expressed in: Substrates Inducers Inhibitors Liver Ethanol Ethanol Disulfiram Lung Acetaminophen Isoniazid Kidney Dapsone Lympocytes Caffeine Theophylline Benzene 30
  31. 31. Biotransformation of Xenobiotics... Major P450 Enzymes in Humans CYP3A4 Expressed Substrates Inducers I nhibitors in: Liver; Acetaminophen Rifampin Ketoconazole; Kidney; Carbamazepine Carbamazepine Ritonavir; Intestine; Cyclosporine Phenobarbital Grapefruit juice; Most Dapsone Phenytoin Troleandomycin abundant Digitoxin P450 Diltiazem enzyme in Diazepam liver Erythromycin Etoposide Lidocaine Loratadine Midazolam Lovasatin Nifedipine Rapamycin Taxol Verapamil 31
  32. 32. Biotransformation of Xenobiotics... Major P450 Enzymes in Humans CYP4A9/ 11 Expressed Substrates Inducers Inhibitors in: Liver Fatty acids and ??? ??? derivaties; Catalzyes  - and  1-hyroxylation 32
  33. 33. Biotransformation of Xenobiotics... 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) 33
  34. 34. Biotransformation of Xenobiotics... 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 34
  35. 35. Biotransformation of Xenobiotics... 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) 35
  36. 36. Biotransformation of Xenobiotics... 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 36
  37. 37. Glucuronidation & geneticBiotransformation of Xenobiotics... 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 37
  38. 38. Glucuronidation & -Biotransformation of Xenobiotics... glucuronidase • Conjugates excreted in bile or urine (MW)  -glucuronidase from gut microflora cleaves glucuronic acid • Aglycone can be reabsorbed & undergo enterohepatic recycling 38
  39. 39. Biotransformation of Xenobiotics... 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 39
  40. 40. Biotransformation of Xenobiotics... 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) 40
  41. 41. Biotransformation of Xenobiotics... 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 41
  42. 42. Biotransformation of Xenobiotics... 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) 42
  43. 43. Biotransformation of Xenobiotics... 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) 43
  44. 44. Methylation & geneticBiotransformation of Xenobiotics... 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 44
  45. 45. Biotransformation of Xenobiotics... 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 45
  46. 46. Acetylation & geneticBiotransformation of Xenobiotics... 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 46
  47. 47. Biotransformation of Xenobiotics... 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 47
  48. 48. Biotransformation of Xenobiotics... 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 48
  49. 49. Biotransformation of Xenobiotics... 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 49
  50. 50. Biotransformation of Xenobiotics... 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 50
  51. 51. Biotransformation of Xenobiotics... Glutathione Conjugation • Conjugation of N-acetylbenzoquinoneimine (activated metabolite of acetaminophen) • O-demethylation of organophosphates • Activation of trinitroglycerin – Products are oxidized glutathione (GSSG), dinitroglycerin, NO (vasodilator) • Reduction of hydroperoxides – Prostaglandin metabolism 51
  52. 52. Biotransformation of Xenobiotics... Glutathione Conjugation • Four classes of soluble glutathione-S- transferase ( , , ,  ) • Distinct microsomal and cytosolic glutathione- S-transferases • Genetic polymorphism 52
  53. 53. Biotransformation of Xenobiotics... Glutathione-S-transferase • Inducers (include 3-methylcholanthrene, phenobarbital, corticosteroids, anti-oxidants) • Overexpression of enzyme leads to resistance (e.g., insects to DDT, corn to atrazine, cancer cells to chemotherapy) • Species specificity – Aflatoxin B1 not carcinogenic in mice which can conjugate with glutathione very rapidly 53
  54. 54. Biotransformation of Xenobiotics... Glutathione Conjugation • Excretion of glutathione conjugates – Excreted intact in bile – Converted to mercapturic acids in kidney, excreted in urine • Enzymes involved are -glutamyltranspeptidase, aminopeptidase M • Activation of xenobiotics following GSH conjugation – Four mechanisms identified 54
  55. 55. FDA-CDER Guidances forBiotransformation of Xenobiotics... Industry • Recommendations, not regulations • Discuss aspects of drug development • Used in context of planning drug development to achieve marketing approval • Among guidances are those dealing with in vitro and in vivo drug interaction studies 55
  56. 56. Biotransformation of Xenobiotics... In vitro guidance • CDER Guidance for Industry: Drug Metabolism/Drug Interaction Studies in the Drug Development Process: Studies in Vitro, April 1997, CLIN 3 • Availability: – www.fda.gov/cder/guidance/index.htm 56
  57. 57. Biotransformation of Xenobiotics... In vitro guidance: assumptions • Circulating concentrations of parent drug and/or active metabolites are effectors of drug actions • Clearance is principle regulator of drug concentration • Large differences in blood levels can occur because of individual differences • Assay development critical 57
  58. 58. Biotransformation of Xenobiotics... In vitro guidance: techniques/approaches • Identify a drug’s major metabolic pathways • Anticipate drug interactions • Recommended methods – Human liver microsomes – rCYP450s expressed in various cell lines – Intact liver systems – Effects of specific inhibitors – Effects of antibodies on metabolism 58
  59. 59. Biotransformation of Xenobiotics... In vitro guidance: techniques/approaches • Guidance focuses on P450 enzymes • Other hepatic enzymes not as well- characterized • Gastrointestinal drug metabolism is discussed • Metabolism studies in animals (preclinical phase) should be conducted early in drug development 59
  60. 60. Biotransformation of Xenobiotics... In vitro guidance: techniques/approaches • Correlation between in vitro and in vivo studies • Should use in vitro concentrations that approximate in vivo plasma concentrations • Should be used in combination with in vivo studies; e.g., a mass balance study may show that metabolism makes small contribution to elimination pathways 60
  61. 61. Biotransformation of Xenobiotics... In vitro guidance: techniques/approaches • Can rule out a particular pathway • If in vitro studies suggest a potential interaction, should consider investigation in vivo ***When a difference arises between in vivo and in vitro findings, in vivo should take precedence*** 61
  62. 62. In vitro guidance: timing ofBiotransformation of Xenobiotics... studies • Early understanding of metabolism can help in designing clinical regimens • Best to complete in vitro studies prior to start of Phase III 62
  63. 63. Biotransformation of Xenobiotics... In vitro guidance: labeling • In vivo findings should take precedence in drug product labeling • If it is necessary to include in vitro information, should explicitly state conditions of extrapolation to in vivo • Assumption: if a drug is a substrate for a particular enzyme, then certain interactions may be anticipated 63
  64. 64. Biotransformation of Xenobiotics... References • Casarett and Doull’s Toxicology, The Basic Sciences of Poisons, 5th Edition, Klassen, Amdur & Doull (eds), Macmillan Publishing Co. • CDER Guidance for Industry: Drug Metabolism/Drug Interaction Studies in the Drug Development Process: Studies in Vitro, April 1997, CLIN 3 • Davit B, Reynolds K, Yuan R et al. FDA evaluations using in vitro metabolism to predict and interpret in vivo metabolic drug-drug interactions: impact on labeling. J Clin Pharmacol 1999 Sep;39(9):899-910 64

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