1. 1 Drug Metabolism Clinical Pharmacology Spring Course 2006 M. E. Blair Holbein, Ph.D. Clinical Pharmacologist Presbyterian Hospital

2. 2 Drug Metabolism - History  “Xenobiotic metabolism” established by Richard Tecwyn Williams  First paper with an identified “metabolite” in Nature 1931  Wrote first book on the Detoxification Mechanisms” 1959  Focus on elimination of foreign compounds  Proposed a delineation of:  Phase I (oxidation, reduction, hydrolysis) biotransformations as primary covalent chemical modifications to administered compound  Phase II (conjugation) with an endogenous polar species  To either parent drug  Phase I product(s)

3. 3 Biotransformations  Phase I  Oxidation  Cytochrome P450 monooxygenase system  Flavin-containing monooxygenase system  Alcohol dehydrogenase and aldehyde dehydrogenase  Monoamine oxidase (Co-oxidation by peroxidases)  Reduction  NADPH-cytochrome P450 reductase  Reduced (ferrous) cytochrome P450  Hydroloysis  Esterases and amidases  Epoxide hydrolase  Phase II  Glutathione S-transferases  Mercapturic acid biosynthesis  UDP-Glucoron(os)yltranasferases  N-Acetyltransferases  Amino acid N-acyl transferases  Sulfotransferases

4. 4 Biotransformations  Phase I  Oxidation  Cytochrome P450 monooxygenase system  Flavin-containing monooxygenase system  Alcohol dehydrogenase and aldehyde dehydrogenase  Monoamine oxidase (Co-oxidation by peroxidases)  Reduction  NADPH-cytochrome P450 reductase  Reduced (ferrous) cytochrome P450  Hydroloysis  Esterases and amidases  Epoxide hydrolase  Phase II  Glutathione S-transferases  Mercapturic acid biosynthesis  UDP-Glucoron(os)yltranasferases  N-Acetyltransferases  Amino acid N-acyl transferases  Sulfotransferases

5. 5 Biotransformations  Phase I  Oxidation  Cytochrome P450 monooxygenase system  Flavin-containing monooxygenase system  Alcohol dehydrogenase and aldehyde dehydrogenase  Monoamine oxidase (Co-oxidation by peroxidases)  Reduction  NADPH-cytochrome P450 reductase  Reduced (ferrous) cytochrome P450  Hydroloysis  Esterases and amidases  Epoxide hydrolase  Phase II  Glutathione S-transferases  Mercapturic acid biosynthesis  UDP-Glucoron(os)yltranasferases  N-Acetyltransferases  Amino acid N-acyl transferases  Sulfotransferases

6. 6 Drug Metabolism - Determinants of Activity  Inhibition of enzyme activity  Patterns  Competitive  Noncompetitive  Uncompetitive  Effects not mediated by enzyme activity, e.g. free fraction, membrane effects, etc.  Inducibility  Rate-limitations  Substrates  First-pass metabolism, high-extraction drugs  Co-factors  Turnover  Polymorphism  Predictability of in vivo effects based on in vitro data is highly variable

7. 7 Kinetic equations for inhibition of metabolizing enzymes

8. 8 Drug Metabolism - Determinants of Activity  Inhibition of enzyme activity  Patterns  Competitive  Noncompetitive  Uncompetitive  Effects not mediated by enzyme activity, e.g. free fraction, membrane effects, etc.  Inducibility  Rate-limitations  Substrates  First-pass metabolism, high-extraction drugs  Co-factors  Turnover  Polymorphism  Predictability of in vivo effects based on in vitro data is highly variable

9. 9 Biotransformations  Phase I  Oxidation  Cytochrome P450 monooxygenase system  Flavin-containing monooxygenase system  Alcohol dehydrogenase and aldehyde dehydrogenase  Monoamine oxidase (Co-oxidation by peroxidases)  Reduction  NADPH-cytochrome P450 reductase  Reduced (ferrous) cytochrome P450  Hydroloysis  Esterases and amidases  Epoxide hydrolase  Phase II  Glutathione S-transferases  Mercapturic acid biosynthesis  UDP-Glucoron(os)yltranasferases  N-Acetyltransferases  Amino acid N-acyl transferases  Sulfotransferases

10. 10 Phase I Oxidation: Cytochrome P450 Isoenzymes Background:  Huge superfamily of highly versatile enzymes (over 3800 sequences identified)  Found in the genomes of virtually all organisms  Heme-containing proteins named for the absorption band at 450 nm when combined with carbon monoxide  NADP(H) used with molecular oxygen to produce oxidation of a variety of compounds:  Xenobiotics  Endobiotics  In prokaryotes, P450s are soluble proteins.  In eukaryotes, they are usually bound to the endoplasmic reticulum or inner mitochondrial membranes.  Human drug metabolism primarily in the endoplasmic reticulum of hepatocytes. Also in the small intestine, kidney, lung and brain.  More than thirty (30) CYP human isoenzymes have been identified.

11. 11 Catalytic reaction cycle CYP450 and the oxidation of xenobiotics e-e- e-e- CYP Reductase NADP + NADP Fe 2+ CYP Fe 3+ CYP DRUG O2O2 Fe 2+ CYP 2H + H2OH2O Fe 3+ CYP DRUG OH O2O2 DRUG OH

12. 12 Catalytic reaction cycle involving cytochrome P450 in the oxidation of xenobiotics Oxidized Drug + NADP + + H2O Drug + NADPH + H + + O2

13. 13 CYP450 Mediated Chemical Transformation  Hydroxylation  Aliphatic  Aromatic  N-Dealkylation, O-Dealkylation, S-Dealkylation  Oxidative Deamination  Dehalogenation  N-Oxidation  S-Oxidation

14. 14 CYP Mediated Oxidation  Aliphatic Hydroxylation RCH 2 CH 3 OH RCHCH 3 Ex: Hydroxylation of ibuprofen

15. 15 CYP Mediated Oxidation  Aromatic Hydroxylation Ex: Hydroxylation of acetanilide to 4-hydroxyacetanilide

16. 16 CYP Mediated Oxidation  Aromatic Hydroxylation  Directly through asymmetric oxygen transfer  Through an unstable arene oxide intermediate  Predictability  Influence of environment Ex:Hydroxylation of aromatic carbon atoms

17. 17 CYP Mediated Oxidation  Aromatic Hydroxylation  Results in several oxidized metabolites Ex:Metabolism of phenytoin

18. 18 CYP Mediated Oxidation  Dealkation (N-, O-, S-) Ex: N-demethylation of ethylmorphine

19. 19 CYP Mediated Oxidation  N-demethylation generates formaldehyde as a by-product

20. 20 CYP Mediated Oxidation  Dealkation (N-, O-, S-) Ex: N-demethylation and hydroxylation of propranolol

21. 21 CYP Mediated Oxidation  Oxidative Deamination Ex: General mechanism for oxidative deamination

22. 22 CYP Mediated Oxidation  Oxidative Deamination Ex: Deamination of amphetamine to inactive ketone

23. 23 CYP Mediated Oxidation  Dehalogenation Ex: Dehalogenation generates reactive free radicals. Metabolism of carbon tetrachloride generates oxidized lipids

24. 24 CYP Mediated Oxidation  N-Oxidation may produce toxic by-products

25. 25 CYP Mediated Oxidation  N-Oxidation Ex: N-oxidation of Dapsone

26. 26 CYP Mediated Oxidation  S-Oxidation Ex: General scheme Ex: CYP3A and Flavin monooxygenase produce same metabolite

27. 27 CYP Mediated Oxidation  S-Oxidation Ex: A-oxidation of tazofelone

28. 28 Drug Metabolism CYP450  Cytochrome P450 system responsible for the majority of oxidative reactions  Significant polymorphism in many.  CYP2C9, CYP2C19, and CYP2D6—can be even be genetically absent!  Drugs may be metabolized by a single isoenzyme  Desipramine/CYP2D6; indinavir/CYP3A4; midazolam/CYP3A; caffeine/CYP1A2; omeprazole/CYP2C19  Drugs may be metabolized by multiple isoenzymes  Most drugs metabolized by more than one isozyme  Imipramine: CYP2D6, CYP1A2, CYP3A4, CYP2C19  If co-administered with CYP450 inhibitor, some isozymes may “pick up slack” for inhibited isozyme.  Drugs may be metabolized by several different enzyme systems; e.g. CYP450 and MFO.  This enzyme system notably susceptible to induction.  Inherent turnover; highly variable response

29. 29 Cytochrome P450 (CYP) Isoenzymes  All CYP isoenzymes in the same family have at least 40% structural similarity, and those in the same subfamily have at least 60% structural similarity.  Nomenclature ex: CYP2D6  Root: cytochrome P450 CYP  Genetic Family: CYP2  Genetic Subfamily: CYP2D  Specific Gene: CYP2D6  NOTE that this nomenclature is genetically based; it has NO functional implication Phase I Oxidation: Cytochrome P450 (CYP) Isoenzymes

30. 30 Proportion of Drugs Metabolized by CYP450 Enzymes in Humans

31. 31 Cytochrome P450 3A4,5,7  Largest number of drugs metabolized  Present in the largest amount in the liver.  Present in GI tract  Not polymorphic  Inherent activity varies widely  Activity has been shown to predominate in the gut.  Substrates:  Most calcium channel blockers: nifedipine, amlodipine; HMG Co A  Most benzodiazepines: diazepam, midazolam  Most HIV protease inhibitors: indinavir, ritonavir  Most HMG-CoA-reductase inhibitors: atorvastatin, lovastatin  Cyclosporine, tacrolimus  Most non-sedating antihistamines  Cisapride  Macrolide antibiotics: clarithromycin, erythromycin  Chlorpheniramine;  Also: haloperidol, buspirone; sildenafil, tamoxifen, trazodone, vincristine

32. Wilkinson, G. R. N Engl J Med 2005;352:2211-2221 Mechanism of Induction of CYP3A4-Mediated Metabolism of Drug Substrates (Panel A) The Resulting Reduced Plasma Drug Concentration (Panel B)

33. 33 Cytochrome P450 2D6  Second largest number of substrates.  Polymorphic distribution  Majority of the population is characterized as an extensive or even ultra-extensive metabolizer.  Approximately 7% of the U.S. Caucasian population and 1-2% of African or Asian inheritance have a genetic defect in CYP2D6 that results in a poor metabolizer phenotype.  Substrates include: many beta-blockers – metoprolol, timolol, amitriptylline, imipramine, paroxetine, haloperidol, risperidone, thioridazine, codeine, dextromethorphan, ondansetron, tamoxifen, tramadol  Inhibited by: amiodarone, chlorpheniramine, cimetidine, fluoxetine, ritonavir

34. Common Drug Substrates and Clinically Important Inhibitors of CYP2D6 Wilkinson, G. R. N Engl J Med 2005;352:2211-2221

35. 35 Cytochrome P450 2C9  Note: Absent in 1% of Caucasian and African- Americans.  Substrates include: many NSAIDs – ibuprofen, tolbutamide, glipizide, irbesartan, losartan, celecoxib, fluvastatin, phenytoin, sulfamethoxazole, tamoxifen, tolbutamide, warfarin  Inhibited by: fluconazole, isoniazid, ticlopidine  Induced by: rifampin

36. 36 Cytochrome P450 1A2  Substrates include: theophylline, caffeine, imipramine, clozapine  Inhibited by: many fluoroquinolone antibiotics, fluvoxamine, cimetidine  Induced by: smoking tobacco

37. 37

38. 38 Copyright restrictions may apply. Cornelis, M. C. et al. JAMA 2006;295:1135-1141. Coffee Intake and Relative Risk of Myocardial Infarction by CYP1A2 Genotype

39. 39 Cytochrome P450 2C19  Note: Absent in 20-30% of Asians, 3-5% of Caucasians  Substrates include: omeprazole, diazepam, phenytoin, phenobarbitone, amitriptylline, clomipramine, cyclophosphamide, progesterone  Inhibited by: fluoxetine, fluvoxamine, ketoconazole, lansoprazole, omeprazole, ticlopidine

40. 40 Cytochrome P450 2B6  Substrates include: bupropion, cyclophosphamide, efavirenz, methadone  Inhibited by: thiotepa  Induced by: phenobarbital, rifampin

41. 41 Cytochrome P450 2E1  Substrates include: acetaminophen

42. 42 Cytochrome P450 2C8  Substrates; paclitaxel, torsemide, amodiaquine, cerivastatin, repaglinide  Inhibited by: trimethoprim, quercetin, glitazones, gemfibrozil, montelukast  Induced by: rifampin

43. 43 Biotransformations  Phase I  Oxidation Cytochrome P450 monooxygenase system  Flavin-containing monooxygenase system  Alcohol dehydrogenase and aldehyde dehydrogenase  Monoamine oxidase (Co-oxidation by peroxidases)  Reduction  NADPH-cytochrome P450 reductase  Reduced (ferrous) cytochrome P450  Hydroloysis  Esterases and amidases  Epoxide hydrolase  Phase II  Glutathione S-transferases  Mercapturic acid biosynthesis  UDP-Glucoron(os)yltranasferases  N-Acetyltransferases  Amino acid N-acyl transferases  Sulfotransferases

44. 44 Non-CYP Mediated Chemical Transformation  Hydrolysis  Reduction  Oxidations  Flavine monooxygenases  Monoamine and diamine oxidases  Alcohol and aldehyde dehydrogenase

45. 45 Non-CYP Mediated Biotransformation  Hydrolysis  Esterases, amidases and proteases  Non-microsomal (cytosolic)  Widely distributed in most tissues

46. 46 Non-CYP Mediated Biotransformation  Reduction Ex: Reduction of side-chain of digoxin produces inactive metabolite

47. 47 Non-CYP Mediated Chemical Transformation  Hydrolysis  Reduction  Oxidations  Flavine monooxygenases  Monoamine and diamine oxidases  Alcohol and aldehyde dehydrogenase

48. 48 Flavine Monooxygenases  Wide variety of substrates  First isolated from pig liver  Originally termed N-oxidase (or Ziegler’s enzyme)  Products are generally polar, non-toxic compounds  Some generation of reactive intermediates, esp. S-oxides

49. 49 Flavine Monooxygenases Isoenzymes  Six genes in mammals  Nomenclature based on sequence homology  FMO1: major human fetal liver; adult kidney  FMO2: lung (most species)  FMO3: major adult liver form; major form in brain  Interindividual variability  FMO4: atypical  FMO5: trace  FMO6: reported  Question of inducibility (vs. P450)  Genetic polymorphism

50. 50 Flavine Monooxygenases Reactions  S-Oxygenation  Spironolactone  Cimetidine  Stereoselective: FMO3 forms the (+) enantiomer and FMO1 forms (- ) enantiomer  N-Oxygenation  Imipramine  Nicotine

51. 51 Flavine Monooxygenases Mechanism  Requires O 2 and NADPH  Unlike P450 which forms oxidizing intermediate AFTER binding substrate, FMO exists in “preloaded” state and will oxygenate any lipophilic substrate that binds with it.  Individual FMOs have broader substrate range than individual CYP

52. 52 Flavine Monooxygenase Cycle FMO Cycle NADP + + H 2 O Enz | Fl ox DRUG DRUG - O NADPH Enz | FlH 2 + NADP + O2O2 Enz | FlHOH Enz | FlOOH

53. 53 Catalytic reaction cycle CYP450 and the oxidation of xenobiotics e-e- e-e- CYP Reductase NADP + NADP Fe 2+ CYP Fe 3+ CYP DRUG O2O2 Fe 2+ CYP 2H + H2OH2O Fe 3+ CYP DRUG OH O2O2 DRUG OH

54. 54 Flavine Monooxygenase Cycle FMO Cycle NADP + + H 2 O Enz | Fl ox DRUG DRUG - O NADPH Enz | FlH 2 + NADP + O2O2 Enz | FlHOH Enz | FlOOH

55. 55 Non-CYP Mediated Oxidation  Oxidation: Flavine Monooxygenases Ex: N-Oxidation of nicotine, catalyzed by FMO3

56. 56 Non-CYP Mediated Oxidation  Oxidation: Flavine Monooxygenases Ex: S-Oxidation of cimetidine, catalyzed by FMO3

57. 57 Non-CYP Mediated Chemical Transformation  Hydrolysis  Reduction  Oxidations  Flavine monooxygenases  Monoamine and diamine oxidases  Alcohol and aldehyde dehydrogenase

58. 58 Non-CYP Mediated Oxidation  Oxidation: Monoamine Oxidases  Mitochondrial enzymes  Deaminate endogenous neurotransmitters  Dopamine  Serotonin  Norepinephrine  Epinephrine  Same type of products as other oxidizing enzymes  Distinguish source enzyme of metabolites by other means  Found in liver, kidney, intestine, brain

59. 59 Non-CYP Mediated Oxidation  Oxidation: Diamine Oxidases  Endogenous amines  Histamine  Polyamines  Putrescine  Cadaverine  Amines converted to aldehydes (in presence of O 2 )  Contribute to oxidation of some drugs  Found in liver, intestine, placenta

60. 60 Non-CYP Mediated Chemical Transformation  Hydrolysis  Reduction  Oxidations  Flavine monooxygenases  Monoamine and diamine oxidases  Alcohol and aldehyde dehydrogenase

61. 61 Non-CYP Mediated Oxidation  Alcohol and Aldehyde Dehydrogenases Ex: Products of alcohol dehydrogenase are substrates for aldehyde dehydrogenase.

62. 62 Non-CYP Mediated Oxidation  Alcohol and Aldehyde Dehydrogenases Ex: Products of alcohol dehydrogenase are substrates for aldehyde dehydrogenase.

63. Relative Contribution to Drug Metabolism - Phase I Evans & Relling Science 1999

64. 64 Phase II Biotransformation: Conjugation: Glucuronidation, Sulfation, Acetylation  Addition of hydrophilic groups (glucuronic acid, sulfate, glycine, or acetyl) onto the drug or drug metabolite  Catalyzed by a group of enzymes called transferases.  Located in cytosol  Microsomal enzyme: Uridine diphosphate glucuronosyltransferase (UGTs)

65. 65 Biotransformations  Phase I  Oxidation  Cytochrome P450 monooxygenase system  Flavin-containing monooxygenase system  Alcohol dehydrogenase and aldehyde dehydrogenase  Monoamine oxidase (Co-oxidation by peroxidases)  Reduction  NADPH-cytochrome P450 reductase  Reduced (ferrous) cytochrome P450  Hydroloysis  Esterases and amidases  Epoxide hydrolase  Phase II  Glutathione S-transferases  UDP-Glucoron(os)yltranasferases  N-Acetyltransferases  Amino acid N-acyl transferases  Sulfotransferases

66. 66 Phase II Biotransformations (Conjugations)  Glutathione  Catalyzed by glutathione-S-transferases  Cytosolic and microsomal  Detoxification of electrophilic (and potentially carcinogenic) molecules

67. 67 UDP-Glucuronosyltransferase (UGT)  Catalyses conjugation of glucuronic acid with a substrate with a suitable functional group  The most important (quantitatively) conjugation step  Substrates  Xenobiotics (drugs, dietary chemicals, carcinogens, environmental pollutants)  Endobiotics (steroid hormones, bilirubin, bile acids, fatty acids)  Altered activity important (toxicology, pharmacologically)  Microsomal location in endoplasmic reticulum on opposite side of membrane from CYP  Transporter functions for cofactors  Polymorphic (at least two families)  Rare disorders associated with genetic abnormalities  Criglar-Najjar Syndromes (types1,2)  Absence of bilirubin conjugation enzyme and marked unconjugated hyperbilirubinemia & jaundice  Gilbert Syndrome  Partial block in bilirubin conjugation; benign elevation in total and unconjugated bilirubin

68. 68 Phase II Biotransformations (Conjugations)  Glucuronidation Ex: N- and O- linked glucuronide formation markedly enhances the polarity and water solubility.

69. 69 Phase II Biotransformations (Conjugations)  Glucuronides can be generated from a variety of substrates

70. 70 Phase II Biotransformations (Conjugations)  Sulfation General pathway for enzymatic sulfation

71. 71 Phase II Biotransformations (Conjugations)  Sulfation Ex: Minoxidil

72. 72 Phase II Biotransformations (Conjugations)  Acetylation Ex: Acetyl transferase donates the acyl group from Coenzyme A to drug substrates

73. 73 Phase II Biotransformations (Conjugations)  Acetylation Ex: Isoniazid inactivation by acetylation

74. 74 Phase II Biotransformations (Conjugations)  Hydroxylation and acetylation Ex: Reactive nitrenium ions may be produced in the metabolism of aromatic amines through hydroxylation and acetylation

75. 75 Biotransformations  Phase I  Oxidation  Cytochrome P450 monooxygenase system  Flavin-containing monooxygenase system  Alcohol dehydrogenase and aldehyde dehydrogenase  Monoamine oxidase (Co-oxidation by peroxidases)  Reduction  NADPH-cytochrome P450 reductase  Reduced (ferrous) cytochrome P450  Hydroloysis  Esterases and amidases  Epoxide hydrolase  Phase II  Glutathione S-transferases  UDP-Glucoron(os)yltranasferases  N-Acetyltransferases  Amino acid N-acyl transferases  Sulfotransferases

76. 76 Questions?  Blair Holbein, Ph.D., BCAP  Presbyterian Hospital of Dallas  Email: bholbein@hcin.netbholbein@hcin.net  Website: http://phdres.caregate.nethttp://phdres.caregate.net  Annotated bibliography

77. 77 References  Wright JM. Drug Interactions.  In: Carruthers SG, Hoffman BB, et al.s, ed. Melmon and Morrelli’s Clinical Pharmacology: Basic Principles in Therapeutics, 4th ed. New York 2000 :McGraw-Hill.  Markey SM.Pathways of Drug Metabolism  In: Atkinson AJ, Daniels CE, Dedrick RL, et al., ed. Principles of Clinical Pharmacology, New York 2001: Academic Press.