1. Drug Metabolism S.P. Markey Laboratory of Neurotoxicology NIMH, NIH Nov. 14, 2002

2. Evolution of Drug Metabolism As a Science Post WWII Pioneers R.T. Williams – Great Britain –1942, worked on the metabolism on TNT with regard to toxicity in munitions workers; due to the war he assembled teams to work on metabolism of sulfonamides, benzene, aniline, acetanilide, phenacetin, and stilbesterol –Developed concept of Phase 1 & Phase 2 Reactions. Biotransformation involves metabolic oxygenation, reduction, or hydrolysis; result in changes in biological activity (increased or decreased) Second phase, conjugation, in almost all cases resulted in detoxication.

3. Evolution of Drug Metabolism As a Science Post WWII Pioneers B.B. Brodie, U.S. –NYU and Laboratory of Industrial Hygiene, NYC 1949 – Metabolic fate of acetanilide and phenacetin in man (with J. Axelrod) –1950s, NIH – pioneering studies on all aspects of drug metabolism; esp. reserpine, serotonin;hexobarbital tolerance –1952 – R.T. Williams spent 6 months at NIH; subsequently many students went between both labs (Dick Adamson, Jim Gillette, and Sidney Udenfriend) –1950s, Brodie lab developed the spectrophotofluorimeter (R. Bowman)

4. Drug Metabolism Hepatic microsomal enzymes (oxidation, conjugation) Extrahepatic microsomal enzymes (oxidation, conjugation) (oxidation, conjugation) Hepatic non-microsomal enzymes (acetylation, sulfation,GSH, alcohol/aldehyde dehydrogenase, hydrolysis, ox/red)

5. Liver Microsomal System Oxidative Reactions: Cytochrome P450 mediated Examples –Formation of an inactive polar metabolite Phenobarbital –Formation of an active metabolite By Design: Purine & pyrimidine chemotherapy prodrugs Inadvertent: terfenadine – fexofenadine –Formation of a toxic metabolite Acetaminophen – NAPQI

6. Electron flow in microsomal drug oxidizing system CO hh CYP-Fe +2 Drug CO O2O2 e-e- e-e- 2H + H2OH2O Drug CYP R-Ase NADPH NADP + OH Drug CYP Fe +3 PC Drug CYP Fe +2 Drug CYP Fe +2 Drug O2O2 CYP Fe +3 OH Drug

7. Cytochrome P450 Isoforms (CYPs) - An Overview NADPH + H + + O 2 + Drug  NADP + + H 2 O + Oxidized Drug Carbon monoxide binds to the reduced Fe(II) heme and absorbs at 450 nm (origin of enzyme family name) CYP monooxygenase enzyme family is major catalyst of drug and endogenous compound oxidations in liver, kidney, G.I. tract, skin, lungs Oxidative reactions require the CYP heme protein, the reductase, NADPH, phosphatidylcholine and molecular oxygen CYPs are in smooth endoplasmic reticulum in close association with NADPH-CYP reductase in 10/1 ratio The reductase serves as the electron source for the oxidative reaction cycle

8. CYP Families Twelve CYP gene families have been identified in humans, and the categories are based upon protein sequence homology Most of the drug metabolizing enzymes are in CYP 1, 2, & 3 families. CYPs have molecular weights of 45-60 kDa. Frequently, two or more enzymes can catalyze the same type of oxidation, indicating redundant and broad substrate specificity. CYP3A4 is very common to the metabolism of many drugs; its presence in the GI tract is responsible for poor oral availabilty of many drugs

9. CYP Nomenclature Families - CYP plus arabic numeral (>40% homology of amino acid sequence, eg. CYP1) Subfamily - 40-55% homology of amino acid sequence; eg. CYP1A Subfamily - additional arabic numeral when more than 1 subfamily has been identified; eg. CYP1A2 Italics indicate gene (CYP1A2); regular font for enzyme

10. CYP Tables Human CYPs - variability and importance in drug metabolismHuman CYPs Isoforms in metabolism of clinically important drugsIsoforms Factors that influence CYP activityFactors Drugs that inhibit CYPsDrugs that inhibit Non-Nitrogenous CYP inhibitorsNon-Nitrogenous Extrahepatic CYPsExtrahepatic

11. Human Liver Drug CYPs S. Rendic & F.J. DiCarlo, Drug Metab Rev 29:413-80, 1997

12. Factors Influencing Activity and Level of CYP Enzymes S. Rendic & F. J. Di Carlo Drug Metab Rev 29: 413-580, 1997 Red indicates enzymes important in drug metabolism

13. Participation of the CYP Enzymes in Metabolism of Some Clinically Important Drugs S. Rendic & F.J. Di Carlo, Drug Metab Rev 29:413-580, 1997

14. Participation of the CYP Enzymes in Metabolism of Some Clinically Important Drugs (cont’d) S. Rendic & F.J. Di Carlo, Drug Metab Rev 29:413-580, 1997

15. Drugs that Inhibit Drug Metabolism by Forming Complexes with CYPs Modified from: A. Alvares and W.B. Pratt, Pathways of Drug Metabolism in Principles of Drug Metabolism (Eds. W.B. Pratt, P.Taylor) 3 rd Edition, 1990

16. Non-nitrogenous Substances that Effect Drug Metabolism by Forming Complexes with CYPs Grapefruit juice - CYP 3A4 inhibitor; highly variable effects; unknown constituents –D.G. Bailey, et al.; Br J Clin Pharmacol 1998, 46:101-110 Isosafrole, safrole - CYP1A1, CYP1A2 inhibitor; found in root beer, perfume Piperonyl butoxide & alcohol -CYP1A1, CYP1A2 inducer; insecticide constituent

17. Overheard Conversation At a B&B breakfast table, after grapefruit juice was served, someone remarked “A friend read the package insert with her prescription and the fine print warned against drinking grapefruit juice…is this true? Should it be avoided with all medications? How about grapefruit itself? How about orange juice?”

18. Effect of Grapefruit Juice on Felodipine Plasma Concentration 5mg tablet with juice without Review- D.G. Bailey, et al.; Br J Clin Pharmacol 1998, 46:101-110

19. Grapefruit Juice Facts GJ or G (not OJ) elevates plasma peak drug concentration, not elimination t 1/2 GJ reduced metabolite/parent drug AUC ratio GJ caused 62% reduction in small bowel enterocyte 3A4 and 3A5 protein; liver not as markedly effected (i.v. pharmacokinetics unchanged) GJ effects last ~4 h, require new enzyme synthesis Effect cumulative (up to 5x C max ) and highly variable among individuals depending upon 3A4 small bowel basal levels

20. Human Drug Metabolizing CYPs Located in Extrahepatic Tissues S. Rendic & F.J. DiCarlo, Drug Metab Rev 29:413-80, 1997

21. Human Drug Metabolizing CYPs Located in Extrahepatic Tissues (cont’d) S. Rendic & F.J. DiCarlo, Drug Metab Rev 29:413-80, 1997

22. CYP Biotransformations Chemically diverse small molecules are converted, generally to more polar compounds Reactions include: –Aliphatic hydroxylation, aromatic hydroxylationAliphatic hydroxylationaromatic hydroxylation –Dealkylation (N-,O-, S-)Dealkylation (N-,O-, S-) –N-oxidation, S-oxidationN-oxidation, S-oxidation –DeaminationDeamination –DehalogenationDehalogenation

23. Examples: ibuprofen, pentobarbital

24. ibuprofen

25. pentobarbital

26. Examples: acetanilide, phenytoin, propranolol Endogenous substrates: steroid hormones (not aromatic amino acids) Aromatic Hydroxylation

27. phenytoin Arene epoxide intermediate produces multiple products

28. propranolol

29. N (or O, S)-Dealkylation N-demethylation generates formaldehyde

30. ethylmorphine N-demethylation favored over O-deakylation

31. propranolol

32. 6-methylthiopurine

33. Examples: chlorpheniramine, trimethylamine Examples: chlorpromazine, cimetidine

34. chlorpromazine chlorpheniramine

35. Examples: amphetamine, diazepam Deamination

36. amphetamine

37. Example: carbon tetrachloride, others include. halothane, methoxyflurane

38. Non-CYP Drug Biotransformations Oxidations Hydrolyses Conjugation (Phase 2 Rxs) –Major Conjugation Reactions Glucuronidation (high capacity)Glucuronidation Sulfation (low capacity)Sulfation Acetylation (variable capacity)Acetylation Examples:Procainamide, Isoniazid –Other Conjugation Reactions: O-Methylation, S- Methylation, Amino Acid Conjugation (glycine, taurine, glutathione) –Many conjugation enzymes exhibit polymorphism

39. Non-CYP drug oxidations Monoamine Oxidase (MAO), Diamine Oxidase (DAO) - MAO (mitochondrial) oxidatively deaminates endogenous substrates including neurotransmitters (dopamine, serotonin, norepinephrine, epinephrine); drugs designed to inhibit MAO used to effect balance of CNS neurotransmitters (L-DOPA); MPTP converted to toxin MPP+ through MAO-B. DAO substrates include histamine and polyamines. Alcohol & Aldehyde Dehydrogenase - non-specific enzymes found in soluble fraction of liver; ethanol metabolism Xanthine Oxidase - converts hypoxanthine to xanthine, and then to uric acid. Drug substrates include theophylline, 6- mercaptopurine. Allopurinol is substrate and inhibitor of xanthine oxidase; delays metabolism of other substrates; effective for treatment of gout.

40. Non-CYP drug oxidations Flavin Monooxygenases –Family of enzymes that catalyze oxygenation of nitrogen, phosphorus, sulfur – particularly facile formation of N-oxides –Different FMO isoforms have been isolated from liver, lung (D. Ziegler, 1993, Ann Rev Pharmacol Toxicol 33:179-199) –Complete structures defined (Review: J. Cashman, 1995, Chem Res Toxicol 8:165-181) –Require molecular oxygen, NADPH, flavin adenosine dinucleotide (FAD) –Single point (loose) enzyme-substrate contact with reactive hydroperoxyflavin monoxoygenating agent –FMOs are heat labile and metal-free, unlike CYPs –Factors affecting FMOs (diet, drugs, sex) not as highly studied as CYPs

41. FMO Oxidations

42. Hydrolysis Reactions Esters Example: aspirin (others include procaine, clofibrate)

43. Hydrolysis Reactions Amides Example:lidocaine; others include peptide drugs

44. Conjugation Reactions Glucuronidation Liver has several soluble UDP-Gluc-transferases

45. Glucuronic acid conjugation to phenols, 3°-amines, aromatic amines

46. Conjugation Reactions Sulfation Examples: ethanol, p-hydroxyacetanilide, 3-hydroxycoumarin (PAPS, 3’-phosphoadenosine- 5’-phosphosulfate) +

47. Sulfation may produce active metabolite

48. Conjugation Reactions Acetylation Examples: Procainamide, isoniazid, sulfanilimide, histamine NAT enzyme is found in many tissues, including liver

49. Procainamide Unchanged in Urine, 59% 3% 24% Fast 17% Slow Unchanged in Urine, 85% NAPA 0.3% 1%

50. Procainamide trace metabolite non-enzymatic Lupus?

51. Drug Conjugation Example: Isoniazid - N-acetyltransferase First line drug in the treatment of TB; normally given at a does of 5 mg/kg, max. 300 mg/day for period of 9 months Rapid and slow acetylators first seen in TB patients; t 1/2 for fast is 70 min; t 1/2 for slow is 180 min N-acetyltransferase (NAT2 isoform) is in liver, gut Peripheral neuropathy (about 2% patients; higher doses produce effects in 10-20%) seen in slow acetylators (reversible with pyridoxine) Hepatotoxicity also seen, esp. in older patients

52. NAT2 minor IsoniazidN-Acetylisoniazid

53. N-Acetylation may trigger nitrenium ion formation

54. Additional Effects on Drug Metabolism Species Differences –Major differences in different species have been recognized for many years (R.T. Williams). Phenylbutazone half-life is 3 h in rabbit, ~6 h in rat, guinea pig, and dog and 3 days in humans. Induction –Two major categories of CYP inducers Phenobarbital is prototype of one group - enhances metabolism of wide variety of substrates by causing proliferation of SER and CYP in liver cells. Polycylic aromatic hydrocarbons are second type of inducer (ex: benzo[a]pyrene). –Induction appears to be environmental adaptive response of organism –Orphan Nuclear Receptors (PXR, CAR) are regulators of drug metabolizing gene expression

55. PXR and CAR Protect Against Xenobiotics xenobiotics PXR RXR CAR cytoplasm nucleus xenoprotection target genes S.A. Kliewer

56. CYP3A Inducers Activate Human, Rabbit, and Rat PXR rifampicin PCN dexamethasone RU486 clotrimazole Reporter activity (fold) troglitazone 135791113151719 tamoxifen Cell-based reporter assay S.A. Kliewer

57. CYP3A Regulation xenobiotics (e.g., drugs) HO-xenobiotics endobiotics (e.g., steroids) HO-endobiotics CYP3A CYP3A xenobiotics liver intestine XRE ? Protect against xenobiotics Cause drug-drug interactions rifampicin PCN dexamethasone RU486 clotrimazole troglitazone tamoxifen Expressed in liver and intestine Activated by xenobiotics Bind to Xenobiotic Response Elements S.A. Kliewer et al. Endo Rev 23:687, 2002

58. Pregnane X Receptor (PXR) human PXRLigandLigandDNADNA mouse PXR 77%77% 96%96% rat PXR 76%76% 96%96% 82%82% 94%94% rabbit PXR PXR is one of Nuclear Receptor (NR) family of ligand-activated transcription factors. Named on basis of activation by natural and synthetic C21 steroids (pregnanes), including pregnenolone 16  -carbonitrile (PCN) Cloned due to homology with other nuclear receptors Highly active in liver and intestine Binds as heterodimer with retinoic acid receptor (RXR) S.A. Kliewer

59. Constitutive Androstane Receptor (CAR) Highly expressed in liver and intestine Binds response elements as RXR heterodimer High basal transcriptional activity without ligand Sequestered in cytoplasm Activated by xenobiotics –phenobarbital, TCPOBOP (1,4-bis[2-(3,5- dichloropyridyloxy)]benzene) CAR PXR S.A. Kliewer

60. Plasticity in the PXR Binding Pocket hyperforin SR12813 Volume: 1280 Å 3 1544 Å 3 S.A. Kliewer

61. PXR Structure Large, elliptical hydrophobic cavity The cavity changes shape to accommodate different ligands PXR is ideally suited to function as xeno-sensor xenobiotics PXR RXR xenobiotic metabolism S.A. Kliewer

62. PXR and CAR Regulate Overlapping Genes PCN (PXR) Phase I enzymes Cyp3a11 Cyp2b10 Aldh1a1 Aldh1a7 Phase II enzymes Ugt1a1 Gst-a1 Transporters Mrp2 Mrp3 Oatp2 (3.5x) (12x) (2.1x) (1.6x) (2.8x) (16x) (3.0x) (9.2x) TCPOBOP (CAR) (3.4x) (110x) (1.9x) (15x) (2.0x) (1.9x) Liver RNA S.A. Kliewer

63. Acetaminophen Acetanilide – 1886 – accidentally discovered antipyretic; excessively toxic (methemoglobinemia); para-aminophenol and derivatives were tested. Phenacetin introduced in 1887, and extensively used in analgesic mixtures until implicated in analgesic abuse nephropathy; 1946, Lester reported conjugated para-aminophenol as major metabolite of acetanilide 1948-49 Brodie and Axelrod recognized acetaminophen as the major active metabolite in phenacetin CAR modulates acetaminophen toxicity [Science (Oct 11) 298:422, 2002]

64. Acetaminophen and p-Aminophenols Acetanilide, 1886 (accidental discovery of antipyretic activity; high toxicity) Phenacetin or acetophenetidin, 1887 (nephrotoxic, methemoglobinemia) Acetaminophen, 1893 Recognized as active metabolite of acetanilide and phenacetin in 1948 (Brodie &Axelrod); popular in US since 1955 70-90% 75-80%

65. Acetominophen Metabolism ~60% ~35% CYP2E1* CYP1A2 CYP3A4 NAPQI N-acetyl-p-benzoquinone imine *induced by ethanol, isoniazid Protein adducts, Oxidative stress Toxicity

66. Acetaminophen overdose results in more calls to poison control centers in the United States than overdose with any other pharmacologic substance. The American Liver Foundation reports that 35% of cases of severe liver failure are caused by acetaminophen poisoning which may require organ transplantation. N-acetyl cysteine is an effective antidote, especially if administered within 10 h of ingestion [NEJM 319:1557- 1562, 1988] Addition of N-acetyl cysteine to acetaminophen tablets proposed to prevent liver toxicity. [British Medical Journal, Vol. 323, Sept. 15, 2001] Acetaminophen Toxicity

67. Acetaminophen Protein Adducts CYP2E HS-Protein H 2 N- Protein S.D. Nelson, Drug Metab. Rev. 27: 147-177 (1995) J.L. Holtzman, Drug Metab. Rev. 27: 277-297 (1995)

68. NAPQI toxicity linked to CAR activation, GSH depletion Glutathione S-Transferase (GST Pi) CAR Phenobarb TCPOBOP GST Pi androstanol toxicity oxidative stress mechanism ?

69. Protective effect. Liver cells die (pale areas) when exposed to high doses of acetaminophen (left), but a CAR inhibitor protects against such damage (right). Jun Zhang, * Wendong Huang, * Steven S. Chua, Ping Wei, David D. Moore Science, October 11, 298:422, 2002

70. Acetaminophen toxicity mechanism Mice nulled for glutathione S-transferase are resistant to acetaminophen toxicity –equal amounts of acetaminophen protein adducts formed in null and wild type suggesting protein adducts may not be toxic –hepatic GSH lowered in wild type (but not in KO) after acetaminophen CAR nulled mice are also resistant to acetaminophen toxicity –hepatic GSH lowered in wild type (but not in KO) after acetaminophen –CAR-humanized mice demonstrate same toxicity response N-acetyl cysteine is an effective agent to block GSH depletion and rescue from liver damaging toxicity NAPQI-protein adduction or NAPQI-GSH depletion-oxidative stress....to be continued

71. Terfenadine (Seldane © )

72. Terfenadine in the News DHHS/FDA: Terfenadine; Proposal to Withdraw Approval of Two New Drug Applications –Federal Register 62, January 14, 1997 Hoechst Marion Roussel To Promote Switch From Seldane to Allegra –Independent News Service, January 14, 1997 Citing Its Side Effects, F.D.A. Weighs Ban on Allergy Drug –The New York Times, January 14, 1997 FDA Wants Drug Seldane Off Market –The Washington Post, January 14, 1997 Hoechst’s First Quarter Results Below Forecasts –Independent News Service, May 7, 1997

73. Terfenadine Developed in 1980s as a 2nd generation H1- antihistamine; from introduction in 1985, prescriptions > 16 million in 1991 First generation antihistamines are lipophilic ethylamine derivatives that readily penetrate the CNS and placenta - objective of 2nd generation is minimal CNS effects (non-sedating), not crossing the blood brain barrier; longer acting Cardiac side-effects are serious - inhibition of potassium channels by unmetabolized parent drug causes prolongation of QT interval leading to life threatening arrythmia (torsades de pointes); first recognized at USUHS in 1989 (Monahan BP et al, JAMA 1990; 264:2788-2790.) Drugs or substances inhibiting terfenadine metabolism (grapefruit juice, ketoconazole, itraconazole, antimicrobials) or liver dysfunction exacerbate the side effects

74. Terfenadine Metabolism CYP3A4 Terfenadine (Seldane) Fexofenadine (Allegra)

75. Drug Metabolism - WWW Information Resources http://www.icgeb.trieste.it/p450/ –Directory of P450 Containing Systems; comprehensive web site regarding all aspects of chemical structure (sequence and 3D) of P450 proteins from all species; steroid ligands; links to related sites including leading researchers on P450 http://www.panvera.com/tech/dmeguide/index.html –Drug Metabolism Resource Guide - catalog with useful information and characteristics of natural and recombinant drug metabolizing enzymes; assay methods http://www.netsci.org/Science/Special/feature06.html –Site contains essay “The emerging role of ADME in optimizing drug discovery and design” RJ Guttendorf, Parke-Davis http://www.fda.gov/cder/guidance/ –Site contains many useful documents regarding drug metabolism and FDA recommendations including "Drug Metabolism/Drug Interaction Studies in the Drug Development Process: Studies in Vitro", FDA Guidance for Industry.