1. Lectures • Wednesday, October 8th • Drug Metabolism Overview • Phase I Drug Metabolism • Monday, October 13th • Phase II Drug Metabolism • Intrinsic Clearance • Wednesday, October 15th • In vitro Experimental Methods • In vivo Experimental Methods

2. Drug Metabolism: Phase II Dr. Arthur G. Roberts

3. Outline • Introduction • UDP-glucuronosyltransferases (UGTs) • Sulfotransferases (SULTs) • Glutathione-S-Transferases (GSTs) • N-acetyltransferases (NATs) • Methyltransferases • Catechol O-Methyltransferases (COMTs) • Histamine Methyltransferases (HMTs) • ThiopurineMethyltransferases (TPMTs)

4. Pathways of Drug Metabolism Drugs Oxidation and Reduction Conjugation

5. UGTs TPMT COMT HMT STs GST-A GST-P GST-T GST-M NAT2 NAT1 others Fractional Contribution HMT COMT STs TPMT GST-A GST-P UGTs GST-T GST-M NAT2 NAT1 others HMT: histamine methyltransferase; TPMT: thiopurine methyltransferase; COMT: catechol O-methyltransferase; UGT: Uridine Glucuronosyl-S-Transferases; ST: Sulfotransferase; GST: Glutathione-S-Transferases Evans and Relling, Science (1999)

6. UDP-glucuronosyltransferases (UGTs) Membrane UDP = N-terminal C-terminal glucuronic acid (GA)

7. Conjugates to nucleophilic groups such as amines, amides, thiols, carboxylic acids and alcohols/phenols Includes some unusual substrates (e.g. b di-ketone)

8. Tissue Location (Shipkova, 2005 Clin. Chim. Acta)

9. Subcellular Location UDP-N-acetylglucosamine UDP Glucuronic Acid Smooth ER Smooth ER, rough ER, golgi apparatus and nuclear membrane (Clark, 1994)

10. Substrate Specificity • Promiscuous • Drug-drug interaction • UGT-UGT Interaction

11. Anticancer Drugs Other Drugs UGT1A1 Inhibitors

12. Endogenous Substrates steroids

13. Drug-Drug Interaction in UGTs Substrate not metabolized by UGT1A1 Zidovudine (a.k.a AZT) Estradiol (hormone) Ethinylestradiol (contraceptive) site of glucuronidation

14. UGT-UGT Interaction Serotonin Morphine Kurkela, M., Patana, A. S., Mackenzie, P. I., Court, M. H., Tate, C. G., Hirvonen, J., Goldman, A., & Finel, M. (2007) Pharmacogenetics and genomics17, 115-126.

15. Families • UGT1A • Planar and bulky phenols (e.g. estrogens), amines and unsaturated carboxylic acids • UGT1A1: bilirubin and morphine • UGT2B • drugs, steroids, bile acids and unsaturated carboxylic acids

17. Morphine

18. Regulation: Induction AhR=Aryl Hydrocarbon Receptor CAR=ConstituitiveAndrostane Receptor PXR=Pregnane-X-Receptor XRE=Xenobiotic Response Elements PBREM=Phenobarbital Response Elements PXRRE=PXR Response Elements PXR CAR AhR PXRRE PBREM XRE UGT1A/2B UGT1A/2B UGT1A (Xie, 2004)

19. Diseases • Hyperbilirubinemia • Gilbert’s syndrome (20-30% UGT1A1 Activity) • Crigler-Najjar syndrome • Type I (0% UGT1A1 Activity) • Type II (10% UGT1A1 Activity) • Gray Baby Syndrome (Chloramphenicol) • Neonatal Jaundice • UGT1A1*28/*28 • light therapy • Autoimmune Hepatitis

20. D C B A E Chloramphenicol (Broad Spectrum Antibiotic)

21. Metabolic Switching Drugs Toxic Oxidation Conjugation

22. Sulfotranferases (SULTs) PAPS (Petrotchenko, 2001 FEBS Letters)

23. Reaction/Mechanism PAPS synthase

24. Common Substrates

25. Resulting Products OSO3 NSO3 OSO3 NOSO3 OSO3 *Note: This molecule is hydroxylated first.

26. Subcellular Location • Cytosolic • Homodimers/Heterodimers • Membrane-bound • Golgi Apparatus

27. Families and General Substrate Specificities • SULT1 • Phenols, Catechols, Benzylic Alcohols, Amines, Estrogens • SULT2 • Steroids

28. SO3 SULT1 or SULT2? 1-hydroxymethylpyrene (Environmental pollutant)

29. Glutathione-S-Transferases GST A1-1 GST A4-4 % exchanged >50% 35-50% 20-35% <20% Hou, L., Honaker, M. T., Shireman, L. M., Balogh, L. M., Roberts, A. G., Ng, K. C., Nath, A., & Atkins, W. M. (2007) J. Biol. Chem.282, 23264-23274.

30. General Reaction (GSH) (GS-Conjugate) (Townsend, 2003 Oncogene)

31. Reactions epoxides R R R R halogenated alkanes GS GS isothiocyanates isocyanates O quinones o-quinones

32. More Reactions X X EWG EWG EWG X- Nucleophilic Aromatic Substitution EWG EWG EWG EWG = strong electron withdrawing group; X= halide or other good leaving group GS GS Michael Addition spontaneous thiolysis H2O GS GSH + HO spontaneous EWG EWG

33. SG OH Carbamazepine Epoxide Metabolite

34. GS Sulforaphane (From vegetables- Anticancer, Antimicrobial)

35. SG HCl chlorobutane

36. SG NAPQI (n-acetyl-p-benzoquinone imine)

37. Families and Tissue Locations • alpha (A) - Liver • kappa (K) • mu (M) – brain, lung , testes, lymphoblasts, skeletal muscle • omega (O) • pi (P) – all tissues except Liver • sigma (S) – macrophages, placenta and fat • prostaglandin metabolism • theta (T) • zeta (Z)

38. Location • Tissue • Everywhere • Subcellular • Cytosolic, Mitochondrial and Microsomal

39. Regulation • GST A, M and P • electrophiles that produce reactive oxygen • antioxidant response element

40. Genetics (Often Missing) • GST M1 • <50% Caucasians and East Asians • >90% Polynesians and Micronesians • >50% Africans • absence increases risk for lung and bladder cancer • GST T1 • 20% missing in Caucasians • 50% missing in Asians • 40% missing in Africans

41. Glutathione conjugate metabolism Urine

42. N-acetyltransferases (NAT) Acetyl-CoA

43. General Reaction NAT R R

44. Reactions (Sim, 2008 Toxicology)

45. Families, Location and General Substrate Specificities • NAT1 • everywhere • folate degradation • breast cancer • NAT2 • Liver and Gut • sulfamethazine and arylhydrazine • isoniazid induced neurotoxicity and hydralazine-induced lupus (slow metabolizers) sulfamethazine arylhydrazine (Sim, 2008 Toxicology)

46. NAT1 and Folate Degradation folate folatecatabolite NAT1 acetylated folatecatabolite

47. Substrate Specificities p-aminobenzoic acid (PABA) 2-aminofluorene (2-AF) hydralazine (HDZ) (Sim, 2008 Toxicology)

48. Sulfamethoxazole (antibiotic)

49. hydralazine (smooth muscle relaxant)

50. Genetics: Polymorphic Variants NAT2 NAT1 (Sim, 2008 Toxicology)