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    PHASE II DRUG METABOLISM Fall 2011 Philip C. Smith, Ph.D. Room 1309, Kerr Hall 919 962-0095 Outline:

    i. References

    ii. Definitions

    iii. Common features of Phase II metabolism

    iv. Sulfotransferases

    v. N-Acetyltransferases

    vi. Aminoacid conjugation

    vii. Glutathione conjugation

    This lecture material is for use as instructional material at the University of North Carolina. No part of this material may be reproduced or transmitted electronically without permission in writing from the author.

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    Phase II Metabolism of Drugs - References Books:Pearson PG, Wienkers LC, editors, Handbook of Drug Metabolism, second edition, Informa Healthcare, New York, 2009. Testa B, Krmer SD. The biochemistry of drug metabolism--an introduction: part 4. reactions of conjugation and their enzymes. Chem Biodivers

    Gonzales, FJ, Tukey RH, Drug Metabolism, In: Goodman and Gillmans The Pharmacological Basis of Therapeutics. McGraw-Hill, New York, 2006.

    . 5:2171-336 (2008). [Available online]

    Sies, H, Packer, L. eds. Phase II Conjugation Enzymes and Transport Sytstems. Methods in Enzymology V.400, Academic Press, New York, 2005.

    Kauffman, F.C., editor, Conjugation-deconjugation reactions in drug metabolism and toxicity. Springer Verlag, New York, 1994.

    Mulder, G.J., Conjugation reactions in drug metabolism. Taylor and Francis, New York, 1990.

    Mulder, G.J., Sulfation of drugs and related compounds, CRC Press, Boca Raton, FL, 1981.

    Testa, B. and Jenner, P., Drug metabolism: Chemical and biochemical aspects. Marcel Dekker, New York, 1976.

    Dutton, G.J., Glucuronidation of drugs and other compounds, CRC Press, Boca Raton, FL, 1980. Reviews/Articles: Guillemette C, Lvesque E, Harvey M, Bellemare J, Menard V. UGT genomic diversity: beyond gene

    duplication. Drug Metab RevIshii Y, Nurrochmad A, Yamada H.

    . 42(1):24-44 (2010). Modulation of UDP-glucuronosyltransferase activity by endogenous

    compounds. Drug Metab Pharmacokinet. 25(2):134-48 (2010). Walker K, Ginsberg G, Hattis D, Johns DO, Guyton KZ, Sonawane B. Genetic polymorphism in N-

    Acetyltransferase (NAT): Population distribution of NAT1 and NAT2 activity. J Toxicol Environ Health B Crit Rev. 12:440-72 (2009).

    Hebbring SJ, Moyer AM, Weinshilboum RM. Sulfotransferase gene copy number variation: pharmacogenetics and function. Cytogenet Genome Res. 123 :205-10 (2008).

    Nowell S, Falany CN. Pharmacogenetics of human cytosolic sulfotransferases. OncogeneWang LQ, James MO. Inhibition of sulfotransferases by xenobiotics. Current Drug Metab. 7: 83-104 (2006).

    . 25:1673-8 (2006).

    Zhou J, Zhang J, Xie W. Xenobiotic nuclear receptor mediated regulation of UDP-glucuronosyltransferases. Current Drug Metab. 6: 289-298 (2005).

    Maruo Y, Iwai M, Mori A, Sato H, Takeuchi Y. Polymorphism of UDP-glucuronosyltransferases and drug metabolism. Current Drug Metab. 6: 91-99 (2005).

    Glatt H, Meinl W. Pharmacogenetics of soluble sulfotransferases (SULTs). Naunyn Schmiedebergs Arch Pharmacol. 369:55-68 (2004).

    Chapman E, Best MD, Hanson SR, Wong CH. Sulfotransferases: structure, mechanism, biological activity, inhibition, and synthetic utility. Angew Chem Int Ed Engl. 43: 3526-3548 (2004).

    Coughtrie MW. Sulfation through the looking glass--recent advances in sulfotransferase research for the curious. Pharmacogenomics J. 2:297-308 (2002).

    Thomae BA, Eckloff BW, Freimuth RR, Wieben ED and Weinshilboum RM. Human suflotransferase SULT2A1 pharmacogenetics: Genotype-to-phenotype studies. Pharmacogenomics J 2: 48-56 (2002).

    Hein DW. Molecular genetics and function of NAT1 and NAT2: role in aromatic amine metabolism and carcinogenesis. Mutation Res. 506-507: 65-77 (2002).

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    Conjugation Reactions Some Definitions Glucuronidation GSH conjugation Sulfation Amino acid conjugation Acetylation Methylation

    Definition of Phase II metabolism: Phase II metabolism is terminology coined by RT Williams, whereby a compound is

    first subject to oxidation, reduction or hydrolysis which may be associated with bioactivation, and then the functional group created is conjugated to a less toxic or inactive compound. (The product not always the less toxic or inactive species).

    Phase I Phase II, UGT Toluene Benzoic acid Phase II Glycine aminotransferase

    Hippuric acid Urine

    Many compounds or drugs contain functional groups that can be directly conjugated and thus do not require Phase I metabolism to create handles for conjugation.

    Phase III metabolism is sometimes used to refer to efflux via transporters. Q. Then should uptake via transporters be Phase 0? Q. Which is rate-limiting?

    Conjugation does not always result in less toxicity or inactivation, eg. Morphine-6-glucuronide is pcol active

    N-acetyl procainamide is pcol active GSH conjugates of haloalkenes are nephrotoxic via -lyase Acyl glucuronides are reactive, binding covalently to proteins

    Of 52 pcol active metabolites in a 1985 review, only two were conjugates and those were acetylated products (Sutfin and Jusko, in Drug Metabolism and Disposition: Considerations in Clinical Pharmacology, Wilkinson and Rawlins, ed. MTP Press, Boston, 1985).

    COOH COO-glucuronide


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    Common features of Phase II metabolism i. Coupling of a conjugate:

    Increase in molecular weight of the product. Conjugate MW pKa range. glucuronidation 176 3 - 3.5 glycine conjugation 57 3.5 - 4.0 sulfation 81 < 1 glutathione conjugation 289 2.1, 3.5 methylation 14 neutral N-acetylation 42 neutral

    Increase or decrease in lipophilicity of the product. a. Acidic conjugate may increase binding to albumin, thus decreasing V (eg. acetaminophen and triamterene sulfates).

    b. Increased polarity of conjugate may limit passive partitioning into cells, thus decreasing V.

    eg. acetaminophen Vss 52 L in sheep (Wang and Benet) sulfate metab. 12 L glucuronide metab. 10 L

    b. Acetylation and methylation effect on V is less unpredictable. eg. Procainamide Vss 1.9 L/kg (Goodman & Gilman, 9th Ed.) NAPA 1.4 L/kg

    HN C CH3



    O O


    HN C CH3










    C O

    HN C CH3







    H2N CHN C2H4 N




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    Conjugated metabolites often have higher clearance than the parent drug, due in part to active excretion into urine and bile.

    Q. Would a metabolite be expected to have a half-life longer or shorter than that of the parent drug? (Consider the half-life equation and the rate-limiting step when there is a sequential series of steps).

    t1/2 = ln 2 * V / CL ii. Co-substrate synthesis and availability Co-substrate depletion. Well documented for sulfation (Levy et al.), glutathione and glycine conjugation (Levy,


    Gregus et al. Drug Metab. Dispos. 20: 234 (1992). - Glycine and CoA depletion in rats. Usually we do not have a good estimate of what co-substrate levels are in vivo at the site of

    metabolism. As bi-substrate enzymes, co-substrate conc. influences metabolic rate.

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    iv. Effect of conjugation on directing metabolite excretion. Phase II conjugation often creates anionic metabolites that are then efficiently excreted into the bile via MRP2, other transporters. Active secretion of organic acid metabolites in the renal proximal tubules also enhances excretion.

    mono- and Bile di-glucuronides Bilirubin

    e.g. Gunn rat which lacks UGT1*1 develops unconjugated hyperbilirubinemia. Th