Abstract
Glucuronidation catalyzed by the UDP-glucuronosyltransferases (UGTs) is a major pathway for drug metabolism and elimination in humans. Identification of the UGTs responsible for glucuronidation of existing and novel drugs will assist in the prediction of adverse reactions resulting from drug-drug interactions or genetic polymorphism. An integrated approach is proposed for UGT reaction phenotyping using recombinant enzymes and human liver microsomes. Described methods include screening of recombinant UGTs for activity, comparative enzyme kinetic analysis, correlations with isoform-selective marker activities, and chemical inhibition. The primary focus is on identification of the well-characterized hepatic UGTs, including UGTs 1A1, 1A4, 1A6, 1A9, 2B7, and 2B15, although a similar approach potentially could be used for the study of extrahepatic tissues, such as the kidney and gastrointestinal tract.
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Fisher, M. B., Paine, M. F., Strelevitz, T. J., and Wrighton, S. A. (2001) The role of hepatic and extrahepatic UDP-glucuronosyltransferases in human drug metabolism. Drug Metab. Rev. 33, 273–97.
Rodrigues, A. D. (1999) Integrated cytochrome P450 reaction phenotyping: attempting to bridge the gap between cDNA-expressed cytochromes P450 and native human liver microsomes. Biochem. Pharmacol. 57, 465–480.
Venkatakrishnan, K., von Moltke, L. L., and Greenblatt, D. J. (2001) Human drug metabolism and the cytochromes P450: application and relevance of in vitro models. J. Clin. Pharmacol. 41, 1149–1179.
Iyer, L., Hall, D., Das, S., Mortell, M. A., Ramirez, J., Kim, S., et al. (1999) Phenotype-genotype correlation of in vitro SN-38 (active metabolite of irinotecan) and bilirubin glucuronidation in human liver tissue with UGT1A1 promoter polymorphism. Clin. Pharmacol. Ther. 65, 576–582.
Soars, M. G., Mattiuz, E. L., Jackson, D. A., Kulanthaivel, P., Ehlhardt, W. J., and Wrighton, S. A. (2002) Biosynthesis of drug glucuronides for use as authentic standards. J. Pharmacol. Toxicol. Methods 47, 161–168.
Fisher, M. B., Campanale, K., Ackermann, B. L., VandenBranden, M., and Wrighton, S. A. (2000) In vitro glucuronidation using human liver microsomes and the pore-forming peptide alamethicin. Drug Metab. Dispos. 28, 560–566.
Kemp, D. C., Fan, P. W., and Stevens, J. C. (2002) Characterization of raloxifene glucuronidation in vitro: contribution of intestinal metabolism to presystemic clearance. Drug Metab. Dispos. 30, 694–700.
Alkharfy, K. M., and Frye, R. F. (2001) High-performance liquid chromatographic assay for acetaminophen glucuronide in human liver microsomes. J. Chromatogr. B Biomed. Sci. Appl. 753, 303–308.
Turgeon, D., Chouinard, S., Belanger, P., Picard, S., Labbe, J. F., Borgeat, P., et al. (2003) Glucuronidation of arachidonic and linoleic acid metabolites by human UDP-glucuronosyltransferases. J. Lipid Res. 44, 1182–1191.
Levesque, E., Turgeon, D., Carrier, J. S., Montminy, V., Beaulieu, M., and Belanger, A. (2001) Isolation and characterization of the UGT2B28 cDNA encoding a novel human steroid conjugating UDP-glucuronosyltransferase. Biochemistry 40, 3869–3881.
Krishnaswamy, S., Duan, S. X., von Moltke, L. L., Greenblatt, D. J., and Court, M. H. (2003) Validation of serotonin (5-hydroxtryptamine) as an in vitro substrate probe for human UDP-glucuronosyltransferase (UGT) 1A6. Drug Metab. Dispos. 31, 133–139.
Congiu, M., Mashford, M. L., Slavin, J. L., and Desmond, P. V. (2002) UDP glucuronosyltransferase mRNA levels in human liver disease. Drug Metab. Dispos. 30, 129–134.
Patten, C. J., Code, E. L., Dehal, S. S., Gange, P. V., and Crespi, C. L. (2001) Analysis of UGT enzyme levels in human liver microsomes using form specific anti-peptide antibodies, probe substrate activities and recombinant UGT enzymes [abstract]. Drug Metab. Rev. 33, 165.
Dehal, S. S., Gange, P. V., Crespi, C. L., and Patten, C. J. (2001) Characterization of a probe substrate and an inhibitor of UDP-glucuronosyltransferase 1A4 activity in human liver microsomes and cDNA-expressed UGT-enzymes [abstract]. Drug Metab. Rev. 33, 162.
Court, M. H., Duan, S. X., Guillemette, C., Journault, K., Krishnaswamy, S., Von Moltke, L. L., et al. (2002) Stereoselective conjugation of oxazepam by human UDP-glucuronosyltransferases (UGTs): S-oxazepam is glucuronidated by UGT2B15, while R-oxazepam is glucuronidated by UGT2B7 and UGT1A9. Drug Metab. Dispos. 30, 1257–1265.
Williams, J. A., Ring, B. J., Cantrell, V. E., Campanale, K., Jones, D. R., Hall, S. D., et al. (2002) Differential modulation of UDP-glucuronosyltransferase 1A1 (UGT1A1)-catalyzed estradiol-3-glucuronidation by the addition of UGT1A1 substrates and other compounds to human liver microsomes. Drug Metab. Dispos. 30, 1266–1273.
Boase, S. and Miners, J. O. (2002) In vitro-in vivo correlations for drugs eliminated by glucuronidation: investigations with the model substrate zidovudine. Br. J. Clin. Pharmacol. 54, 493–503.
Author information
Authors and Affiliations
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2004 Humana Press Inc., Totowa, NJ
About this protocol
Cite this protocol
Court, M.H. (2004). In Vitro Identification of UDP-Glucuronosyltransferases (UGTs) Involved in Drug Metabolism. In: Yan, Z., Caldwell, G.W. (eds) Optimization in Drug Discovery. Methods in Pharmacology and Toxicology. Humana Press. https://doi.org/10.1385/1-59259-800-5:185
Download citation
DOI: https://doi.org/10.1385/1-59259-800-5:185
Publisher Name: Humana Press
Print ISBN: 978-1-58829-332-9
Online ISBN: 978-1-59259-800-7
eBook Packages: Springer Protocols