DMD Large equally mixed donor pool

Home Help [Feedback] [For Subscribers] [Archive] [Search] --
QUICK SEARCH:   [advanced]


     

Drug Metabolism and Disposition Fast Forward
First published on March 23, 2005; DOI: 10.1124/dmd.105.004333

This Article
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
dmd.105.004333v1
33/6/812    most recent
Right arrow Submit a response
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Author home page(s):
Annalise Di Marco
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Di Marco, A.
Right arrow Articles by Laufer, R.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Di Marco, A.
Right arrow Articles by Laufer, R.


Received for publication February 18, 2005.
Revised March 18, 2005.
Accepted for publication March 18, 2005.

Determination of drug glucuronidation and UDP-glucuronosyltransferase selectivity using a 96-well non-HPLC radiometric assay

Annalise Di Marco 1, Michelle D'Antoni 2, Silvia Attaccalite 2, Pietro Carotenuto 2, Ralph Laufer 3*

1 IRBM - MRL Rome 2 IRBM-MRL Rome 3 Istituto di Ricerche di Biologia Molecolare (IRBM)

* Address correspondence to: E-mail: ralph_laufer{at}merck.com

Abstract

A rapid and sensitive radiometric assay for UDP-glucuronosyltransferase (UGT) is described. UGT substrates are incubated in 96-well plates with microsomes in the presence of [14C]UDPGA, and [14C]-labelled glucuronidation products are separated from the unreacted nucleotide sugar by solid phase extraction using 96-well extraction plates. The assay was validated with 15 structurally diverse UGT substrates containing acidic, phenolic and hydroxyl reacting groups. Glucuronidation velocities for these compounds were determined using human, rat, and dog liver microsomes, and reaction kinetics was studied with 1-naphthol and 4-methylumbelliferone. Results obtained with the new assay confirmed the previously reported rank order of glucuronidation velocity of several typical UGT substrates and the finding that glucuronidation of most of these compounds is significantly faster in dog than in human liver microsomes. UGT specificity of 5 compounds was determined using recombinant human UGTs. The major UGT isoforms identified were UGT1A6, UGT1A7, and UGT1A9 for 4-methylumbelliferone, UGT1A6 and UGT1A8 for 1-naphthol, UGT2B7 for naloxone, UGT1A3 and UGT2B7 for ketoprofen, and UGT1A4 for trifluoperazine. Identical results were obtained with a conventional HPLC method coupled to mass spectrometric detection. The new assay should prove to be valuable for rapidly benchmarking recombinant UGTs and microsomal preparations from different species and tissues, for identifying high turnover compounds during drug discovery, and for reaction phenotyping studies.


Key words: glucuronidation, phase II drug metabolism, UDP glucuronyltransferases


This article has been cited by other articles:


Home page
 Drug Metab. Dispos.Home page
H.-X. Liu, Y. Liu, J.-W. Zhang, W. Li, H.-T. Liu, and L. Yang
UDP-Glucuronosyltransferase 1A6 Is the Major Isozyme Responsible for Protocatechuic Aldehyde Glucuronidation in Human Liver Microsomes
Drug Metab. Dispos., August 1, 2008; 36(8): 1562 - 1569.
[Abstract] [Full Text] [PDF]

Home page
 Drug Metab. Dispos.Home page
K. P. Kanebratt and T. B. Andersson
Evaluation of HepaRG Cells as an in Vitro Model for Human Drug Metabolism Studies
Drug Metab. Dispos., July 1, 2008; 36(7): 1444 - 1452.
[Abstract] [Full Text] [PDF]

Home page
 Drug Metab. Dispos.Home page
V. Uchaipichat, P. I. Mackenzie, D. J. Elliot, and J. O. Miners
SELECTIVITY OF SUBSTRATE (TRIFLUOPERAZINE) AND INHIBITOR (AMITRIPTYLINE, ANDROSTERONE, CANRENOIC ACID, HECOGENIN, PHENYLBUTAZONE, QUINIDINE, QUININE, AND SULFINPYRAZONE) "PROBES" FOR HUMAN UDP-GLUCURONOSYLTRANSFERASES
Drug Metab. Dispos., March 1, 2006; 34(3): 449 - 456.
[Abstract] [Full Text] [PDF]


Home Help [Feedback] [For Subscribers] [Archive] [Search] --
All ASPET Journals Molecular Pharmacology Pharmacological Reviews
Molecular Interventions Drug Metabolism and Disposition

Copyright © 2005 by the American Society for Pharmacology and Experimental Therapeutics.