Accelerated Communication 3 *-AZIDO-3 *-DEOXYTHIMIDINE ( AZT ) IS GLUCURONIDATED BY HUMAN UDP-GLUCURONOSYLTRANSFERASE 2 B 7 ( UGT 2 B 7 )

3*-Azido-3*-deoxythymidine (AZT) is frequently prescribed to patients infected with the human immunodeficiency virus. After absorption, AZT is rapidly metabolized into 3*-azido-3*-deoxy-5*-glucuronylthymidine by UDP-glucuronosyltransferase (UGT) enzymes. Using labeled [C]UDP-glucuronic acid and microsomal preparations from human kidney 293 cells stably expressing the different human UGT2B isoenzymes, it was demonstrated that AZT glucuronidation is catalyzed specifically by human UGT2B7. The identity of the metabolite formed was confirmed as AZT-G by liquid chromatography coupled with mass spectrometry. UGT2B7 is encoded by a polymorphic gene and kinetic analysis of AZT glucuronidation by the two allelic variants UGT2B7(H) and UGT2B7(Y), yielded apparent Km values of 91.0 and 80.1 mM, respectively. Normalization to protein levels yielded glucuronidation efficiency ratios (Vmax/Km) of 21.3 and 11.0 ml z min z mg protein for UGT2B7(H) and UGT2B7(Y), respectively. It remains possible that other UGT enzymes are also involved in AZT conjugation; however, the glucuronidation of AZT by UGT2B7, which is a UGT2B protein expressed in the liver, is consistent with previous findings and supports the physiological relevance of this enzyme in AZT conjugation. Drug therapy for HIV and AIDS infection generally includes treatment with nucleoside analogs, which act as HIV-reverse transcriptase (HIV-RT) inhibitors (Patterson et al., 1997). 3 9-Azido-39-deoxythymidine (AZT) is the most studied member of the 29,39-dideoxynucleoside family of compounds, which has demonstrated clinical efficacy with decreased mortality and morbidity in patients infected with HIV (Fischl et al., 1987; Patterson et al., 1997). The mechanism of HIV-RT inhibition is based on the catabolic phosphorylation of the molecule, generating AZT-59-triphosphate (AZT-TP), which is an analog of thymidine-59-triphosphate, and acts as a competitor for the HIV-RT and a DNA-chain terminator (Mitsuya et al., 1990). However, toxic effects such as bone marrow suppression, vomiting, nausea (Mitsuya et al., 1990), circulating erythrocytes and neutrophil suppression (Moore et al., 1995), and mitochondrial myopathy (Mitsuya et al., 1990; Izuta et al., 1991), which are thought to be due to the interaction of AZT-TP with mammalian DNA polymerases b andg (Izuta et al., 1991), are associated with this agent. Before it can exert its antiviral effect by inhibiting HIV-RT, AZT must be phosphorylated by cellular enzymes to azidothymidine triphosphate (Fig. 1) (Lavie et al., 1998). This activation is catalyzed by successive kinases, beginning by the formation of AZT-monophosphate by thymidine kinase. The bottleneck of AZT activation lies in the second phosphorylation step, catalyzed by thymidylate kinase (Lavie et al., 1998). Cells treated with AZT accumulate the toxic AZT-monophosphate whereas only low concentrations of the active AZT-triphosphorylated (AZT-TP) are measured (2–10% of total phosphate metabolites) (Barry et al., 1996). The alternate pathway of AZT elimination from the body occurs primarily by glucuronidation (Good et al., 1990; Moore et al., 1995) with UDP-glucuronosyltransferase (UGT) enzymes (EC 2.4.1.17), which catalyze the transfer of the glucuronide moiety from UDP-glucuronic acid (UDPGA) to small hydrophobic molecules. The resulting compounds are generally more water-soluble, less toxic, and easier to excrete into the bile or urine (Dutton, 1980). Based on divergent evolution, the mammalian UGT proteins have been categorized into two major families, UGT1 and UGT2 (Mackenzie et al., 1997). It has been thought that UGT1A enzymes were This work was supported by the Medical Research Council of Canada (A.B. and D.W.H.), the Fonds de la Recherche en Santé du Québec (A.B. and D.W.H.), Endorecherche and the National Institutes of Health (GM26221 to T.R.T.). Olivier Barbier and David Turgeon are holders of scholarships from La Fondation de l’Université Laval, and Caroline Girard is holder of a Summer Research Fellowship from the American Endocrine Society. 1 These authors contributed equally to this work. 2 Abbreviations used are: HIV-RT, HIV-reverse transcriptase; AZT, 39-azido39-deoxythimidine; AZT-G, 39-azido-39-deoxy-59-glucuronylthymidine; AZT-TP, AZT-59-triphosphate; UDPGA, UDP-glucuronic acid; HK293, human kidney 293; UGT, UDP-glucuronosyltransferase; LC-MS/MS, liquid chromatography coupled with mass spectrometry. Send reprint requests to: Dr. Alain Bélanger or Dr. Dean W. Hum, Laboratory of Molecular Endocrinology, CHUL Research Center, 2705 Laurier Blvd., Quebec G1V 4G2, Canada. E-mail: Alain. Bélanger@crchul.ulaval.ca or Dean.Hum@crchul . ulaval.ca 0090-9556/00/2805-0497–502$03.00/0 DRUG METABOLISM AND DISPOSITION Vol. 28, No. 5 Copyright © 2000 by The American Society for Pharmacology and Experimental Therapeutics Printed in U.S.A. DMD 28:497–502, 2000 /1869/823193 497 at A PE T Jornals on July 8, 2017 dm d.aspurnals. org D ow nladed from