Glucuronidation of Active Tamoxifen Metabolites by the Human UDP Glucuronosyltransferases

Abstract

ABSTRACT:Tamoxifen (TAM) is an antiestrogen that has been widely used in the treatment and prevention of breast cancer in women. One of the major mechanisms of metabolism and elimination of TAM and its major active metabolites 4-hydroxytamoxifen (4-OH-TAM) and 4-OH-N-desmethyl-TAM (endoxifen; 4-hydroxy-N-desmethyl-tamoxifen) is via glucuronidation. Although limited studies have been performed characterizing the glucuronidation of 4-OH-TAM, no studies have been performed on endoxifen. In the present study, characterization of the glucuronidating activities of human UDP glucuronosyltransferases (UGTs) against isomers of 4-OH-TAM and endoxifen was performed. Using homogenates of individual UGT-overexpressing cell lines, UGTs 2B7 ϳ 1A8 > UGT1A10 exhibited the highest overall O-glucuronidating activity against trans-4-OH-TAM as determined by V max /K M , with the hepatic enzyme UGT2B7 exhibiting the highest binding affinity and lowest K M (3.7 M). As determined by V max /K M , UGT1A10 exhibited the highest overall O-glucuronidating activity against cis-4-OH-TAM, 10-fold higher than the next-most active UGTs 1A1 and 2B7, but with UGT1A7 exhibiting the lowest K M . Although both N-and O-glucuronidation occurred for 4-OH-TAM in human liver microsomes, only O-glucuronidating activity was observed for endoxifen; no endoxifen-N-glucuronidation was observed for any UGT tested. UGTs 1A10 ϳ 1A8 > UGT2B7 exhibited the highest overall glucuronidating activities as determined by V max /K M for trans-endoxifen, with the extrahepatic enzyme UGT1A10 exhibiting the highest binding affinity and lowest K M (39.9 M). Similar to that observed for cis-4-OH-TAM, UGT1A10 also exhibited the highest activity for cis-endoxifen. These data suggest that several UGTs, including UGTs 1A10, 2B7, and 1A8 play an important role in the metabolism of 4-OH-TAM and endoxifen.

Functional Characterization of Low-Prevalence Missense Polymorphisms in the UDP-Glucuronosyltransferase 1A9 Gene

“…11-OH-DB[a,l]P conditions were 78% buffer A with a linear gradient up to 75% buffer B at a flow rate of 0.5 ml/min. Untransfected HEK293 cells were used as a negative control, and putative glucuronide peaks were confirmed first dmd.aspetjournals.org using ␤-glucuronidase and then liquid chromatography-mass spectrometry as described previously (Dellinger et al, 2006;Sun et al, 2007). Experiments were always performed in triplicate as independent assays.…”

“…The rate of glucuronidation by UGT1A9-overexpressing cell microsomes was determined essentially as described previously (Fang et al, 2002;Wiener et al, 2004;Al-Zoughool and Talaska, 2005;Dellinger et al, 2006Dellinger et al, , 2007. For glucuronidation rate determinations, substrate concentrations, microsomal protein levels, and incubation times for individual assays were chosen to maximize levels of detection within a linear range of uptake and were similar to established protocols (Fang et al, 2002;Wiener et al, 2004;Dellinger et al, 2007;Sun et al, 2007). For O-glucuronidated substrates, kinetic analysis against 3-OH-B[a]P was performed using UGT1A9-overexpressing cell microsomes (1 g of protein) preincubated with alamethicin (50 g/mg protein) for 10 min on ice.…”

“…Stable UGT1A9-overexpressing cell lines were generated as described previously Sun et al, 2007). In brief, the individual UGT1A9 variants were transfected into HEK293 cells (purchased from American Type Culture Collection; Manassas, VA) by electroporation.…”

The Development of Drug Metabolism Research as Expressed in the Publications of ASPET: Part 3, 1984–2008

“…With the multiplicity of enzymes, unique compound specificities, and potential for inhibition and induction, the development of any new drug candidate demands a detailed overview of these phase II enzymes. For example, the major form(s) responsible for conjugation of gemfibrozil (2B7) (Mano et al, 2007), tamoxifen active metabolites (1A10, 2B7, 1A8) (Falany et al, 2006;Sun et al, 2007), and troglitazone (liver 1A1, intestine 1A8, 1A10) (Watanabe et al, 2002) illustrate selectivity within the glucuronosyltransferase family. Tissue-and gender-specific expression of glucuronosyltransferases has recently been explored in mice (Buckley and Klaassen, 2007).…”

Prevalent breast cancer patients with a homozygous mutant status for CYP2D6*4: response and biomarkers in tamoxifen users

“…CYP3A4 and CYP3A5 are the most important for the demethylation, and CYP2D6 is the most important for the hydroxylation reactions [6,7]. Sulfotransferases and UDP-glucuronosyltransferases are important for increasing the solubility and facilitate the excretion of tamoxifen and its metabolites [8,9].…”