Selectivity in the Efflux of Glucuronides by Human Transporters: MRP4 Is Highly Active toward 4‑Methylumbelliferone and 1‑Naphthol Glucuronides, while MRP3 Exhibits Stereoselective Propranolol Glucuronide Transport

Xenobiotic and endobiotic glucuronides, which are generated in hepatic and intestinal epithelial cells, are excreted via efflux transporters. Multidrug resistance proteins 2–4 (MRP2–MRP4) and the breast cancer resistance protein (BCRP) are efflux transporters that are expressed in these polarized cells, on either the basolateral or apical membranes. Their localization, along with expression levels, affects the glucuronide excretion pathways. We have studied the transport of three planar cyclic glucuronides and glucuronides of the two propranolol enantiomers, by the vesicular transport assay, using vesicles from baculovirus-infected insect cells expressing human MRP2, MRP3, MRP4, or BCRP. The transport of estradiol-17β-glucuronide by recombinant MRP2–4 and BCRP, as demonstrated by kinetic values, were within the ranges previously reported. Our results revealed high transport rates and apparent affinity of MRP4 toward the glucuronides of 4-methylumbelliferone, 1-naphthol, and 1-hydroxypyrene (<i>K</i>_m values of 168, 13, and 3 μM, respectively) in comparison to MRP3 (<i>K</i>_m values of 278, 98, and 8 μM, respectively). MRP3 exhibited lower rates, but stereoselective transport of propranolol glucuronides, with higher affinity toward the <i>R</i>-enantiomer than the <i>S</i>-enantiomer (<i>K</i>_m values 154 vs 434 μM). The glucuronide of propranolol <i>R</i>-enantiomer was not significantly transported by either MRP2, MRP4, or BCRP. Of the tested small glucuronides in this study, BCRP transported only 1-hydroxypyrene glucuronide, at very high rates and high apparent affinity (<i>V</i>_max and <i>K</i>_m values of 4400 pmol/mg/min and 11 μM). The transport activity of MRP2 with all of the studied small glucuronides was relatively very low, even though it transported the reference compound, estradiol-17β-glucuronide, at a high rate (<i>V</i>_max = 3500 pmol/mg/min). Our results provide new information, at the molecular level, of efflux transport of the tested glucuronides, which could explain their disposition in vivo, as well as provide new tools for in vitro studies of MRP3, MRP4, and BCRP

UGT1A10 Is a High Activity and Important Extrahepatic Enzyme: Why Has Its Role in Intestinal Glucuronidation Been Frequently Underestimated?

The aim of this work was to highlight a considerable and broad problem in UGT1A10 activity assessment that has led to underestimation of its role in intestinal glucuronidation of drugs and other xenobiotics. The reason appears to be poor activity of the commercial UGT1A10 that is used by many laboratories, and here we have tested it by comparison with our recombinant His-tagged UGT1A10 (designated as UGT1A10-H), both expressed in insect cells. The glucuronidation rates of morphine, estradiol, estrone, SN-38, diclofenac, 4-methylumbelliferone, 7-amino-4-methylcoumarin, <i>N</i>-(3-carboxypropyl)-4-hydroxy-1,8-naphthalimide, and bavachinin were assayed. The results revealed that the activity of commercial UGT1A10 was low, very low, and in the cases of morphine, estrone, 7-methyl-4-aminocoumarin, and bavachinin it was below the detection limit. On the other hand, under the same conditions, UGT1A10-H exhibited high glucuronidation rates toward all these compounds. Moreover, using estradiol, morphine, and estrone, in the presence and absence of suitable inhibitors, nilotinib or atractylenolide I, it was demonstrated that UGT1A10-H, but not the commercial UGT1A10, provides a good tool to study the role of native UGT1A10 in the human intestine. The results also suggest that much of the data in the literature on UGT1A10 activity may have to be re-evaluated

Enzyme kinetics of UGT1A8-catalyzed glucuronidation of 17β-estradiol (A), entacapone (B), 1-naphthol (C), and 4-MU (D) in the absence and presence of BSA.

<p>The reactions with 17β-estradiol were analyzed for the formation of 17β-estradiol-3-β-D-glucuronide. The glucuronidation rates are presented as the average value ± S.E., and are expression level-normalized values. The concentrations of substrates were corrected for binding to 0.1% BSA. The determined enzyme kinetic parameters are presented in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0054767#pone-0054767-t002" target="_blank">Table 2</a>. See <i>Materials and Methods</i> for all further details.</p

Enzyme kinetics of UGT2B15-catalyzed glucuronidation of 17α-estradiol (A) and 4-MU (B), in the absence and presence of BSA.

<p>The reaction with 17α-estradiol was analyzed for the formation of 17α-estradiol-3-β-D-glucuronide. The glucuronidation rates are presented as the average value ± S.E. These values are not expression normalized because we used commercial UGT2B15 without appropriate His-tag. The concentrations of substrates were corrected for binding to 0.1% BSA. The determined enzyme kinetic parameters are presented in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0054767#pone-0054767-t003" target="_blank">Table 3</a>. See <i>Materials and Methods</i> for all further details.</p

Enzyme kinetics of UGT1A7-catalyzed glucuronidation of entacapone (A) and 4-MU (B) in the absence and presence of BSA.

<p>The glucuronidation rates are presented as the average value ± S.E., and are expression level-normalized values (see <i>Materials and Methods</i>). The concentrations of substrates were corrected for binding to 0.1% BSA. The determined enzyme kinetic parameters are presented in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0054767#pone-0054767-t002" target="_blank">Table 2</a>.</p

Enzyme kinetic parameters of UGTs 1A1, 1A6, 1A7, 1A8, and 1A10-catalyzed glucuronidation in the absence and presence of BSA.

<p>The glucuronidation rates are presented as expression level-normalized values ± S.E. The 95% CI are presented in the parenthesis. The p-values were calculated using the extra sum-of-squares F-test (see <i>Materials and Methods</i> for all details).</p><p>MM, Michaelis-Menten; SI, substrate inhibition; HE, Hill equation;</p>*<p>P<0.05;</p>**<p>P<0.01;</p>***<p>P<0.001.</p

Analytical conditions in the separation and quantification of glucuronides.

1<p>LOD, limit of detection; LOQ, limit of quantification; values are calculated assuming maximal injection volume;</p>2<p>The UV signal for was correlated with fluoresence for enhanced sensitivity.</p

Enzyme kinetics of UGT2B7-catalyzed glucuronidation of 17α-estradiol (A), 17β-estradiol (B), and 4-MU (C), in the absence and presence of BSA.

<p>The reactions with 17α-estradiol and 17β-estradiol were analyzed for the formation of 17α-estradiol-17-β-D-glucuronide and 17β-estradiol-17-β-D-glucuronide, respectively. The glucuronidation rates are presented as the average value ± S.E., and are expression level-normalized values. The concentrations of substrates were corrected for binding to 0.1% BSA. The determined enzyme kinetic parameters are presented in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0054767#pone-0054767-t003" target="_blank">Table 3</a>. See <i>Materials and Methods</i> for all further details.</p

The chemical structures of the substrates that were used in this study.

<p>In 17α- and 17β-estradiol both the 3-OH and the 17-OH can be conjugated, mostly by different UGTs (see Figs. 2 and 3). In the case of entacapone, the glucuronidation occurs on hydroxy group in position 3.</p

Enzyme kinetics of UGT2B17-catalyzed glucuronidation of 17β-estradiol (A), 1-naphthol (B), and 4-MU (C), in the absence and presence of BSA.

<p>The reaction with 17β-estradiol was analyzed for the formation of 17β-estradiol-17-β-D-glucuronide. The glucuronidation rates are presented as the average value ± S.E., and are expression level-normalized values. The concentrations of substrates were corrected for binding to 0.1% BSA. The determined enzyme kinetic parameters are presented in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0054767#pone-0054767-t003" target="_blank">Table 3</a>. See <i>Materials and Methods</i> for all further details.</p