Selectivity in the Efflux of Glucuronides by Human ... - ACS Publications

Article pubs.acs.org/molecularpharmaceutics

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 Erkka Jar̈ vinen,† Johanna Troberg,† Heidi Kidron,‡ and Moshe Finel*,† †

Division of Pharmaceutical Chemistry and Technology, and ‡Division of Pharmaceutical Biosciences, Faculty of Pharmacy, University of Helsinki, 00014 University of Helsinki, Finland S Supporting Information *

ABSTRACT: 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 4methylumbelliferone, 1-naphthol, and 1-hydroxypyrene (Km values of 168, 13, and 3 μM, respectively) in comparison to MRP3 (Km values of 278, 98, and 8 μM, respectively). MRP3 exhibited lower rates, but stereoselective transport of propranolol glucuronides, with higher affinity toward the R-enantiomer than the S-enantiomer (Km values 154 vs 434 μM). The glucuronide of propranolol R-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 (Vmax and Km 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 (Vmax = 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. KEYWORDS: hydroxypyrene, 1-naphthol, MRP2/ABCC2, ABCG2/BCRP, MRP3/ABCC3, MRP4/ABCC4

1. INTRODUCTION Drugs, other xenobiotics, and endobiotics are often excreted from the human body as metabolites that result from oxidation or conjugation reactions (phases I and II, respectively), or their combination.1 Conjugation with glucuronic acid, called glucuronidation, which is catalyzed by one or several of the UDP-glucuronosyltransferase (UGT) enzymes, is a prevalent metabolic pathway for many small xenobiotics.2 These conjugates, commonly called glucuronides, are more hydrophilic than their parent compounds and mostly do not cross cell membranes passively.3,4 As a result, they could accumulate inside cells to higher concentrations than in the blood circulation, unless they are efficiently transported across the membranes by efflux transporters. Glucuronides are actively excreted from cells through transporter proteins in the plasma membrane, such as the ATP-binding cassette (ABC) trans© 2017 American Chemical Society

porters. Thereby, these transporters can substantially affect the disposition of drugs and their metabolites, e.g., by contributing to enterohepatic circulation. The ABC transporter family contains several pharmacologically and pharmacokinetically relevant efflux transporters that transport drugs and their metabolites across cell membranes.5,6 Multidrug resistance proteins 2, 3, and 4 (MRP2−MRP4 or ABCC2−ABCC4) and the breast cancer resistance protein (BCRP or ABCG2) transport anionic compounds such as glucuronides.3,6 These transporters are expressed in the three main tissues that metabolize and excrete drugs and their Received: Revised: Accepted: Published: 3299

May 3, August August August

2017 24, 2017 29, 2017 29, 2017 DOI: 10.1021/acs.molpharmaceut.7b00366 Mol. Pharmaceutics 2017, 14, 3299−3311

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Molecular Pharmaceutics metabolites, namely, liver, intestine, and kidney.5,6 MRP2 and BCRP are localized on apical membranes in hepatocytes and intestinal epithelia, excreting their substrates into bile and intestine, respectively. MRP3 is found on the basolateral membranes of the respective cells, contributing to systemic exposure of its substrates. Both MRP2 and MRP4 are localized on apical membranes of proximal tubular cells in the kidney, contributing to active tubular secretion of organic anions, but in hepatocytes MRP4 is found on the basolateral membranes. Based on experiments in the intestinal cell line Caco-27 and Abcc4 knockout mice studies,8 MRP4 is likely to be expressed also in the intestine and to be localized on the basolateral membrane of the epithelia. There is an emerging need to understand the cellular mechanisms that determine apical versus basolateral transport since these processes have large effects on the disposition of drugs and their metabolites.4,9 It has recently been described that glucuronides could interact with multiple organic anion transporters, including both uptake and efflux transporters.4 Some glucuronides are found in the blood circulation at higher levels than their parent molecules, suggesting an important role for basolateral membrane efflux transporters. Glucuronides are excreted from the circulation either extensively through urine, such as paracetamol10 and morphine glucuronides,11 or via bile and feces, like raloxifene12 and ezetimibe glucuronides.13 In some cases, such as the recently described pradigastat glucuronide, the glucuronidation is the main metabolic pathway and it is entirely excreted via bile and feces, while its plasma and urine levels are negligible, indicating major contribution of hepatic apical transporters.14 Several glucuronide conjugates are substrates for MRP2, MRP3, and MRP4 and some for BCRP.3 However, only a few studies have directly compared glucuronide transport by these human transporters, attempting to characterize the differences and similarities between all of them. Propranolol is a beta blocker that is mostly used to treat cardiovascular diseases.15 It undergoes extensive metabolism in humans, where the major pathway is oxidation and subsequent glucuronidation of the oxidation metabolites.16 However, a fraction of the drug, about 15−20%, is metabolized by direct glucuronidation. Administered propranolol is a mixture of two enantiomers, R and S, and in humans their stereoselective glucuronidation results in different amounts of (R)- and (S)propranolol glucuronides.17,18 These glucuronides have 5- to 10-fold higher plasma concentrations than propranolol and are excreted via urine, mostly by glomerular filtration.19 One of the most widely used compounds for studying UGT activities is 4-methylumbelliferone (7-hydroxy-4-methylcoumarin, 4-MU).20 In addition, 4-MU has been used as a medication for bile therapy21 and hence comprehensive human pharmacokinetic and metabolism data are available for it.22 In humans, it is almost completely (95%) metabolized to the glucuronide conjugate (4-MUG), and this glucuronide is rapidly eliminated via urine. In animals, previous studies with perfused livers from Abcc3 and Abcc4 knockout mice indicated a major role of Mrp3, but not Mrp4, in the hepatic disposition of 4-MUG.23 The major phase I oxidative metabolites of the environmental pollutants naphthalene and pyrene are 1-naphthol and 1-hydroxypyrene, respectively.24 No comprehensive human pharmacokinetic or metabolism data are available for them, but based on in vitro experiments with human liver microsomes, both compounds are converted to the respective glucuronides

at high rates.20,25 In addition, glucuronide metabolites of both compounds have been determined in human urine and, therefore, could indicate polycyclic aromatic hydrocarbon exposure.24 In this study, we have examined the in vitro transport of 4MUG, 1-naphthol glucuronide (1-NPG), 1-hydroxypyrene glucuronide (1-HPG), and (R)- and (S)-propranolol glucuronides (R-PRG and S-PRG) by vesicles containing the recombinant human efflux transporters MRP2, MRP3, MRP4, or BCRP, that were expressed in baculovirus infected insect cells. These five glucuronides (Figure 1) are structurally related and are both in vivo and in vitro relevant model compounds, important issues when attempting to explore differences and

Figure 1. Structures of the glucuronide compounds. Chemical structures of the six glucuronides that were used as substrates for the efflux transporters in this study. The glucuronide conjugate moiety is represented as Gluc. 3300

DOI: 10.1021/acs.molpharmaceut.7b00366 Mol. Pharmaceutics 2017, 14, 3299−3311

Article

Molecular Pharmaceutics

glucuronides was confirmed by comparison of their retention times in HPLC with earlier synthesized and characterized standards.26 In addition, the chromatographic purity was characterized using fluorescence and UV-HPLC, which indicated that it was 97% and 99% for S-PRG and R-PRG, respectively. 2.3. Cloning of ABCC4 cDNA and Construction of ABCC3 and ABCC4 cDNA Containing Expression Vectors. The ABCC4 cDNA was amplified from a commercial sample of normal human kidney cDNA, purchased from BioChain (Newark, CA, USA), using an oligonucleotide from the 5′end that carries a BssHII restriction site upstream of the start codon and an oligonucleotide from the 3′-end with a SpeI restriction site downstream of the stop codon. The amplified PCR product and the pFastBac1 (FB) vector were digested with BssHII and SpeI, and the purified DNA fragments were ligated. The resulting plasmid, MRP4-FB, was isolated from the few ampicillin-resistant colonies, and the cDNA of the entire coding sequence of MRP4 within it was sequenced for verification and comparison to the GenBank accession no. NM_005845. The human ABCC3 cDNA (in pGEM plasmid) was a generous gift from Prof. Piet Borst, The Netherlands Cancer Institute, to the Centre for Drug Research, Division of Pharmaceutical Biosciences, Faculty of Pharmacy, University of Helsinki. To express MRP3 in baculovirus-infected insect cells, the cDNA of ABCC3 in pGEM was amplified as two separate fragments. A shorter 5′ region fragment, about 600 bp in length, was amplified using an oligonucleotide from the flank region to which an MfeI site was inserted and an oligonucleotide from the internal unique SalI site. The other, longer fragment, the 3′ region of the cDNA, was amplified using the complementary oligonucleotide from the internal SalI site and an oligonucleotide from the 3′-end flank sequence with a new SpeI site. Finally, the two amplified fragments, digested with either MfeI and SalI or SalI and SpeI, were coligated into the FB vector that was digested with EcoRI and SpeI. The cDNA of the entire coding sequence of MRP3 in the MRP3-FB plasmid, isolated from the resulting colonies, was sequenced for verification and comparison to GenBank accession no. NM_003786. To express the human recombinant MRP3 and MRP4 in insect cells, baculovirus stocks were prepared and amplified according to the Bac-to-Bac methodology (Thermo Fisher Scientific, Waltham, MA, USA). 2.4. Expression and Vesicle Preparation of MRP2, MRP3, MRP4, and BCRP. Expression and vesicle preparation of the transporters was essentially done as previously described.27−29 Hence, Sf 9 insect cells (from Spodoptera f rugiperda) were cultured in HyClone SFX-insect medium supplemented with 5% fetal bovine serum, both from GE Healthcare Life Sciences (South Logan, UT, USA). Cells (1.6 million per mL) in suspension were infected with optimized amounts of recombinant baculovirus per transporter, or baculovirus containing no cDNA (control preparations, CtrlM for MRPs and CtrlB for BCRP, see below), and cultured for 3 days before harvesting and storage at −20 °C. For vesicle preparations, the harvested frozen cell pellets were thawed by suspending in cold buffer (50 mM Tris hydrochloride pH 7.0 and 300 mM mannitol) containing protease inhibitors and incubated on ice for 45 min. The cells were then collected by centrifugation at 1200g for 10 min, followed by suspending the pellet in TME-buffer (50 mM Tris hydrochloride pH 7.0, 50

similarities in transport activity between these human transporters. In combination with the known cellular location of the transporters, we aim to improve understanding of glucuronide metabolite disposition in humans.

2. MATERIALS AND METHODS 2.1. Chemicals and Solvents. Sodium salts of 1-NPG and E2-17G, 4-MUG and Ko143 hydrates, and GSH were acquired from Sigma-Aldrich (St. Louis, MO, USA). The 1-HPG was previously synthesized in our laboratory,25 and for this study its purity was confirmed to be >99%, based on fluorescence and UV-HPLC analyses. Analytical grade solvents (acetonitrile, ammonium hydroxide water solution, dichloromethane, DMSO, formic acid, and methanol) were also obtained from Sigma-Aldrich. Water-soluble cholesterol/RAMEB (randomly substituted methyl-β-cyclodextrin) was from CycloLab (Budapest, Hungary). Complete Ultra (EDTA-free) and Pierce Mini (EDTA-free) protease inhibitor cocktail tablets were from Roche Diagnostics (Mannheim, Germany) and Thermo Fischer Scientific (Rockford, IL, USA), respectively. Mouse monoclonal antibodies against MRP3 (M3II-21) and MRP4 (F-6), as well as goat anti-mouse IgG-HRP conjugated antibody, were purchased from Santa Cruz Biotechnologies (Santa Cruz, CA, USA). The water for assays and analyses was purified with a Milli-Q water purification system and filtered through a 0.22 μM filter (Merck Millipore, Darmstadt, Germany). All other chemicals and reagents were obtained from known commercial suppliers. 2.2. Enzyme-Assisted Synthesis of R-PRG and S-PRG. A method was previously developed in our laboratory for enzyme-assisted synthesis of R-PRG and S-PRG, using recombinant UGTs that were expressed in our laboratory.26 For this study, the method was further optimized for enhanced yields. Two larger scale biosynthesis reactions were carried out separately, incubating UGT1A9 with (S)-propranolol and UGT1A10 with (R)-propranolol, in small conical flasks under conditions of gentle agitation at +37 °C for either 72 h (UGT1A9) or 48 h (UGT1A10). The S-PRG synthesis reaction was carried out in a total volume of 25 mL and contained 2 mM substrate, 28 mg of total protein of UGT1A9 membrane homogenate, 100 mM Tris pH 7.5, 10 mM MgCl2, and 4 mM uridine 5′-diphosphoglucuronic acid. The synthesis reaction for R-PRG contained 2 mM substrate, 50 mg of total protein of UGT1A10 membrane homogenate, 50 mM phosphate buffer pH 7.4, 10 mM MgCl2, and 4 mM uridine 5′-diphosphoglucuronic acid, in a total volume of 35 mL. The reactions were terminated by centrifugation to remove insoluble protein, followed by alkalization of the supernatant with sodium hydroxide and extracting it three times with equal amounts of dichloromethane. The water phases were acidified with formic acid and loaded onto SPE cartridges (Oasis HLB, 200 mg, 6 mL from Waters, Milford, MA, USA), which were subsequently washed with mixtures of 0−30% methanol in water containing 2% formic acid. The glucuronides were then eluted with 60% methanol in 0.5% ammonia−water solution. The fractions were concentrated under vacuum and subjected to HPLC purification, and the collected purified fractions containing the glucuronides were combined and freeze-dried. The residues were solvated by a mixture of water, methanol, and trace ammonia, transferred to preweighed vials, concentrated, and subsequently kept under vacuum overnight to give ammonium salts of the glucuronides. The final yields were 24% and 18% of S-PRG and R-PRG, respectively. The identity of the 3301


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the quenching buffer. The studied compounds were finally eluted from the retained vesicles by applying 100 μL of 1:1 methanol:0.2% formic acid water into each well, incubating for 30−60 min at room temperature with gentle shaking, and subsequently centrifuging for 2 min at 3000g. The resulting samples were subjected to HPLC quantification (see section 2.7). Substrate-concentration dependent transport assays (kinetic analyses) were conducted as described above, but using seven or eight different substrate concentrations. The incubation times for the kinetic analyses are reported in the figure legends and were selected based on predetermined linear range of the transport for each compound and transporter combination (Figure S1). The ATP-dependent transport was calculated by subtracting the rates in the absence of ATP (−ATP values) from the rates in the presence of ATP (+ATP values) and fitting the resulting values to either the Michaelis−Menten equation (v = Vmax [S]/([S] + Km)) or the Hill equation (v = V max [S] h /(S 50 h + [S] h ), where [S] is the substrate concentration, S50 is the concentration producing half-maximal reaction rate, and h is the Hill coefficient, using least-squares fit. Goodness of the fit was inspected in each case by Eadie− Hofstee transformation of the experimental data and the coefficient of determination (R2). The single substrate concentration assays were performed in two independent experiments, while other data was from a single experiment. Each vesicular transport assay experiment, whether single substrate concentration or kinetic assay, included three replicates of both +ATP and −ATP samples. The transport values are reported in pmol of substrate retained in the vesicles per total vesicle protein per incubation time (pmol/mg/min). Means of the data were calculated and are presented with ±SD as error bars. Statistical significance for the differences between +ATP and −ATP values and the effect of Ko143, the BCRP inhibitor, was evaluated using the two-tailed nonparametric Mann−Whitney U-test. A p value 70% of the orally administered 1-naphthol was excreted via urine and half of this consisted of 1-NPG.44 In perfused rat intestine experiments, when 1-naphthol was administered, 80− 90% of 1-NPG was excreted to the basolateral side.45−47 In addition, it was found in rats that the renal clearance of 1-NPG exceeded more than 5-fold glomerular filtration and it was not changed in the strain that lacked Mrp2, indicating major contribution of renal apical transporters such as Mrp4.47 This, in line with our human efflux transporter results (Figures 3B, 3E, 4B, 4E, and 6B), could indicate that hepatic and intestinal basolateral transporters, such as MRP3/Mrp3 and MRP4/ Mrp4, as well as renal apical transporters, such as MRP4/Mrp4, play important roles in 1-NPG disposition in vivo. On the other hand, in rat livers it was shown, using Mrp2 deletion strain, that some bile excretion exists and knockout of this apical transporter abolished it.47 Human originated Caco-2 cells excreted 1-NPG, formed from 1-naphthol, into the basolateral compartment at severalfold higher amounts than to the apical compartment.48 In addition, it was found in vesicle assays that the transport activity of human MRP4 was substantially inhibited by 1NPG.49 Overall these findings are in line with our new results (Figures 3B, 3E, and 6B), according to which 1-NPG is a good substrate for the human MRP3 and, particularly, MRP4 due to its low Km value for this transporter (Table 1). Hence, both transporters are likely to be involved in the excretion of 1-NPG from human tissues in vivo, such as intestine, liver, and kidney. The above results also suggest that 1-NPG could serve as a good substrate for in vitro assays of MRP4. 4.3. Transport of 1-HPG. The main metabolite found in human urine after exposure to pyrene is 1-HPG.50 1Hydroxypyrene (1-HP) and, especially, 1-HPG are considered as biomarkers for environmental polycyclic aromatic hydrocarbon exposure, similarly to 1-naphthol and its glucuronide.24 No comprehensive in vivo data on human metabolism or

Table 1. Kinetic Constants of the Tested Transporters and Substratesa Transporter

Km, μM (95% CI)

Vmax, pmol/mg/min (95% CI)

R2

4-MUG MRP3 MRP4

278 (205−350) 168 (147−189)

MRP3 MRP4

98 (58−138) 13 (9−16)

MRP3 MRP4 BCRP

8.1 (6.3−9.9) 3.2 (2.8−4.2) 11 (9−12)

MRP3

154 (127−182)

191 (163-218) 778 (730−826)

0.96 0.98

192 (161−223) 1110 (1050−1160)

0.80 0.74

481 (451−512) 1510 (1370−1640) 4430 (4250−4600)

0.94 0.96 0.98

110 (101−119)

0.97

118 (89−146)

0.90

3520 (3280−3730)

0.99

163 (134−192) 243 (208−278) 678 (636−720)

0.80 0.90 0.98

1-NPG

1-HPG

R-PRG S-PRG MRP3 MRP2 MRP3 MRP4 BCRP

434 (240−629) E2-17G S50b = 124 (113−135) hb = 2.3 (2.0−2.6) 36 (20−52) 67 (46−87) 58 (49−66)

a The experimental data from Figures 2 and 6 were fitted using the Michaelis−Menten equation, with the exception of E2-17G transport by MRP2, where the Hill equation was used. The derived kinetic constants of the best fits are reported, with confidence intervals (95% CI) for the fitting of the curves, in the parentheses. bS50 is the concentration producing the half-maximal reaction rate, and h is the Hill coefficient, both in the Hill equation.

intracellular accumulation of the glucuronide.23 On the other hand, it was shown in experiments with everted mouse intestinal sacs that efflux of 4-MUG is higher into the apical compartment than the basolateral side, along the intestine, when 4-MU was administered.36 However, when everted intestinal sacs were prepared from Abcc3−/− mice, the basolateral efflux of 4-MUG was substantially reduced. In rat, on the other hand, it was shown that 4-MUG was preferentially excreted into bile in perfused livers, and that this excretion was reduced to minimal in rat livers lacking Mrp2.37 In addition, in a study of isolated perfused rat kidneys, 4-MUG was preferentially excreted to the perfusate rather than to the urine, but the urinary excretion was 4-fold higher than glomerular filtration rate.38 The findings from animal studies could be divided into two groups, mouse results and rat results. While 4-MUG is primarily excreted by transporters on the basolateral membrane in liver and intestine in both humans and mice, it is excreted apically (i.e., into bile) in rats. The mouse results and our findings in this study agree on the role of MRP3 in 4-MUG transport, but disagree on the contribution of MRP4. Whether the latter difference is due to difference in substrate specificity between the mouse Mrp4 and the human MRP4, or originates from low expression level(s) of that transporter in the rodent liver, remains to be explored. In any case, animal experiments should be interpreted with care when building assumptions and models about the activity of the human efflux transporters and their substrate specificities. It should be added that, in the human cell line Caco-2, the intracellularly generated 4-MUG was excreted at more than 5fold higher amounts to the basolateral side, in comparison to 3308


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clearance of R-PRG17 were higher in comparison to S-PRG. Nonetheless, it is difficult to predict the effect of stereoselective transport by MRP3 on the disposition of S-PRG and R-PRG. In addition, glucuronidation of propranolol in the human intestine could result in different enantiomeric ratio of the glucuronides in comparison to the metabolism in liver and thus affect their excretion rate.26 In conclusion, the human MRP4 and MRP3 transport small planar glucuronides such as 1-NPG, 4-MUG, and 1-HPG. MRP3 was the major transporter of R-PRG and S-PRG, of which the R-enantiomer was transported at higher apparent affinity. On the other hand, MRP2 transported the five tested glucuronides very weakly or not at all, whereas BCRP-mediated transport of 1-NPG, 4-MUG, R-PRG, and S-PRG was low or negligible, while its transport of 1-HPG was at high affinity and capacity. Our results may help to explain in vivo findings on the disposition of these compounds, as well as the functions and differences of the human efflux transporters MRP2, MRP3, MRP4, and BCRP that are involved in the transport of glucuronide conjugates in human tissues.

excretion of 1-HPG are available, but data from several animal studies are present. In rats, when pyrene was administered orally or parenterally, 1-HP was excreted via both urine and feces, of which the latter predominated.51−53 In bile-cannulated rats, biliary excretion of 1-HP was 3- to 5-fold higher than urine excretion.51,52 Most of the biliary and urinary 1-HP was in conjugated forms, such as 1HPG, whereas practically all of the fecal 1-HP was unconjugated.51,53 Nevertheless, it was found that along the rat small intestine the amount of 1-HPG was very high, but it declined toward the large intestine, probably due to hydrolysis of the glucuronide by gut bacteria. This suggests that the rat apical transporters, such as Mrp2 and Bcrp, transported 1-HPG from the liver and intestine.53 In polarized Caco-2 cells, pyrene was administered to the monolayer of cells, and 1-HPG was analyzed from both the basolateral and apical compartments.54 Unfortunately, the amounts of formed glucuronide conjugate in these cells was very small in comparison to the sulfate conjugate of 1-HP. Nevertheless, a substantially higher fraction, around 6-fold, of 1HPG was excreted into the apical than basolateral compartment. In addition, the apical excretion was inhibited significantly by the rather specific BCRP inhibitor, Ko143. Hence, our results on 1-HPG transport (Figures 4C, 4F, and 6C) agree with findings from both rat studies and human originating Caco-2 cell line. Their outcome is that BCRP/Bcrp probably plays a major role in the tissue excretion of 1-HPG, particularly in the intestine and liver. In addition, MRP3/Mrp3 and MRP4/Mrp4 (Figures 3C, 3F, and 6C) could contribute to systemic exposure and subsequent urinary excretion of 1-HPG from the intestine and liver. 4.4. Transport of Propranolol Glucuronides. Although propranolol glucuronides are not the main metabolites for propranolol, they are interesting compounds because in humans they exist in the blood circulation at severalfold higher concentrations than propranolol and are subsequently excreted into urine.16,17 Excretion of the propranolol glucuronides is practically completed within 24 h following propranolol administration,16 and there is no indication of fecal excretion of any drug-related material.15 This suggests that the excretion of propranolol glucuronides from metabolizing tissues (liver and intestine) is to the blood circulation. Our results with the expressed human efflux transporters (Figures 5 and 6D) are somehow in line with these in vivo human findings; propranolol glucuronides are mainly excreted via basolateral transporters, such as MRP3, and no substantially apical excretion in liver and subsequent enterohepatic circulation exists. Our results are also supported by the finding that in patients with renal insufficiency there is around 20-fold accumulation of propranolol glucuronides in comparison to healthy people, which indicates no other major excretion pathway for these glucuronides than excretion into urine.55 In addition, there is no active tubular secretion of propranolol glucuronides in kidney17,19 The finding here, that MRP3 transports R-PRG with higher affinity than S-PRG (Figure 6D and Table 1), is interesting but not exceptional among the MRPs. It was previously reported that the inhibition efficiency of MRP4 by nonsteroidal antiinflammatory drug glucuronides could vary by up to 20-fold between two enantiomers of the same drug.56 Propranolol undergoes stereoselective glucuronidation in humans, and more S-PRG is excreted via urine than R-PRG.18 However, it was found that the excretion rate into urine18 and the plasma

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.molpharmaceut.7b00366.

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Linearity of transport versus time for all of the transporter−substrate combinations that were included in the kinetic analyses (PDF)

AUTHOR INFORMATION

Corresponding Author

*Division of Pharmaceutical Chemistry and Technology, Faculty of Pharmacy, University of Helsinki, 00014 University of Helsinki, Finland. Tel.: +358 50 4480748. E-mail: moshe. finel@helsinki.fi. ORCID

Moshe Finel: 0000-0003-1775-854X Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS

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ABBREVIATIONS USED

We would like to thank Johanna Mosorin, Noora Sjöstedt, and Feng Deng for skillful help in the cloning, expression, and transporter vesicle preparations. The funding of the University of Helsinki Doctoral Program in Drug Research, Sigrid Juselius Foundation (Grant No. 4704583), and the Academy of Finland (Grants No. 12600101 and 1292779) are acknowledged.

1-HPG, 1-hydroxypyrene glucuronide; 1-HP, 1-hydroxypyrene; 1-NPG, 1-naphthol glucuronide; 4-MUG, 4-methylumbelliferone glucuronide or 7-hydroxy-4-methylcoumarin glucuronide; 4-MU, 4-methylumbelliferone; ABC, ATP-binding cassette transporter; BCRP, breast cancer resistance protein; E2-17G, estradiol-17β-glucuronide; MRP, multidrug resistance protein; R-PRG, (R)-propranolol glucuronide; S-PRG, (S)-propranolol glucuronide; UGT, UDP-glucuronosyltransferase 3309


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