Xianbin Tian

Evaluation of the Role of Multidrug Resistance-Associated Protein (Mrp) 3 and Mrp4 in Hepatic Basolateral Excretion of Sulfate and Glucuronide Metabolites of Acetaminophen, 4-Methylumbelliferone, and Harmol in Abcc3–/– and Abcc4–/– Mice

Although glucuronide and sulfate conjugates of many drugs and endogenous compounds undergo appreciable hepatic basolateral excretion into sinusoidal blood, the mechanisms that govern basolateral translocation of these hydrophilic metabolites have not been completely elucidated. In the present study, the involvement in this process of Mrp3 and Mrp4, two basolateral efflux transporters, was evaluated by analyzing the hepatic basolateral excretion of the glucuronide and sulfate metabolites of acetaminophen, 4-methylumbelliferone, and harmol in Abcc3–/– and Abcc4–/– mice using a cassette dosing approach. In the livers of Abcc3–/– and Abcc4–/– mice, the basolateral excretory clearance of acetaminophen sulfate was reduced ∼20 and ∼20%, 4-methylumbelliferyl sulfate was reduced ∼50 and ∼65%, and harmol sulfate was decreased ∼30 and ∼45%, respectively. The basolateral excretory clearance of acetaminophen glucuronide, 4-methylumbelliferyl glucuronide, and harmol glucuronide was reduced by ∼96, ∼85, and ∼40%, respectively, in the livers of Abcc3–/– mice. In contrast, basolateral excretory clearance of these glucuronide conjugates was unaffected by the absence of Mrp4. These results provide the first direct evidence that Mrp3 and Mrp4 participate in the hepatic basolateral excretion of sulfate conjugates, although additional mechanism(s) are likely involved. In addition, they reveal that Mrp3 mediates the hepatic basolateral excretion of diverse glucuronide conjugates.

The important role of Bcrp (Abcg2) in the biliary excretion of sulfate and glucuronide metabolites of acetaminophen, 4-methylumbelliferone, and harmol in mice.

The role of Mrp2, Bcrp, and P-glycoprotein in the biliary excretion of acetaminophen sulfate (AS) and glucuronide (AG), 4-methylumbelliferyl sulfate (4MUS) and glucuronide (4MUG), and harmol sulfate (HS) and glucuronide (HG) was studied in Abcc2(-/-), Abcg2(-/-), and Abcb1a(-/-)/Abcb1b(-/-) mouse livers perfused with the respective parent compounds using a cassette dosing approach. Biliary clearance of the sulfate conjugates was significantly decreased in Bcrp-deficient mouse livers, resulting in negligible biliary excretion of AS, 4MUS, and HS. It is noteworthy that the most profound decrease in the biliary clearance of the glucuronide conjugates was observed in Bcrp-deficient mouse livers, although the biliary clearance of 4MUG was also ∼35% lower in Mrp2-deficient mouse livers. As expected, biliary excretion of conjugates was not impaired in P-glycoprotein-deficient livers. An appreciable increase in perfusate recovery due to a shift in the directionality of metabolite excretion, from bile to perfusate, was noted in knockout mice only for conjugates whose biliary clearance constituted an appreciable (≥37%) fraction of total hepatic excretory clearance (i.e., 4MUS, HG, and HS). Biliary clearance of AG, AS, and 4MUG constituted a small fraction of total hepatic excretory clearance, so an appreciable increase in perfusate recovery of these metabolites was not observed in knockout mice despite markedly decreased biliary excretion. Unlike in rats, where sulfate and glucuronide conjugates were excreted into bile predominantly by Mrp2, mouse Bcrp mediated the biliary excretion of sulfate metabolites and also played a major role in the biliary excretion of the glucuronide metabolites, with some minor contribution from mouse Mrp2.

Ritonavir, Saquinavir, and Efavirenz, but Not Nevirapine, Inhibit Bile Acid Transport in Human and Rat Hepatocytes

Human immunodeficiency virus-infected patients on antiretroviral drug therapy frequently experience hepatotoxicity, the underlying mechanism of which is poorly understood. Hepatotoxicity from other compounds such as bosentan and troglitazone has been attributed, in part, to inhibition of hepatocyte bile acid excretion. This work tested the hypothesis that antiretroviral drugs modulate hepatic bile acid transport. Ritonavir (28 μM), saquinavir (15 μM), and efavirenz (32 μM) inhibited [3H]taurocholate transport in bile salt export pump expressing Sf9-derived membrane vesicles by 90, 71, and 33%, respectively. In sandwich-cultured human hepatocytes, the biliary excretion index (BEI) of [3H]taurocholate was maximally decreased 59% by ritonavir, 39% by saquinavir, and 20% by efavirenz. Likewise, in sandwich-cultured rat hepatocytes, the BEI of [3H]taurocholate was decreased 100% by ritonavir and 94% by saquinavir. Sodium-dependent and -independent initial uptake rates of [3H]taurocholate in suspended rat hepatocytes were significantly decreased by ritonavir, saquinavir, and efavirenz. [3H]Taurocholate transport by recombinant NTCP and Ntcp was inhibited by ritonavir (IC50 = 2.1 and 6.4 μM in human and rat, respectively), saquinavir (IC50 = 6.7 and 20 μM, respectively), and efavirenz (IC50 = 43 and 97 μM, respectively). Nevirapine (75 μM) had no effect on bile acid transport in any model system. In conclusion, ritonavir, saquinavir, and efavirenz, but not nevirapine, inhibited both the hepatic uptake and biliary excretion of taurocholate.

Differential Involvement of Mrp2 (Abcc2) and Bcrp (Abcg2) in Biliary Excretion of 4-Methylumbelliferyl Glucuronide and Sulfate in the Rat

The hepatic excretion of hydrophilic conjugates, end products of phase II metabolism, is not completely understood. In the present studies, transport mechanism(s) responsible for the biliary excretion of 4-methylumbelliferyl glucuronide (4MUG) and 4-methylumbelliferyl sulfate (4MUS) were studied. Isolated perfused livers (IPLs) from Mrp2-deficient (TR-) Wistar rats were used to examine the role of Mrp2 in the biliary excretion of 4MUG and 4MUS. After a 30-μmol dose of 4-methylumbelliferone, cumulative biliary excretion of 4MUG was extensive in wild-type rat IPLs (25 ± 3 μmol) but was negligible in TR- livers (0.4 ± 0.1 μmol); coadministration of the Bcrp and P-glycoprotein inhibitor GF120918 [N-(4-[2-(1,2,3,4-tetrahydro-6,7-dimethoxy-2-isoquinolinyl)ethyl]-phenyl)-9,10-dihydro-5-methoxy-9-oxo-4-acridine carboxamide] had no effect on 4MUG biliary excretion in wild-type rat IPLs. In contrast, biliary excretion of 4MUS was partially maintained in Mrp2-deficient rat IPLs. Recovery of 4MUS in bile was ∼50 to 60% lower in both control TR- (149 ± 8 nmol) and wild-type IPLs with GF120918 coadministration (176 ± 30 nmol) relative to wild-type control livers (378 ± 37 nmol) and was nearly abolished in TR- IPLs in the presence of GF120918 (13 ± 8 nmol). These changes were the result of decreased rate constants governing 4MUG and 4MUS biliary excretion. In vitro assays and perfused livers from Bcrp and P-glycoprotein gene-knockout mice indicated that 4MUS did not interact with P-glycoprotein but was transported by Bcrp in a GF120918-sensitive manner. In the rat liver, Mrp2 mediates the biliary excretion of 4MUG, whereas both Mrp2 and Bcrp contribute almost equally to the transport of 4MUS into bile.

Altered hepatobiliary disposition of 5 (and 6)-carboxy-2',7'-dichlorofluorescein in Abcg2 (Bcrp1) and Abcc2 (Mrp2) knockout mice

This study characterized the hepatobiliary disposition of 5 (and 6)-carboxy-2′,7′-dichlorofluorescein (CDF), a model Abcc2/Mrp2 (canalicular) and Abcc3/Mrp3 (basolateral) substrate, in perfused livers from male C57BL/6 wild-type, Abcg2–/–, and Abcc2–/– mice. After single-pass liver perfusion with 1 μM CDF diacetate for 30 min and an additional 30-min perfusion with CDF-free buffer, cumulative biliary excretion of CDF in Abcg2–/– mice was significantly higher than in wild-type mice (65 ± 6 and 47 ± 15% of dose, respectively, p < 0.05), whereas CDF recovery in bile of Abcc2–/– mice was negligible. Cumulative recovery of CDF in perfusate was significantly higher in Abcc2–/– (90 ± 8% of dose) relative to wild-type (35 ± 11% of dose) mice. Compartmental pharmacokinetic analysis revealed that the rate constant for CDF biliary excretion was significantly increased in Abcg2–/– (0.061 ± 0.005 min–1) compared with wild-type (0.039 ± 0.011 min–1) mice. The rate constant governing the basolateral excretion of CDF was ∼4-fold higher in Abcc2–/– (0.12 ± 0.02 min–1) relative to wild-type (0.030 ± 0.011 min–1) mice but was not altered in Abcg2–/– (0.031 ± 0.004 min–1) mice. Hepatic Abcc3 protein levels, determined by immunoblot analysis, were ∼60% higher in Abcc2–/– mice than in wild-type mice. In contrast, neither Abcc3 protein levels nor Abcc2 mRNA levels were altered in Abcg2–/– relative to wild-type mice. These data in knockout mouse models demonstrate that loss of expression and function of one canalicular transport protein may change the route and/or extent of excretion into bile or perfusate because of alterations in the function of other basolateral or canalicular transport proteins.

Impact of basolateral multidrug resistance-associated protein (Mrp) 3 and Mrp4 on the hepatobiliary disposition of fexofenadine in perfused mouse livers.

The disposition of fexofenadine, a commonly used antihistamine drug, is governed primarily by active transport. Biliary excretion of the parent compound is the major route of systemic clearance. Previous studies demonstrated that fexofenadine hepatic uptake is mediated by organic anion transporting polypeptides. Recently, we showed that in mice fexofenadine is excreted into bile primarily by multidrug resistance-associated protein (Mrp) 2 (Abcc2). In the present study, the roles of Mrp3 (Abcc3) and Mrp4 (Abcc4) in the hepatobiliary disposition of fexofenadine were examined in knockout mice using in situ liver perfusion. Compared with that in wild-type mice, basolateral excretion of fexofenadine was impaired, resulting in a ∼50% decrease in perfusate recovery in Abcc3-/- mice; in contrast, fexofenadine hepatobiliary disposition was unaltered in Abcc4-/- mice. As expected, in Abcc2-/- mice, fexofenadine was redirected from the canalicular to the basolateral membrane for excretion. In Abcc2-/-/Abcc3-/- double-knockout mice, fexofenadine biliary excretion was impaired, but perfusate recovery was similar to that in wild-type mice and more than 2-fold higher than that in Abcc3-/- mice, presumably due to compensatory basolateral transport mechanism(s). These results demonstrate that multiple transport proteins are involved in the hepatobiliary disposition of fexofenadine. In addition to Mrp2 and Mrp3, other transport proteins play an important role in the biliary and hepatic basolateral excretion of this zwitterionic drug.

Multidrug resistance-associated protein 2 is primarily responsible for the biliary excretion of fexofenadine in mice.

Previous studies implicated P-glycoprotein (P-gp) as the major transport protein responsible for the biliary excretion of fexofenadine (FEX). However, FEX biliary excretion was not impaired in P-gp- or breast cancer resistance protein (Bcrp)-knockout mice or multidrug resistance-associated protein 2 (Mrp2)-deficient rats. The present study tested the hypothesis that species differences exist in the transport protein primarily responsible for FEX biliary excretion between mice and rats. Livers from Mrp2-knockout (Mrp2KO) mice and Mrp2-deficient (TR-) rats were perfused in a single-pass manner with 0.5 μM FEX. N-(4-[2-(1,2,3,4-Tetrahydro-6,7-dimethoxy-2-isoquinolinyl)ethyl]-phenyl)-9,10-dihydro-5-methoxy-9-oxo-4-acridine carboxamide (GF120918) (10 μM) was employed to inhibit P-gp and Bcrp. The biliary excretion rate of FEX was decreased 85% in Mrp2KO relative to wild-type mice (18.4 ± 2.2 versus 122 ± 34 pmol/min/g liver). In mice, more than 50% of FEX unbound intrinsic biliary clearance (\batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(\mathrm{CL}_{\mathrm{bile},{\ }\mathrm{int}}^{{^\prime}}\) \end{document} = 3.0 ml/h/g liver) could be attributed to Mrp2 (Mrp2-dependent \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(\mathrm{CL}_{\mathrm{bile},{\ }\mathrm{int}}^{{^\prime}}\) \end{document} ∼ 1.7 ml/h/g liver), with P-gp and Bcrp playing a minor role (P-gp- and Bcrp-dependent \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(\mathrm{CL}_{\mathrm{bile},{\ }\mathrm{int}}^{{^\prime}}\) \end{document} ∼ 0.3 ml/h/g liver). Approximately one third of FEX \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(\mathrm{CL}_{\mathrm{bile},{\ }\mathrm{int}}^{{^\prime}}\) \end{document} was attributed to unidentified mechanisms in mice. In contrast to mice, FEX biliary excretion rate (245 ± 38 and 250 ± 25 pmol/min/g liver) and \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(\mathrm{CL}_{\mathrm{bile},{\ }\mathrm{int}}^{{^\prime}}\) \end{document} (9.72 ± 2.47 and 6.49 ± 0.68 ml/h/g liver) were comparable between TR- and control Wistar rats, respectively, suggesting that unidentified transport mechanism(s) can completely compensate for the loss of Mrp2 function in rats. Mrp2 clearly plays a major role in FEX biliary excretion in mice. In conclusion, remarkable species differences exist in FEX hepatobiliary transport mechanisms.

Roles of P-glycoprotein, Bcrp, and Mrp2 in biliary excretion of spiramycin in mice

ABSTRACT The multidrug resistance proteins P-glycoprotein (P-gp), breast cancer resistance protein (Bcrp), and multidrug resistance-associated protein 2 (Mrp2) are the three major canalicular transport proteins responsible for the biliary excretion of most drugs and metabolites. Previous in vitro studies demonstrated that P-gp transported macrolide antibiotics, including spiramycin, which is eliminated primarily by biliary excretion. Bcrp was proposed to be the primary pathway for spiramycin secretion into breast milk. In the present study, the contributions of P-gp, Bcrp, and Mrp2 to the biliary excretion of spiramycin were examined in single-pass perfused livers of male C57BL/6 wild-type, Bcrp-knockout, and Mrp2-knockout mice in the presence or absence of GF120918 (GW918), a P-gp and Bcrp inhibitor. Spiramycin was infused to achieve steady-state conditions, followed by a washout period, and parameters governing spiramycin hepatobiliary disposition were recovered by using pharmacokinetic modeling. In the absence of GW918, the rate constant governing spiramycin biliary excretion was decreased in Mrp2− knockout mice (0.0013 ± 0.0009 min−1) relative to wild-type mice (0.0124 ± 0.0096 min−1). These data are consistent with the ∼8-fold decrease in the recovery of spiramycin in the bile of Mrp2-knockout mice and suggest that Mrp2 is the major canalicular transport protein responsible for spiramycin biliary excretion. Interestingly, biliary recovery of spiramycin in Bcrp-knockout mice was increased in both the absence and presence of GW918 compared to wild-type mice. GW918 significantly decreased the rate constant for spiramycin biliary excretion and the rate constant for basolateral efflux of spiramycin. In conclusion, the biliary excretion of spiramycin in mice is mediated primarily by Mrp2 with a modest P-gp component.

The Contribution of Intestinal UDP-Glucuronosyltransferases in Modulating 7-Ethyl-10-hydroxy-camptothecin (SN-38)-Induced Gastrointestinal Toxicity in Rats

Life-threatening diarrhea afflicts a considerable percentage of patients treated with irinotecan, an anticancer agent with effects elicited through its active metabolite 7-ethyl-10-hydroxycamptothecin (SN-38). The primary detoxification pathway for SN-38 is glucuronidation. The purpose of this study was to evaluate the role that intestinal UDP-glucuronosyltransferases (UGTs) have from hepatic UGTs in modulating this diarrhea. To investigate this, Gunn rats devoid of UGT1A activity were injected with recombinant adenoviral vectors expressing UGT1A1, 1A6, and 1A7, resulting in reconstituted hepatic UGT expression comparable to a heterozygote. Hepatic microsome studies indicated that 4 to 7 days after adenoviral injection, transfected Gunn rats (j/jAV) had SN-38 glucuronide (SN-38G) formation rates three times higher than control heterozygote rats (j+AV). The adenovirus did not impart any glucuronidating capacity to the intestine in j/jAV rats, whereas j+AV rats possessed intestinal UGT function. After the administration of 20 mg/kg/day irinotecan i.p. to j/jAV rats 4 days after adenovirus injection, diarrhea ensued before the fourth irinotecan dose. j+AV rats were spared the diarrhea, and the toxicity was mild compared with the j/jAV rats, as measured by diarrhea scores, weight loss, and histological assessments of the cecum and colon. The pharmacokinetics of irinotecan, SN-38, and SN-38G indicate that the systemic exposure of SN-38 and SN-38G was higher and lower, respectively, in j/jAV rats. Despite this, the biliary excretion of irinotecan and metabolites was similar. Because intestinal UGTs are the main discriminating factor between j/jAV and j+AV rats, their presence seems to be critical for the gastrointestinal protection observed in j+AV rats.

Integration of Preclinical and Clinical Data with Pharmacokinetic Modeling and Simulation to Evaluate Fexofenadine as a Probe for Hepatobiliary Transport Function

PurposeThe suitability of fexofenadine as a probe substrate to assess hepatobiliary transport function in humans was evaluated by pharmacokinetic modeling/simulation and in vitro/in situ studies using chemical modulators.MethodsSimulations based on a pharmacokinetic model developed to describe fexofenadine disposition in humans were conducted to examine the impact of altered hepatobiliary transport on fexofenadine disposition. The effect of GF120918 on fexofenadine disposition was evaluated in human sandwich-cultured hepatocytes (SCH). Additionally, the effect of GF120918, bosentan, and taurocholate on fexofenadine disposition in perfused livers from TR− Wistar rats was examined.ResultsBased on modeling/simulation, fexofenadine systemic exposure was most sensitive to changes in the hepatic uptake rate constant, and did not reflect changes in hepatic exposure due to altered hepatic efflux. GF120918 did not impair fexofenadine biliary excretion in human SCH. GF120918 coadministration significantly decreased Cl’biliary to 27.5% of control in perfused rat livers.ConclusionsSimulations were in agreement with perfused liver data which predicted changes in fexofenadine systemic exposure primarily due to altered hepatic uptake. Fexofenadine is not a suitable probe to assess hepatic efflux function based on systemic concentrations. GF120918-sensitive protein(s) mediate fexofenadine biliary excretion in rat liver, whereas in human hepatocytes multiple efflux proteins are involved in fexofenadine hepatobiliary disposition.