MECHANISMS OF IMPAIRED BILIARY EXCRETION OF ACETAMINOPHEN GLUCURONIDE AFTER ACUTE PHENOBARBITAL TREATMENT OR PHENOBARBITAL PRETREATMENT HAO XIONG, HIROSHI SUZUKI, YUICHI SUGIYAMA, PETER J. MEIER, GARY M. POLLACK, AND KIM L. R. BROUWER Division of Drug Delivery and Disposition, School of Pharmacy, University of North Carolina, Chapel Hill, North Carolina (H.X., G.M.P., K.L.R.B.); Graduate School of Pharmaceutical Sciences, University of Tokyo, Tokyo, Japan (H.S., Y.S.); and Division of Clinical Pharmacology and Toxicology, Department of Medicine, University Hospital Zurich, Zurich, Switzerland (P.J.M.) (Received January 24, 2002; accepted May 28, 2002) This article is available online at http://dmd.aspetjournals.org ABSTRACT: Previous studies have demonstrated that phenobarbital (PB) sig- nificantly impairs the biliary excretion of acetaminophen glucuro- nide (AG) in rats. Studies also suggested that Mrp2 mediates AG biliary excretion, and Mrp3 is involved in AG basolateral export. It was hypothesized that inhibition of Mrp2-mediated AG transport by PB or PB metabolites, and PB induction of Mrp3, may contribute to the impaired biliary excretion of AG by PB. In the present study, the hepatobiliary transport of AG in single-pass isolated perfused Wistar and TR  rat livers was investigated. The AG biliary clear- ance was markedly decreased, and the AG basolateral clearance was significantly increased in TR  rat livers. Uptake of AG by Mrp2 and Mrp3, and inhibition of Mrp2- and Mrp3-mediated transport by PB and major PB metabolites, were investigated with rat Mrp2- or Mrp3-expressing Sf9 cell plasma membrane vesicles (Sf9-PMVs). AG was transported by Mrp3 (K m  0.91 mM). Net ATP-dependent AG uptake into Mrp2-expressing Sf9-PMVs could not be detected directly. However, AG significantly inhibited Mrp2-mediated 5-(and 6)-carboxy-2,7-dichlorofluorescein (CDF) transport. p-Hydroxy- phenobarbital glucuronide (p-OHPBG), but not PB or p-hydroxy- phenobarbital, significantly inhibited Mrp2-mediated CDF trans- port. The IC 50 values for p-OHPBG inhibition of Mrp2-mediated CDF uptake and Mrp3-mediated AG transport were similar (0.68 and 0.46 mM, respectively). PB treatment (80 mg/kg/day  4 days) markedly increased hepatic Mrp3 expression in Wistar rats. In conclusion, inhibition of Mrp2-mediated AG transport by p-OHPBG provided one possible explanation for the impaired biliary excre- tion of AG after acute PB treatment. However, impaired biliary excretion of AG after PB pretreatment may be attributed primarily to the induction of hepatic Mrp3 by PB. Acetaminophen glucuronide (AG 1 ), a monovalent organic anion formed in hepatocytes following acetaminophen (APAP) administra- tion, undergoes both biliary excretion and basolateral export from hepatocytes. Approximately 50% of AG formed in hepatocytes is excreted in bile; the remainder traverses the basolateral membrane into blood and undergoes renal elimination. AG excretion in bile accounts for 7% of the administered APAP dose (100 mg/kg) in rats in vivo (Brouwer and Jones, 1990) and 10% in the isolated perfused rat liver at equivalent APAP concentrations (Studenberg and Brouwer, 1992; Turner and Brouwer, 1997). Pretreatment with phenobarbital (PB), a common enzyme-inducing agent, significantly increased AG formation but impaired AG biliary excretion 3- to 6-fold in the rat in vivo (Brouwer and Jones, 1990) and in the isolated perfused rat liver (Studenberg and Brouwer, 1992). Acute PB treatment also markedly (3-fold) decreased the biliary excretion of AG along with a moder- ate reduction (1.5-fold) in AG formation (Studenberg and Brouwer, 1992). Several distinct mechanisms may be involved in decreased biliary excretion of AG after PB treatment. Previous studies suggested that the decrease in APAP glucuronidation may contribute in part to the impaired biliary excretion of AG after acute PB treatment. Intracel- lular sequestration of AG does not play a role in the reduction in AG biliary excretion after either PB pretreatment or acute PB treatment (Studenberg and Brouwer, 1992, 1993). PB is a well-known enzyme inducer that also induces hepatocyte basolateral transporters, multi- drug resistance-associated protein 3 (Mrp3; Abcc3), and organic anion transporting polypeptide 2 (Oatp2; Slc21a5), in Sprague-Dawley rats (Ogawa et al., 2000; Rausch-Derra et al., 2001). PB pretreatment may This work was supported by National Institutes of Health Grant GM41935. It was presented in part at the American Association of Pharmaceutical Scientists annual meeting, 2001, October 21–25, Denver, CO and was submitted to the Graduate School of the University of North Carolina in partial fulfillment of re- quirements for the Doctor of Philosophy degree in Pharmaceutical Sciences (H.X.). 1 Abbreviations used are: AG, acetaminophen glucuronide; APAP, acetamin- ophen; PB, phenobarbital; Mrp, multidrug resistance-associated protein; p-OHPB, p-hydroxyphenobarbital; p-OHPBG, p-hydroxyphenobarbital glucuro- nide; AS, acetaminophen sulfate; TR - , Mrp2 transport-deficient; PMV, plasma membrane vesicle; TC, taurocholate; CDF, 5-(and 6)-carboxy-2',7'-dichlorofluo- rescein; HPLC, high-performance liquid chromatography; E 2 17G, estradiol 17- glucuronide; GFP, green fluorescence protein; Oatp, organic anion transporting polypeptide. Address correspondence to: Kim L. R. Brouwer, Pharm.D., Ph.D., Division of Drug Delivery and Disposition, School of Pharmacy, CB 7360, Beard Hall, Uni- versity of North Carolina at Chapel Hill, Chapel Hill, NC 27599-7360. E-mail: kbrouwer@unc.edu 0090-9556/02/3009-962–969$7.00 DRUG METABOLISM AND DISPOSITION Vol. 30, No. 9 Copyright © 2002 by The American Society for Pharmacology and Experimental Therapeutics 689/1003958 DMD 30:962–969, 2002 Printed in U.S.A. 962