Membrane Vesicle ABC Transporter Assays for Drug Safety Assessment

Carlo J. van Staden1, Ryan E. Morgan1, Bharath Ramachandran1, Yuan Chen1, Paul H. Lee1, Hisham K. Hamadeh1

1 Amgen, Inc., Thousand Oaks, California
Publication Name:  Current Protocols in Toxicology
Unit Number:  Unit 23.5
DOI:  10.1002/0471140856.tx2305s54
Online Posting Date:  November, 2012
GO TO THE FULL TEXT: PDF or HTML at Wiley Online Library


The use of plasma membrane vesicles that overexpress the bile salt export pump (BSEP) or multidrug resistance−associated protein 2, 3, or 4 (MRP2‐4) with an in vitro vacuum filtration system offers a rapid and reliable means for screening drug candidates for their effects on transporter function in hepatocytes and thus their potential for causing drug‐induced liver injury (DILI). Comparison of transporter activity in the presence and absence of ATP allows for determination of a specific assay window for each transporter. This window is used to determine the degree to which each test compound inhibits transporter activity. This assay battery is helpful for prioritizing and rank‐ordering compounds within a chemical series with respect to each other and in the context of known inhibitors of transporter activity and/or liver injury. This model can be used to influence the drug development process at an early stage and provide rapid feedback regarding the selection of compounds for advancement to in vivo safety evaluations. A detailed protocol for the high‐throughput assessment of ABC transporter function is provided, including specific recommendations for curve‐fitting to help ensure consistent results. Curr. Protoc. Toxicol. 54:23.5.1‐23.5.24. © 2012 by John Wiley & Sons, Inc.

Keywords: bile salt export pump; ATP‐binding cassette transporter; multidrug resistance‐associated protein; cholestasis; drug induced liver injury

PDF or HTML at Wiley Online Library

Table of Contents

  • Introduction
  • Basic Protocol 1: Membrane Vesicle ABC Transporter Assays
  • Basic Protocol 2: Non‐Linear Regression Analysis
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
PDF or HTML at Wiley Online Library


Basic Protocol 1: Membrane Vesicle ABC Transporter Assays

  • Test compounds, each as 10 mM stock in dimethyl sulfoxide (DMSO; for long term storage, maintain at ≤−20°C)
  • Positive control compounds (see recipe): use cyclosporine A alone for BSEP, MRP3, MRP4; cyclosporine A plus indomethacin for MRP2
  • DMSO
  • Assay buffer for ABC transporter of interest (see recipe)
  • Radiolabeled reporter substrate:
    • [3H]taurocholic acid ([3H(G)]‐, 5.0 Ci/mmol, PerkinElmer, for hBSEP)
    • [3H]estradiol‐17‐β‐D‐glucuronide ([estradiol‐6,7‐3H(N)]‐, 30‐60 Ci/mmol, PerkinElmer, for MRP2‐4)
  • 0.5% (v/v) polyethylenimine (PEI; see recipe)
  • 0.2 M Mg2+‐ATP (see recipe)
  • 200 mM L‐glutathione (GSH; Sigma, cat. no. G4251) in distilled water (store up to 6 months at −20°C)
  • Cryopreserved human BSEP, MRP2, MRP3, and/or MRP4 Sf9 membrane vesicles (5 mg/ml protein; GenoMembrane, Life Technologies, cat. nos. GM0001, GM0005, GM0012, GM0021)
  • Unlabeled reporter substrate:
    • 1 mM taurocholic acid (see recipe)
    • 10 mM estradiol‐17‐β‐D‐glucuronide (E 217βG; see recipe)
  • Wash buffer for ABC transporter of interest (see recipe)
  • 70% (v/v) ethanol
  • Scintillation cocktail (e.g., Microscint 20, PerkinElmer)
  • Standard 96‐well round‐bottom, polypropylene microtiter plates (e.g., Corning, cat. no. 3365)
  • Manual or electronic multichannel pipettor with appropriate tips (e.g., Biohit)
  • 96‐well filter plates (e.g., PerkinElmer UniFilter‐96 GF/C microplates, cat. no. 6005174)
  • Automated Print‐and‐Apply barcode instrument for microtiter plates (e.g., Agilent VCode; optional)
  • Heat sealer for microtiter plates (e.g., Agilent PlateLoc)
  • Glass bottle with cap
  • 8‐channel automated bulk reagent dispenser (e.g., Titan Multidrop 384) with cartridge and integrated input and output stackers
  • Vacuum manifold system or other vacuum source
  • Swinging bucket centrifuge with microtiter plate carrier
  • Microtiter plate shaker
  • 96‐well rapid filtration system for filter microplates (e.g., FilterMate‐96 Harvester, PerkinElmer)
  • Large vacuum oven set to 65°C
  • 96‐channel automated dispenser for viscous solutions (e.g., Brandel Micro‐Dispenser)
  • Microtiter plate seals (choose one):
    • Automated sealer for filter microplates (e.g., Tomtec Quadra‐Seal 9600) with roll of clear, non‐fluorescent adhesive seals (e.g., Tomtec, cat. no. 696‐430‐1)
    • White, opaque adhesive seals for bottom of plates (supplied with GF/C filter plates; optional) and clear, non‐fluorescent adhesive seals for top of plates (e.g., TopSeal‐A, cat. No. 6005185, PerkinElmer)
  • Liquid scintillation counter for 96‐well plates (e.g., TopCount NXT, PerkinElmer)
PDF or HTML at Wiley Online Library


  •   FigureFigure 23.5.1 Illustration of hepatocellular transporter organization. Bile acids are synthesized from cholesterol within the hepatocyte. Further reactions may take place, such as conjugation to glycine or taurine (amino acid conjugated and unconjugated bile acids depicted as BS−). Other reactions may render bile acids as phase 2 metabolized species, such as glucuronidated or sulfated bile acids (BS‐C). Bile acids are transported to the bile ducts via BSEP, or by MRP2 if they are BS‐C species. Under cholestatic conditions, MRP3 and MRP4 are described in compensatory mechanisms by which hepatocytes can prevent bile acid accumulation and cytotoxicity by managing basolateral elimination of bile acids into the blood. NTCP is the primary route by which hepatocytes reclaim bile acids from blood; however, the OATPs represent a sodium‐independent means for bile acid uptake. Organic solute transporter subunits α and β (OSTα and OSTβ) also play a role in the basolateral elimination of bile acids, but less is known about their function within hepatocytes. Not all transporters or substrates are depicted in this figure.
  •   FigureFigure 23.5.2 Illustration of a plasma membrane vesicle and principle of assay for radiolabeled substrate uptake. TCA, taurocholic acid.
  •   FigureFigure 23.5.3 Basic principle of membrane vesicle transporter assays.
  •   FigureFigure 23.5.4 Layout of 96‐well test compound plates. (A) The compound dosing plate is used to perform the 1:3 dilution scheme and generate the mother plate (MP). (B) Final compound concentrations (mM) in the MP and daughter plates (DPs). (C) Final compound concentrations (µM) in the assay.
  •   FigureFigure 23.5.5 Titan Multidrop 384.
  •   FigureFigure 23.5.6 Tomtec Quadra‐Seal 9600 automated Unifilter plate sealer.
  •   FigureFigure 23.5.7 Brandel Micro‐Dispenser for dispensing scintillation cocktail to a standard 96‐well microtiter plate.
  •   FigureFigure 23.5.8 (A) Example of curve with no bottom plateau where Maximal Activity of troglitazone on MRP3 transport is <20%. (B) Example of curve with no bottom plateau where Maximal Activity of rosiglitazone on MRP3 transport is >50%. (C) Example of curve with no top plateau in MRP3 assay. (D) Example of a curve going in the opposite direction due to stimulation of E217βG transport across MRP2 in the presence of indomethacin. (E) Example of a curve in which the response data points at the highest tested concentration in the MRP3 assay are going in the opposite direction as a result of exposure to cyclosporine A. (F) Example of a curve in the MRP2 assay in response to cyclosporine A treatment with a well‐defined bottom plateau at a >0% activity level, indicating that the test compound is a partial inhibitor. In some cases, such a curve may indicate solubility limitations. (G) Example of a curve in the MRP4 transporter assay in which the bottom plateau for the test compound is within the noise level for the activity response and hence considered inactive.


Literature Cited

Literature Cited
   Alrefai, W.A. and Gill, R.K. 2007. Bile acid transporters: Structure, function, regulation and pathophysiological implications. Pharm. Res. 24:1803‐1823.
   Amacher, D.E. 2012. The primary role of hepatic metabolism in idiosyncratic drug‐induced liver injury. Expert Opin. Drug Metab. Toxicol. 8:335‐347.
   Belinsky, M.G., Dawson, P.A., Shchaveleva, I., Bain, L.J., Wang, R., Ling, V., Chen, Z.S., Grinberg, A., Westphal, H., Klein‐Szanto, A., Lerro, A., and Kruh, G.D. 2005. Analysis of the in vivo functions of Mrp3. Mol. Pharmacol. 68:160‐168.
   Benet, L.Z., Broccatelli, F., and Oprea, T.I. 2011. BDDCS applied to over 900 drugs. AAPS J. 13:519‐547.
   Borst, P., de Wolf, C., and van de Wetering, K. 2007. Multidrug resistance‐associated proteins 3, 4, and 5. Pflugers Arch. 453:661‐673.
   Dawson, P.A., Lan, T., and Rao, A. 2009. Bile acid transporters. J. Lipid Res. 50:2340‐2357.
   Dawson, S., Stahl, S., Paul, N., Barber, J., and Kenna, J.G. 2012. In vitro inhibition of the bile salt export pump correlates with risk of cholestatic drug‐induced liver injury in humans. Drug Metab. Dispos. 40:130‐138.
   Fattinger, K., Funk, C., Pantze, M., Weber, C., Reichen, J., Stieger, B., and Meier, P.J. 2001. The endothelin antagonist bosentan inhibits the canalicular bile salt export pump: A potential mechanism for hepatic adverse reactions. Clin. Pharmacol. Ther. 69:223‐231.
   Feng, B., Xu, J.J., Bi, Y.A., Mireles, R., Davidson, R., Duignan, D.B., Campbell, S., Kostrubsky, V.E., Dunn, M.C., Smith, A.R., and Wang, H.F. 2009. Role of hepatic transporters in the disposition and hepatotoxicity of a HER2 tyrosine kinase inhibitor CP‐724,714. Toxicol. Sci. 108:492‐500.
   Fomenko, I., Durst, M., and Balaban, D. 2006. Robust regression for high throughput drug screening. Comput. Methods Programs Biomed. 82:31‐37.
   Fouassier, L., Kinnman, N., Lefevre, G., Lasnier, E., Rey, C., Poupon, R., Elferink, R.P., and Housset, C. 2002. Contribution of mrp2 in alterations of canalicular bile formation by the endothelin antagonist bosentan. J. Hepatol. 37:184‐191.
   Funk, C., Ponelle, C., Scheuermann, G., and Pantze, M. 2001. Cholestatic potential of troglitazone as a possible factor contributing to troglitazone‐induced hepatotoxicity: In vivo and in vitro interaction at the canalicular bile salt export pump (Bsep) in the rat. Mol. Pharmacol. 59:627‐635.
   Gradhand, U., Lang, T., Schaeffeler, E., Glaeser, H., Tegude, H., Klein, K., Fritz, P., Jedlitschky, G., Kroemer, H.K., Bachmakov, I., Anwald, B., Kerb, R., Zanger, U.M., Eichelbaum, M., Schwab, M., and Fromm, M.F. 2008. Variability in human hepatic MRP4 expression: Influence of cholestasis and genotype. Pharmacogenomics J. 8:42‐52.
   Hamadeh, H.K., Todd, M., Healy, L., Meyer, J.T., Kwok, A.M., Higgins, M., and Afshari, C.A. 2010. Application of genomics for identification of systemic toxicity triggers associated with VEGF‐R inhibitors. Chem. Res. Toxicol. 23:1025‐1033.
   Heredi‐Szabo, K., Kis, E., Molnar, E., Gyorfi, A., and Krajcsi, P. 2008. Characterization of 5(6)‐carboxy‐2,′7′‐dichlorofluorescein transport by MRP2 and utilization of this substrate as a fluorescent surrogate for LTC4. J. Biomol. Screen. 13:295‐301.
   Heredi‐Szabo, K., Glavinas, H., Kis, E., Mehn, D., Bathori, G., Veres, Z., Kobori, L., von Richter, O., Jemnitz, K., and Krajcsi, P. 2009. Multidrug resistance protein 2‐mediated estradiol‐17beta‐D‐glucuronide transport potentiation: in vitro‐in vivo correlation and species specificity. Drug Metab. Dispos 37:794‐801.
   Hofmann, A.F. 2007. Biliary secretion and excretion in health and disease: current concepts. Ann. Hepatol. 6:15‐27.
   Horikawa, M., Kato, Y., Tyson, C.A., and Sugiyama, Y. 2003. Potential cholestatic activity of various therapeutic agents assessed by bile canalicular membrane vesicles isolated from rats and humans. Drug Metab. Pharmacokinet. 18:16‐22.
   Keppler, D. 2011. Cholestasis and the role of basolateral efflux pumps. Z. Gastroenterol. 49:1553‐1557.
   Kis, E., Ioja, E., Nagy, T., Szente, L., Heredi‐Szabo, K., and Krajcsi, P. 2009. Effect of membrane cholesterol on BSEP/Bsep activity: Species specificity studies for substrates and inhibitors. Drug Metab. Dispos. 37:1878‐1886.
   Kis, E., Ioja, E., Rajnai, Z., Jani, M., Mehn, D., Heredi‐Szabo, K., and Krajcsi, P. 2011. BSEP inhibition—In vitro screens to assess cholestatic potential of drugs. Toxicol. In Vitro epub ahead of print:
   Klaassen, C.D. and Aleksunes, L.M. 2010. Xenobiotic, bile acid, and cholesterol transporters: Function and regulation. Pharmacol. Rev. 62:1‐96.
   Konig, J. 2011. Uptake transporters of the human OATP family: Molecular characteristics, substrates, their role in drug‐drug interactions, and functional consequences of polymorphisms. Handbook Exp. Pharmacol. 201:1‐28.
   Kostrubsky, V.E., Strom, S.C., Hanson, J., Urda, E., Rose, K., Burliegh, J., Zocharski, P., Cai, H., Sinclair, J.F., and Sahi, J. 2003. Evaluation of hepatotoxic potential of drugs by inhibition of bile‐acid transport in cultured primary human hepatocytes and intact rats. Toxicol. Sci. 76:220‐228.
   Kostrubsky, S.E., Strom, S.C., Kalgutkar, A.S., Kulkarni, S., Atherton, J., Mireles, R., Feng, B., Kubik, R., Hanson, J., Urda, E., and Mutlib, A.E. 2006. Inhibition of hepatobiliary transport as a predictive method for clinical hepatotoxicity of nefazodone. Toxicol. Sci. 90:451‐459.
   Lee, K.S., Oh, S.J., Kim, H.M., Lee, K.H., and Kim, S.K. 2011. Assessment of reactive metabolites in drug‐induced liver injury. Arch. Pharm. Res. 34:1879‐1886.
   Leslie, E.M., Watkins, P.B., Kim, R.B., and Brouwer, K.L. 2007. Differential inhibition of rat and human Na+‐dependent taurocholate cotransporting polypeptide (NTCP/SLC10A1) by bosentan: A mechanism for species differences in hepatotoxicity. J. Pharmacol. Exp. Ther. 321:1170‐1178.
   Masubuchi, Y. 2006. Metabolic and non‐metabolic factors determining troglitazone hepatotoxicity: A review. Drug Metab. Pharmacokinet. 21:347‐356.
   McRae, M.P., Lowe, C.M., Tian, X., Bourdet, D.L., Ho, R.H., Leake, B.F., Kim, R.B., Brouwer, K.L., and Kashuba, A.D. 2006. Ritonavir, saquinavir, and efavirenz, but not nevirapine, inhibit bile acid transport in human and rat hepatocytes. J. Pharmacol. Exp. Ther. 318:1068‐1075.
   Mita, S., Suzuki, H., Akita, H., Stieger, B., Meier, P.J., Hofmann, A.F., and Sugiyama, Y. 2005. Vectorial transport of bile salts across MDCK cells expressing both rat Na+‐taurocholate cotransporting polypeptide and rat bile salt export pump. Am. J. Physiol. 288:G159‐G167.
   Morgan, R.E., Trauner, M., van Staden, C.J., Lee, P.H., Ramachandran, B., Eschenberg, M., Afshari, C.A., Qualls, C.W., Jr. Lightfoot‐Dunn, R., and Hamadeh, H.K. 2010. Interference with bile salt export pump function is a susceptibility factor for human liver injury in drug development. Toxicol. Sci. 118:485‐500.
   Motulsky, H.J. and Brown, R.E. 2006. Detecting outliers when fitting data with nonlinear regression ‐ a new method based on robust nonlinear regression and the false discovery rate. BMC Bioinformatics 7:123.
   Nadanaciva, S. and Will, Y. 2009. Current concepts in drug‐induced mitochondrial toxicity. Curr. Protoc. Toxicol. 40:2.15.1‐2.15.9.
   Nies, A.T. and Keppler, D. 2007. The apical conjugate efflux pump ABCC2 (MRP2). Pflugers Arch. 453:643‐659.
   Noe, J., Stieger, B., and Meier, P.J. 2002. Functional expression of the canalicular bile salt export pump of human liver. Gastroenterology 123:1659‐1666.
   Padda, M.S., Sanchez, M., Akhtar, A.J., and Boyer, J.L. 2011. Drug‐induced cholestasis. Hepatology 53:1377‐1387.
   Palmeira, C.M. and Rolo, A.P. 2004. Mitochondrially‐mediated toxicity of bile acids. Toxicology 203:1‐15.
   Pauli‐Magnus, C. and Meier, P.J. 2005. Hepatocellular transporters and cholestasis. J. Clin. Gastroenterol. 39:S103‐S110.
   Pauli‐Magnus, C. and Meier, P.J. 2006. Hepatobiliary transporters and drug‐induced cholestasis. Hepatology 44:778‐787.
   Pedersen, J.M., Matsson, P., Bergstrom, C.A., Norinder, U., Hoogstraate, J., and Artursson, P. 2008. Prediction and identification of drug interactions with the human ATP‐binding cassette transporter multidrug‐resistance associated protein 2 (MRP2; ABCC2). J. Med. Chem. 51:3275‐3287.
   Rius, M., Hummel‐Eisenbeiss, J., Hofmann, A.F., and Keppler, D. 2005. Substrate specificity of human ABCC4 (MRP4)‐mediated cotransport of bile acids and reduced glutathione. Am. J. Physiol. Gastrointest. Liver Physiol. 290:640‐649.
   Rolo, A.P., Palmeira, C.M., Holy, J.M., and Wallace, K.B. 2004. Role of mitochondrial dysfunction in combined bile acid‐induced cytotoxicity: The switch between apoptosis and necrosis. Toxicol. Sci. 79:196‐204.
   Russel, F.G., Koenderink, J.B., and Masereeuw, R. 2008. Multidrug resistance protein 4 (MRP4/ABCC4): A versatile efflux transporter for drugs and signalling molecules. Trends Pharmacol. Sci. 29:200‐207.
   Saito, H., Osumi, M., Hirano, H., Shin, W., Nakamura, R., and Ishikawa, T. 2009. Technical pitfalls and improvements for high‐speed screening and QSAR analysis to predict inhibitors of the human bile salt export pump (ABCB11/BSEP). AAPS J. 11:581‐589.
   Sakurai, A., Kurata, A., Onishi, Y., Hirano, H., and Ishikawa, T. 2007. Prediction of drug‐induced intrahepatic cholestasis: In vitro screening and QSAR analysis of drugs inhibiting the human bile salt export pump. Expert Opin. Drug Saf. 6:71‐86.
   Snow, K.L. and Moseley, R.H. 2007. Effect of thiazolidinediones on bile acid transport in rat liver. Life Sci. 80:732‐740.
   Stepan, A.F., Walker, D.P., Bauman, J., Price, D.A., Baillie, T.A., Kalgutkar, A.S., and Aleo, M.D. 2011. Structural alert/reactive metabolite concept as applied in medicinal chemistry to mitigate the risk of idiosyncratic drug toxicity: a perspective based on the critical examination of trends in the top 200 drugs marketed in the United States. Chem. Res. Toxicol. 24:1345‐1410.
   Stieger, B. 2009. Recent insights into the function and regulation of the bile salt export pump (ABCB11), Current Opinions in Lipidology 20:176‐181.
   Stieger, B. 2010. Role of the bile salt export pump, BSEP, in acquired forms of cholestasis. Drug Metab. Rev. 42:437‐445.
   Stieger, B., Meier, Y., and Meier, P.J. 2007. The bile salt export pump. Pflugers Arch. 453:611‐620.
   Stine, J.G. and Lewis, J.H. 2011. Drug‐induced liver injury: A summary of recent advances. Expert Opin. Drug Metab. Toxicol. 7:875‐890.
   St‐Pierre, M.V., Kullak‐Ublick, G.A., Hagenbuch, B., and Meier, P.J. 2001. Transport of bile acids in hepatic and non‐hepatic tissues. J. Exp. Biol. 204:1673‐1686.
   Trauner, M. and Boyer, J.L. 2003. Bile salt transporters: molecular characterization, function, and regulation. Physiol. Rev. 83:633‐671.
   Xia, C.Q., Milton, M.N., and Gan, L.S. 2007. Evaluation of drug‐transporter interactions using in vitro and in vivo models. Curr. Drug Metab. 8:341‐363.
   Yoshikado, T., Takada, T., Yamamoto, T., Yamaji, H., Ito, K., Santa, T., Yokota, H., Yatomi, Y., Yoshida, H., Goto, J., Tsuji, S., and Suzuki, H. 2011. Itraconazole‐induced cholestasis: Involvement of the inhibition of bile canalicular phospholipid translocator MDR3/ABCB4. Mol. Pharmacol. 79:241‐250.
   Zamek‐Gliszczynski, M.J., Nezasa, K., Tian, X., Bridges, A.S., Lee, K., Belinsky, M.G., Kruh, G.D., and Brouwer, K.L. 2006. 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. J. Pharmacol. Exp. Ther. 319:1485‐1491.
   Zelcer, N., Huisman, M.T., Reid, G., Wielinga, P., Breedveld, P., Kuil, A., Knipscheer, P., Schellens, J.H., Schinkel, A.H., and Borst, P. 2003. Evidence for two interacting ligand binding sites in human multidrug resistance protein 2 (ATP binding cassette C2). J. Biol. Chem. 278:23538‐23544.
   Zelcer, N., van de Wetering, K., de Waart, R., Scheffer, G.L., Marschall, H.U., Wielinga, P.R., Kuil, A., Kunne, C., Smith, A., van der Valk, M., Wijnholds, J., Elferink, R.O., and Borst, P. 2006. Mice lacking Mrp3 (Abcc3) have normal bile salt transport, but altered hepatic transport of endogenous glucuronides. J. Hepatol. 44:768‐775.
   Zollner, G. and Trauner, M. 2008. Mechanisms of cholestasis. Clin. Liver Dis. 12:1‐26.
   Zollner, G., Wagner, M., and Trauner, M. 2010. Nuclear receptors as drug targets in cholestasis and drug‐induced hepatotoxicity. Pharmacol. Ther. 126:228‐243.
PDF or HTML at Wiley Online Library