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
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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

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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
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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)
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