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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Abstract

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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Materials

Basic Protocol 1: Membrane Vesicle ABC Transporter Assays

  Materials
  • 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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Figures

  •   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.

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Literature Cited

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