Pharmaceutical Research

, Volume 24, Issue 12, pp 2281–2296

Multichannel Liquid Chromatography–Tandem Mass Spectrometry Cocktail Method for Comprehensive Substrate Characterization of Multidrug Resistance-Associated Protein 4 Transporter

  • Yasuo Uchida
  • Junichi Kamiie
  • Sumio Ohtsuki
  • Tetsuya Terasaki
Research Paper

DOI: 10.1007/s11095-007-9453-7

Cite this article as:
Uchida, Y., Kamiie, J., Ohtsuki, S. et al. Pharm Res (2007) 24: 2281. doi:10.1007/s11095-007-9453-7

Abstract

Purpose

To develop a comprehensive substrate-screening method for the ATP-binding cassette (ABC) transporter, and identify new substrates for multidrug resistance-associated protein 4 (MRP4/ABCC4).

Methods

Human MRP4-expressing membrane vesicles were incubated with a mixture of 50 compounds, including methotrexate, a known MRP4 substrate. The amounts transported were simultaneously determined by liquid chromatography–tandem mass spectrometry.

Results

From 49 compounds, 12 were identified as substrate candidates for MRP4 in the first screening. The second screening was performed involving the uptake of mixture using single quadrupole multichannel mode, and the third screening was performed involving the uptake of individual compounds using multiple reaction monitoring multichannel mode. As a result, eight substrate candidates were additionally identified. Subsequently, in the fourth step, osmotic pressure-dependent transport was demonstrated for 18 compounds (cefmetazole, piperacillin, rebamipide, tetracycline, ampicillin, benzylpenicillin, bumetanide, cephalosporin C, enalapril, pipemidic acid, furosemide, ceftazidime, pravastatin, hydrochlorothiazide, sulbactam, baclofen, bezafibrate and alacepril) among the 20 substrate candidates, thereby confirming them as MRP4 substrates. By contrast, the uptakes of meloxicam and nateglinide did not depend on osmolarity, indicating that these compounds were not substrates, but bound to MRP4.

Conclusions

The new comprehensive substrate-screening method for ABC transporters allowed the identification of 18 new substrates for MRP4.

Key words

ATP-binding cassette transporterhuman multidrug resistance-associated protein 4liquid chromatography–tandem mass spectrometrysubstrate screeningvesicle uptake study

Abbreviations

ABC

ATP-binding cassette

ACE

angiotensin-converting enzyme

BBB

blood–brain barrier

BCSFB

blood–cerebrospinal fluid barrier

CMZ

cefmetazole

CNS

central nervous system

DHEAS

dehydroepiandrosterone sulfate

ESI

electrospray ionization

LC–MS

liquid chromatography–mass spectrometry

LC–MS/MS

liquid chromatography–tandem mass spectrometry

MRM

multiple reaction monitoring

MRP

multidrug resistance-associated protein

MTX

methotrexate

MUID

membrane uptake-index difference

OAT

organic anion transporter

Q1

single quadrupole

Copyright information

© Springer Science+Business Media, LLC 2007

Authors and Affiliations

  • Yasuo Uchida
    • 1
  • Junichi Kamiie
    • 1
    • 2
  • Sumio Ohtsuki
    • 1
    • 2
  • Tetsuya Terasaki
    • 1
    • 2
  1. 1.Division of Membrane Transport and Drug Targeting, Graduate School of Pharmaceutical SciencesTohoku UniversitySendaiJapan
  2. 2.SORSTJapan Science and Technology Agency (JST)KawaguchiJapan