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 output: Contribution to journalArticlepeer-review

52 Citations (Scopus)

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.

Original languageEnglish
Pages (from-to)2281-2296
Number of pages16
JournalPharmaceutical research
Volume24
Issue number12
DOIs
Publication statusPublished - 2007 Dec

Keywords

  • ATP-binding cassette transporter
  • Human multidrug resistance-associated protein 4
  • Liquid chromatography-tandem mass spectrometry
  • Substrate screening
  • Vesicle uptake study

ASJC Scopus subject areas

  • Biotechnology
  • Molecular Medicine
  • Pharmacology
  • Pharmaceutical Science
  • Organic Chemistry
  • Pharmacology (medical)

Fingerprint Dive into the research topics of 'Multichannel liquid chromatography-tandem mass spectrometry cocktail method for comprehensive substrate characterization of multidrug resistance-associated protein 4 transporter'. Together they form a unique fingerprint.

Cite this