Skip to main content

Advertisement

Log in

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

  • Research Paper
  • Published:
Pharmaceutical Research Aims and scope Submit manuscript

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.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Scheme 1
Fig. 1
Fig. 2

Similar content being viewed by others

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

References

  1. C. A. Ritter, G. Jedlitschky, H. Meyer zu Schwabedissen, M. Grube, K. Kock, H. K. Kroemer. Cellular export of drugs and signaling molecules by the ATP-binding cassette transporters MRP4 (ABCC4) and MRP5 (ABCC5) Drug Metab. Rev. 37:253–278 (2005).

    Article  PubMed  CAS  Google Scholar 

  2. R. A. van Aubel, P. H. Smeets, J. G. Peters, R. J. Bindels, F. G. Russel. The MRP4/ABCC4 gene encodes a novel apical organic anion transporter in human kidney proximal tubules: putative efflux pump for urinary cAMP and cGMP J. Am. Soc. Nephrol. 13:595–603 (2002).

    PubMed  Google Scholar 

  3. T. Imaoka, H. Kusuhara, M. Adachi, J. D. Schuetz, K. Takeuchi, Y. Sugiyama. Functional involvement of multidrug resistance-associated protein 4 (MRP4/ABCC4) in the renal elimination of the antiviral drugs adefovir and tenofovir Mol. Pharmacol. 71:619–627 (2007).

    Article  PubMed  CAS  Google Scholar 

  4. M. Nishimura, S. Naito. Tissue-specific mRNA expression profiles of human ATP-binding cassette and solute carrier transporter superfamilies Drug Metab. Pharmacokinet. 20:452–477 (2005).

    Article  PubMed  CAS  Google Scholar 

  5. M. Leggas, M. Adachi, G. L. Scheffer, D. Sun, P. Wielinga, G. Du, K. E. Mercer, Y. Zhuang, J. C. Panetta, B. Johnston, R. J. Scheper, C. F.Stewart, J. D. Schuetz. Mrp4 confers resistance to topotecan and protects the brain from chemotherapy Mol. Cell. Biol. 24:7612–7621 (2004).

    Article  PubMed  CAS  Google Scholar 

  6. A. Tsuji, T. Terasaki, I. Tamai, K. Takeda. In vivo evidence for carrier-mediated uptake of beta-lactam antibiotics through organic anion transport systems in rat kidney and liver J. Pharmacol. Exp. Ther. 253:315–320 (1990).

    PubMed  CAS  Google Scholar 

  7. S. K. Mujais, A. Quintanilla, M. Zahid, K. Koch, W. Shaw, T. Gibson. Renal handling of enalaprilat Am. J. Kidney Dis. 19:121–125 (1992).

    PubMed  CAS  Google Scholar 

  8. N. Apiwattanakul, T. Sekine, A. Chairoungdua, Y. Kanai, N. Nakajima, S. Sophasan, H. Endou. Transport properties of nonsteroidal anti-inflammatory drugs by organic anion transporter 1 expressed in Xenopus laevis: oocytes Mol. Pharmacol. 55:847–854 (1999).

    PubMed  CAS  Google Scholar 

  9. H. Hasannejad, M. Takeda, K. Taki, H. J. Shin, E. Babu, P. Jutabha, S. Khamdang, M. Aleboyeh, M. L. Onozato, A. Tojo, A. Enomoto, N. Anzai, S. Narikawa, X. L. Huang, T. Niwa, H. Endou. Interactions of human organic anion transporters with diuretics J. Pharmacol. Exp. Ther. 308:1021–1029 (2004).

    Article  PubMed  CAS  Google Scholar 

  10. A. Saheki, T. Terasaki, I. Tamai, A. Tsuji. In vivo and in vitro blood–brain barrier transport of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors Pharm. Res. 11:305–311 (1994).

    Article  PubMed  CAS  Google Scholar 

  11. Y. Deguchi, K. Inabe, K. Tomiyasu, K. Nozawa, S. Yamada, R. Kimura. Study on brain interstitial fluid distribution and blood–brain barrier transport of baclofen in rats by microdialysis Pharm. Res. 12:1838–1844 (1995).

    Article  PubMed  CAS  Google Scholar 

  12. Y. Deguchi, K. Nozawa, S. Yamada, Y. Yokoyama, R. Kimura. Quantitative evaluation of brain distribution and blood–brain barrier efflux transport of probenecid in rats by microdialysis: possible involvement of the monocarboxylic acid transport system J. Pharmacol. Exp. Ther. 280:551–560 (1997).

    PubMed  CAS  Google Scholar 

  13. R. Nau, F. Sorgel, H. W. Prange. Pharmacokinetic optimisation of the treatment of bacterial central nervous system infections Clin. Pharmacokinet. 35:223–246 (1998).

    Article  PubMed  CAS  Google Scholar 

  14. A. Mannila, J. Rautio, M. Lehtonen, T. Jarvinen, J. Savolainen. Inefficient central nervous system delivery limits the use of ibuprofen in neurodegenerative diseases Eur. J. Pharm. Sci. 24:101–105 (2005).

    Article  PubMed  CAS  Google Scholar 

  15. P. R. Wielinga, G. Reid, E. E. Challa, I. van der Heijden, L. van Deemter, M. de Haas, C. Mol, A. J. Kuil, E. Groeneveld, J. D. Schuetz, C. Brouwer, R. A. De Abreu, J. Wijnholds, J. H. Beijnen, P. Borst. Thiopurine metabolism and identification of the thiopurine metabolites transported by MRP4 and MRP5 overexpressed in human embryonic kidney cells Mol. Pharmacol. 62:1321–1331 (2002).

    Article  PubMed  CAS  Google Scholar 

  16. G. Reid, P. Wielinga, N. Zelcer, I. van der Heijden, A. Kuil, M. de Haas, J. Wijnholds, P. Borst. The human multidrug resistance protein MRP4 functions as a prostaglandin efflux transporter and is inhibited by nonsteroidal antiinflammatory drugs Proc. Natl. Acad. Sci. U. S. A. 100:9244–9249 (2003).

    Article  PubMed  CAS  Google Scholar 

  17. Z. E. Sauna, K. Nandigama, S. V. Ambudkar. Multidrug resistance protein 4 (ABCC4)-mediated ATP hydrolysis: effect of transport substrates and characterization of the post-hydrolysis transition state J. Biol. Chem. 279:48855–48864 (2004).

    Article  PubMed  CAS  Google Scholar 

  18. W. A. Korfmacher. Principles and applications of LC–MS in new drug discovery Drug Discov. Today 10:1357–1367 (2005).

    Article  PubMed  CAS  Google Scholar 

  19. L. Anderson, C. L. Hunter. Quantitative mass spectrometric multiple reaction monitoring assays for major plasma proteins Mol. Cell. Proteomics 5:573–588 (2006).

    Article  PubMed  CAS  Google Scholar 

  20. I. Leier, G. Jedlitschky, U. Buchholz, S. P. Cole, R. G. Deeley, D. Keppler. The MRP gene encodes an ATP-dependent export pump for leukotriene C4 and structurally related conjugates J. Biol. Chem. 269:27807–27810 (1994).

    PubMed  CAS  Google Scholar 

  21. Z. S. Chen, K. Lee, S. Walther, R. B. Raftogianis, M. Kuwano, H. Zeng, G. D. Kruh. Analysis of methotrexate and folate transport by multidrug resistance protein 4 (ABCC4): MRP4 is a component of the methotrexate efflux system Cancer Res. 62:3144–3150 (2002).

    PubMed  CAS  Google Scholar 

  22. K. Yamaoka, Y. Tanigawara, T. Nakagawa, T. Uno. A pharmacokinetic analysis program (multi) for microcomputer J. Pharmacobiodyn. 4:879–885 (1981).

    PubMed  CAS  Google Scholar 

  23. Z. S. Chen, K. Lee, G. D. Kruh. Transport of cyclic nucleotides and estradiol 17-beta-D-glucuronide by multidrug resistance protein 4. Resistance to 6-mercaptopurine and 6-thioguanine J. Biol. Chem. 276:33747–33754 (2001).

    Article  PubMed  CAS  Google Scholar 

  24. M. Hasegawa, H. Kusuhara, M. Adachi, J. D. Schuetz, K. Takeuchi, Y. Sugiyama. Multidrug resistance-associated protein 4 is involved in the urinary excretion of hydrochlorothiazide and furosemide J. Am. Soc. Nephrol. 18:37–45 (2007).

    Article  PubMed  CAS  Google Scholar 

  25. R. A. Van Aubel, P. H. Smeets, J. J. van den Heuvel, F. G. Russel. Human organic anion transporter MRP4 (ABCC4) is an efflux pump for the purine end metabolite urate with multiple allosteric substrate binding sites Am. J. Physiol. Renal Physiol. 288:F327–333 (2005).

    Article  PubMed  Google Scholar 

  26. C. F. Higgins, K. J. Linton. The ATP switch model for ABC transporters Nat. Struct. Mol. Biol. 11:918–926 (2004).

    Article  PubMed  CAS  Google Scholar 

  27. A. A. El-Sheikh, J. J. van den Heuvel, J. B. Koenderink, F. G. Russel. Interaction of nonsteroidal anti-inflammatory drugs with multidrug resistance protein (MRP) 2/ABCC2- and MRP4/ABCC4-mediated methotrexate transport J. Pharmacol. Exp. Ther. 320:229–235 (2007).

    Article  PubMed  CAS  Google Scholar 

  28. O. Fardel, E. Jigorel, M. Le Vee, L. Payen. Physiological, pharmacological and clinical features of the multidrug resistance protein 2 Biomed. Pharmacother. 59:104–114 (2005).

    Article  PubMed  CAS  Google Scholar 

  29. H. Sasabe, A. Tsuji, Y. Sugiyama. Carrier-mediated mechanism for the biliary excretion of the quinolone antibiotic grepafloxacin and its glucuronide in rats J. Pharmacol. Exp. Ther. 284:1033–1039 (1998).

    PubMed  CAS  Google Scholar 

  30. Q. Tian, J. Zhang, S. Y. Chan, T. M. Tan, W. Duan, M. Huang, Y. Z. Zhu, E. Chan, Q. Yu, Y. Q. Nie, P. C. Ho, Q. Li, K. Y. Ng, H. Y. Yang, H. Wei, J. S. Bian, S. F. Zhou. Topotecan is a substrate for multidrug resistance associated protein 4 Curr. Drug Metab. 7:105–118 (2006).

    Article  PubMed  CAS  Google Scholar 

  31. M. Z. Fan, O. Adeola, J. J. Turek, E. K. Asem. Methodological aspects of measuring amino acid uptake in studies with porcine jejunal brush border membrane vesicles Physiol. Res. 48:281–289 (1999).

    PubMed  CAS  Google Scholar 

  32. D. M. Bartholomew, D. E. Van Dyk, S. M. Lau, D. P. Keefe, P. A. Rea, P. V. Viitanen. Alternate energy-dependent pathways for the vacuolar uptake of glucose and glutathione conjugates Plant Physiol. 130:1562–1572 (2002).

    Article  PubMed  CAS  Google Scholar 

  33. J. C. Fernandez-Checa, H. Takikawa, T. Horie, M. Ookhtens, N. Kaplowitz. Canalicular transport of reduced glutathione in normal and mutant Eisai hyperbilirubinemic rats J. Biol. Chem. 267:1667–1673 (1992).

    PubMed  CAS  Google Scholar 

  34. H. Shindo, K. Kawai, T. Maeda, I. Igarashi, M. Tajima, S. Sugawara. Absorption, distribution, excretion and metabolism of a new cephamycin antibiotic, CS-1170, in various animal species Jpn. J. Chemother. 26:S-599–114 (1978).

    CAS  Google Scholar 

  35. Y. Shioya, E. Kashiyama, K. Okada, N. Kusumoto, Y. Abe, M. Uchida, T. Shimizu. Metabolic fate of the anti-ulcer agent, ( ± )-2-(4-chlorobenzoylamino)-3-[2(1H)-quinolinon-4-yl]propionic acid (OPC-12759): Absorption, distribution and excretion in rats and dogs Iyakuhin Kenkyu 20:522–533 (1989).

    CAS  Google Scholar 

  36. M. Ohkawa, M. Orito, T. Sugata, M. Shimamura, M. Sawaki, E. Nakashita, K. Kuroda. Pharmacokinetics of cefmetazole (CS-1170) in patients with normal and impaired renal function Jpn. J. Chemother. 27:78–86 (1979).

    Google Scholar 

  37. S. Kishi, N. Ogawa. Phase I clinical trial of the Anti-ulcer Agent, Proamipide (OPC-12759) J. Adult Dis. 19:355–363 (1989).

    Google Scholar 

  38. S. Ohtsuki, H. Asaba, H. Takanaga, T. Deguchi, K. Hosoya, M. Otagiri, T. Terasaki. Role of blood–brain barrier organic anion transporter 3 (OAT3) in the efflux of indoxyl sulfate, a uremic toxin: its involvement in neurotransmitter metabolite clearance from the brain J. Neurochem. 83:57–66 (2002).

    Article  PubMed  CAS  Google Scholar 

  39. A. T. Nies, G. Jedlitschky, J. Konig, C. Herold-Mende, H. H. Steiner, H. P. Schmitt, D. Keppler. Expression and immunolocalization of the multidrug resistance proteins, MRP1-MRP6 (ABCC1-ABCC6), in human brain Neuroscience 129:349–360 (2004).

    Article  PubMed  CAS  Google Scholar 

  40. Z. Hollo, L. Homolya, T. Hegedus, B. Sarkadi. Transport properties of the multidrug resistance-associated protein (MRP) in human tumour cells FEBS Lett. 383:99–104 (1996).

    Article  PubMed  CAS  Google Scholar 

  41. S. E. Schliamser, O. Cars, S. R. Norrby. Neurotoxicity of beta-lactam antibiotics: predisposing factors and pathogenesis J. Antimicrob. Chemother. 27:405–425 (1991).

    Article  PubMed  CAS  Google Scholar 

  42. R. Kikuchi, H. Kusuhara, D. Sugiyama, Y. Sugiyama. Contribution of organic anion transporter 3 (Slc22a8) to the elimination of p-aminohippuric acid and benzylpenicillin across the blood–brain barrier J. Pharmacol. Exp. Ther. 306:51–58 (2003).

    Article  PubMed  CAS  Google Scholar 

  43. M. Kuroda, H. Kusuhara, H. Endou, Y. Sugiyama. Rapid elimination of cefaclor from the cerebrospinal fluid is mediated by a benzylpenicillin-sensitive mechanism distinct from organic anion transporter 3 J. Pharmacol. Exp. Ther. 314:855–861 (2005).

    Article  PubMed  CAS  Google Scholar 

  44. H. Shen, R. F. Keep, Y. Hu, D. E. Smith. PEPT2 (Slc15a2)-mediated unidirectional transport of cefadroxil from cerebrospinal fluid into choroid plexus J. Pharmacol. Exp. Ther. 315:1101–1108 (2005).

    Article  PubMed  CAS  Google Scholar 

Download references

Acknowledgments

We thank H. Yabuuchi (GenoMembrane, Yokohama, Japan) for kindly supplying the human MRP4-expressing membrane vesicles and the control vesicles. We also thank N. Funayama for secretarial assistance. This study was supported, in part, by a Grant-in-Aid for Scientific Research on Priority Area 17081002 from The Ministry of Education, Culture, Sports, Science and Technology (MEXT), Japan, and by a 21st Century Center of Excellence (COE) Program Grant from the Japan Society for the Promotion of Science. This study was also supported, in part, by the Industrial Technology Research Grant Program from New Energy and the Industrial Technology Development Organization (NEDO) of Japan.

Author information

Authors and Affiliations

Authors

Corresponding author

Correspondence to Tetsuya Terasaki.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Uchida, Y., Kamiie, J., Ohtsuki, S. et al. Multichannel Liquid Chromatography–Tandem Mass Spectrometry Cocktail Method for Comprehensive Substrate Characterization of Multidrug Resistance-Associated Protein 4 Transporter. Pharm Res 24, 2281–2296 (2007). https://doi.org/10.1007/s11095-007-9453-7

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s11095-007-9453-7

Key words

Navigation