Pharmaceutical Research

, Volume 31, Issue 1, pp 136–147 | Cite as

Investigation of Endogenous Compounds for Assessing the Drug Interactions in the Urinary Excretion Involving Multidrug and Toxin Extrusion Proteins

  • Koji Kato
  • Haruyuki Mori
  • Tomoko Kito
  • Miyu Yokochi
  • Sumito Ito
  • Katsuhisa Inoue
  • Atsushi Yonezawa
  • Toshiya Katsura
  • Yuji Kumagai
  • Hiroaki Yuasa
  • Yoshinori Moriyama
  • Ken-ichi Inui
  • Hiroyuki Kusuhara
  • Yuichi SugiyamaEmail author
Research Paper



Multidrug and toxin extrusion proteins (MATEs) are multispecific organic cation transporters mediating the efflux of various cationic drugs into the urine. The present study aimed at identifying endogenous compounds in human plasma and urine specimens as biomarkers to evaluate drug interactions involving MATEs in the kidney without administration of their exogenous probe drugs.


An untargeted metabolomic analysis was performed using urine and plasma samples from healthy volunteers and mice treated with or without the potent MATE inhibitor, pyrimethamine. Plasma and urinary concentrations of candidate markers were measured using liquid chromatography-mass spectrometry. Transport activities were determined in MATE- or OCT2-expressing HEK293 cells. The deuterium-labeled compounds of candidates were administered to mice for pharmacokinetics study.


Urinary excretion of eleven compounds including thiamine and carnitine was significantly lower in the pyrimethamine-treatment group in humans and mice, whereas no endogenous compound was noticeably accumulated in the plasma. The renal clearance of thiamine and carnitine was decreased by 70%–84% and 90%–94% (p < 0.05), respectively, in human. The specific uptake of thiamine was observed in MATE1-, MATE2-K- or OCT2-expressing HEK293 cells with Km of 3.5 ± 1.0, 3.9 ± 0.8 and 59.9 ± 6.7 μM, respectively. The renal clearance of carnitine-d 3 was decreased by 62% in mice treated with pyrimethamine.


Our findings indicate that MATEs account for the efflux of thiamine and perhaps carnitine as well as drugs into the urine. The urinary excretion of thiamine is useful to detect drug interaction involving MATEs in the kidney.


drug interaction metabolomics multidrug and toxin extrusion protein organic cation tubular secretion 



area under the plasma concentration–time curve


brush border membrane


brush border membrane vesicles


renal clearance


total plasma clearance




glomerular filtration rate


liquid chromatography-mass spectrometry


multidrug and toxin extrusion protein




organic cation transporter






urinary excretion amount



We thank K. Taguchi, N. Hagima, S. Kamigaso, and K. Iwata of the Taisho Pharmaceutical Company for their skilled and expert technical assistance.

The clinical study was conducted as the NEDO MicroDose-PJ, sponsored by the New Energy and Industrial Technology Development Organization (NEDO), Japan. This study was supported by a Grant-in-Aid for Scientific Research (S) [Grant 24229002], for Scientific Research (B) [Grant 23390034] and for Challenging Exploratory Research [24659071] from Japan Society for the Promotion of Science, Japan, and Scientific Research on Innovative Areas HD-Physiology [Grant 23136101] from the Ministry of Education, Science, and Culture of Japan. It was also supported by a Grant-in-Aid from The Nakatomi Foundation.

K. Kato and H. Mori are full-time employees of Taisho Pharmaceutical Company. The authors have no conflicts of interest that are directly relevant to this study.


  1. 1.
    Yonezawa A, Inui K. Importance of the multidrug and toxin extrusion MATE/SLC47A family to pharmacokinetics, pharmacodynamics/toxicodynamics and pharmacogenomics. Br J Pharmacol. 2011;164(7):1817–25.PubMedCrossRefGoogle Scholar
  2. 2.
    Moriyama Y, Hiasa M, Matsumoto T, Omote H. Multidrug and toxic compound extrusion (MATE)-type proteins as anchor transporters for the excretion of metabolic waste products and xenobiotics. Xenobiotica. 2008;38(7–8):1107–18.PubMedCrossRefGoogle Scholar
  3. 3.
    Tanihara Y, Masuda S, Sato T, Katsura T, Ogawa O, Inui K. Substrate specificity of MATE1 and MATE2-K, human multidrug and toxin extrusions/H(+)-organic cation antiporters. Biochem Pharmacol. 2007;74(2):359–71.PubMedCrossRefGoogle Scholar
  4. 4.
    Damme K, Nies AT, Schaeffeler E, Schwab M. Mammalian MATE (SLC47A) transport proteins: impact on efflux of endogenous substrates and xenobiotics. Drug Metab Rev. 2011;43(4):499–523.PubMedCrossRefGoogle Scholar
  5. 5.
    Tsuda M, Terada T, Mizuno T, Katsura T, Shimakura J, Inui K. Targeted disruption of the multidrug and toxin extrusion 1 (Mate1) gene in mice reduces renal secretion of metformin. Mol Pharmacol. 2009;75(6):1280–6.PubMedCrossRefGoogle Scholar
  6. 6.
    Kajiwara M, Masuda S, Watanabe S, Terada T, Katsura T, Inui K. Renal tubular secretion of varenicline by multidrug and toxin extrusion (MATE) transporters. Drug Metab Pharmacokinet. 2012;27(6):563–9.PubMedCrossRefGoogle Scholar
  7. 7.
    Nakamura T, Yonezawa A, Hashimoto S, Katsura T, Inui K. Disruption of multidrug and toxin extrusion MATE1 potentiates cisplatin-induced nephrotoxicity. Biochem Pharmacol. 2010;80(11):1762–7.PubMedCrossRefGoogle Scholar
  8. 8.
    Li Q, Peng X, Yang H, Wang H, Shu Y. Deficiency of multidrug and toxin extrusion 1 enhances renal accumulation of paraquat and deteriorates kidney injury in mice. Mol Pharm. 2011;8(6):2476–83.PubMedCentralPubMedCrossRefGoogle Scholar
  9. 9.
    Matsushima S, Maeda K, Inoue K, Ohta KY, Yuasa H, Kondo T, et al. The inhibition of human multidrug and toxin extrusion 1 is involved in the drug-drug interaction caused by cimetidine. Drug Metab Dispos. 2009;37(3):555–9.PubMedCrossRefGoogle Scholar
  10. 10.
    Tsuda M, Terada T, Ueba M, Sato T, Masuda S, Katsura T, et al. Involvement of human multidrug and toxin extrusion 1 in the drug interaction between cimetidine and metformin in renal epithelial cells. J Pharmacol Exp Ther. 2009;329(1):185–91.PubMedCrossRefGoogle Scholar
  11. 11.
    Ito S, Kusuhara H, Kuroiwa Y, Wu C, Moriyama Y, Inoue K, et al. Potent and specific inhibition of mMate1-mediated efflux of type I organic cations in the liver and kidney by pyrimethamine. J Pharmacol Exp Ther. 2010;333(1):341–50.PubMedCrossRefGoogle Scholar
  12. 12.
    Ito S, Kusuhara H, Yokochi M, Toyoshima J, Inoue K, Yuasa H, et al. Competitive inhibition of the luminal efflux by multidrug and toxin extrusions, but not basolateral uptake by organic cation transporter 2, is the likely mechanism underlying the pharmacokinetic drug-drug interactions caused by cimetidine in the kidney. J Pharmacol Exp Ther. 2012;340(2):393–403.PubMedCrossRefGoogle Scholar
  13. 13.
    Kusuhara H, Ito S, Kumagai Y, Jiang M, Shiroshita T, Moriyama Y, et al. Effects of a MATE protein inhibitor, pyrimethamine, on the renal elimination of metformin at oral microdose and at therapeutic dose in healthy subjects. Clin Pharmacol Ther. 2011;89(6):837–44.PubMedCrossRefGoogle Scholar
  14. 14.
    Ito S, Kusuhara H, Kumagai Y, Moriyama Y, Inoue K, Kondo T, et al. N-Methylnicotinamide is an endogenous probe for evaluation of drug-drug interactions involving multidrug and toxin extrusions (MATE1 and MATE2-K). Clin Pharmacol Ther. 2012;92(5):635–41.PubMedCrossRefGoogle Scholar
  15. 15.
    Wikoff WR, Nagle MA, Kouznetsova VL, Tsigelny IF, Nigam SK. Untargeted metabolomics identifies enterobiome metabolites and putative uremic toxins as substrates of organic anion transporter 1 (Oat1). J Proteome Res. 2011;10(6):2842–51.PubMedCentralPubMedCrossRefGoogle Scholar
  16. 16.
    Krumpochova P, Sapthu S, Brouwers JF, de Haas M, de Vos R, Borst P, et al. Transportomics: screening for substrates of ABC transporters in body fluids using vesicular transport assays. FASEB J. 2012;26(2):738–47.PubMedCrossRefGoogle Scholar
  17. 17.
    Kato K, Kusuhara H, Kumagai Y, Ieiri I, Mori H, Ito S, et al. Association of multidrug resistance-associated protein 2 single nucleotide polymorphism rs12762549 with the basal plasma levels of phase II metabolites of isoflavonoids in healthy Japanese individuals. Pharmacogenet Genomics. 2012;22(5):344–54.PubMedGoogle Scholar
  18. 18.
    Pluskal T, Castillo S, Villar-Briones A, Oresic M. MZmine 2: modular framework for processing, visualizing, and analyzing mass spectrometry-based molecular profile data. BMC Bioinforma. 2010;11:395.CrossRefGoogle Scholar
  19. 19.
    Wishart DS, Knox C, Guo AC, Eisner R, Young N, Gautam B, et al. HMDB: a knowledgebase for the human metabolome. Nucleic Acids Res. 2009;D603-10.Google Scholar
  20. 20.
    Kanehisa M, Goto S. KEGG: kyoto encyclopedia of genes and genomes. Nucleic Acids Res. 2000;28(1):27–30.PubMedCentralPubMedCrossRefGoogle Scholar
  21. 21.
    Busch AE, Karbach U, Miska D, Gorboulev V, Akhoundova A, Volk C, et al. Human neurons express the polyspecific cation transporter hOCT2, which translocates monoamine neurotransmitters, amantadine, and memantine. Mol Pharmacol. 1998;54(2):342–52.PubMedGoogle Scholar
  22. 22.
    Hirano M, Maeda K, Shitara Y, Sugiyama Y. Contribution of OATP2 (OATP1B1) and OATP8 (OATP1B3) to the hepatic uptake of pitavastatin in humans. J Pharmacol Exp Ther. 2004;311(1):139–46.PubMedCrossRefGoogle Scholar
  23. 23.
    Lowry OH, Rosebrough NJ, Farr AL, Randall RJ. Protein measurement with the Folin phenol reagent. J Biol Chem. 1951;193(1):265–75.PubMedGoogle Scholar
  24. 24.
    Yamaoka K, Tanigawara Y, Nakagawa T, Uno T. A pharmacokinetic analysis program (multi) for microcomputer. J Pharmacobiodyn. 1981;4(11):879–85.PubMedCrossRefGoogle Scholar
  25. 25.
    US Food and Drug Administration. Guidance for Industry: drug interaction studies—study design, data analysis, implications for dosing, and labeling recommendations (Draft guidance) ( (14 September 2012).
  26. 26.
    Finglas PM. Thiamin. Int J Vitam Nutr Res. 1993;63(4):270–4.PubMedGoogle Scholar
  27. 27.
    Steiber A, Kerner J, Hoppel CL. Carnitine: a nutritional, biosynthetic, and functional perspective. Mol Aspects Med. 2004;25(5–6):455–73.PubMedCrossRefGoogle Scholar
  28. 28.
    Moyer JD, Malinowski N, Ayers O. Salvage of circulating pyrimidine nucleosides by tissues of the mouse. J Biol Chem. 1985;260(5):2812–8.PubMedGoogle Scholar
  29. 29.
    Lynch PL, Young IS. Determination of thiamine by high-performance liquid chromatography. J Chromatogr A. 2000;881(1–2):267–84.PubMedCrossRefGoogle Scholar
  30. 30.
    Bain MA, Milne RW, Evans AM. Disposition and metabolite kinetics of oral L-carnitine in humans. J Clin Pharmacol. 2006;46(10):1163–70.PubMedCrossRefGoogle Scholar
  31. 31.
    Cao Y, Wang YX, Liu CJ, Wang LX, Han ZW, Wang CB. Comparison of pharmacokinetics of L-carnitine, acetyl-L-carnitine and propionyl-L-carnitine after single oral administration of L-carnitine in healthy volunteers. Clin Invest Med. 2009;32(1):E13–9.PubMedGoogle Scholar
  32. 32.
    Hale JT, Bigelow JC, Mathews LA, McCormack JJ. Analytical and pharmacokinetic studies with 5-chloro-2′-deoxycytidine. Biochem Pharmacol. 2002;64(10):1493–502.PubMedCrossRefGoogle Scholar
  33. 33.
    Gastaldi G, Cova E, Verri A, Laforenza U, Faelli A, Rindi G. Transport of thiamin in rat renal brush border membrane vesicles. Kidney Int. 2000;57(5):2043–54.PubMedCrossRefGoogle Scholar
  34. 34.
    Watanabe S, Tsuda M, Terada T, Katsura T, Inui K. Reduced renal clearance of a zwitterionic substrate cephalexin in MATE1-deficient mice. J Pharmacol Exp Ther. 2010;334(2):651–6.PubMedCrossRefGoogle Scholar
  35. 35.
    Ashokkumar B, Vaziri ND, Said HM. Thiamin uptake by the human-derived renal epithelial (HEK-293) cells: cellular and molecular mechanisms. Am J Physiol Renal Physiol. 2006;291(4):F796–805.PubMedCrossRefGoogle Scholar
  36. 36.
    Nezu J, Tamai I, Oku A, Ohashi R, Yabuuchi H, Hashimoto N, et al. Primary systemic carnitine deficiency is caused by mutations in a gene encoding sodium ion-dependent carnitine transporter. Nat Genet. 1999;21(1):91–4.PubMedCrossRefGoogle Scholar
  37. 37.
    Elwi AN, Damaraju VL, Baldwin SA, Young JD, Sawyer MB, Cass CE. Renal nucleoside transporters: physiological and clinical implications. Biochem Cell Biol. 2006;84(6):844–58.PubMedCrossRefGoogle Scholar
  38. 38.
    Weber W, Nitz M, Looby M. Nonlinear kinetics of the thiamine cation in humans: saturation of nonrenal clearance and tubular reabsorption. J Pharmacokinet Biopharm. 1990;18(6):501–23.PubMedCrossRefGoogle Scholar
  39. 39.
    Dutta B, Huang W, Molero M, Kekuda R, Leibach FH, Devoe LD, et al. Cloning of the human thiamine transporter, a member of the folate transporter family. J Biol Chem. 1999;274(45):31925–9.PubMedCrossRefGoogle Scholar
  40. 40.
    Evans AM, Fornasini G. Pharmacokinetics of L-carnitine. Clin Pharmacokinet. 2003;42(11):941–67.PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2013

Authors and Affiliations

  • Koji Kato
    • 1
  • Haruyuki Mori
    • 1
  • Tomoko Kito
    • 2
  • Miyu Yokochi
    • 2
  • Sumito Ito
    • 2
  • Katsuhisa Inoue
    • 3
  • Atsushi Yonezawa
    • 4
  • Toshiya Katsura
    • 4
  • Yuji Kumagai
    • 5
  • Hiroaki Yuasa
    • 3
  • Yoshinori Moriyama
    • 6
  • Ken-ichi Inui
    • 7
  • Hiroyuki Kusuhara
    • 2
  • Yuichi Sugiyama
    • 2
    • 8
    Email author
  1. 1.Drug Safety and Pharmacokinetics LaboratoriesTaisho Pharmaceutical Co. Ltd.SaitamaJapan
  2. 2.Laboratory of Molecular Pharmacokinetics Graduate School of Pharmaceutical SciencesThe University of TokyoTokyoJapan
  3. 3.Department of Biopharmaceutics Graduate School of Pharmaceutical SciencesNagoya City UniversityNagoyaJapan
  4. 4.Department of PharmacyKyoto University HospitalKyotoJapan
  5. 5.Clinical Trial CenterKitasato University East HospitalKanagawaJapan
  6. 6.Department of Membrane BiochemistryOkayama University Graduate School of Medicine, Dentistry, & Pharmaceutical SciencesOkayamaJapan
  7. 7.Kyoto Pharmaceutical UniversityKyotoJapan
  8. 8.Sugiyama Laboratory, RIKEN Innovation Center Research Cluster for InnovationRIKENYokohamaJapan

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