Skip to main content

Investigation of Fluorescent Substrates and Substrate-Dependent Interactions of a Drug Transporter Organic Anion Transporting Polypeptide 2B1 (OATP2B1)



In this study, we investigated organic anion transporting polypeptide 2B1 (OATP2B1)-mediated uptake of fluorescent anions to better identify fluorescent substrates for in vitro OATP2B1 assays. The OATP2B1 is involved in the intestinal absorption and one of the pharmacokinetic determinants of orally administered drugs.


A microplate reader was used to determine the cellular accumulation of the fluorescent compounds into the OATP2B1 or the empty vector-transfected HEK293 cells.


Two types of derivatives were found to be OATP2B1 substrates: heavy halogenated derivatives, such as 4′,5′-dibromofluorescein (DBF), and carboxylated derivatives, such as 5-carboxyfluorescein (5-CF). The DBF and 5-CF were transported in a time and concentration-dependent manner. The DBF was transported at a broad pH (pH 6.5–8.0) while 5-CF was transported at an acidic pH (pH 5.5–6.5). The Km values were 0.818 ± 0.067 μM at pH 7.4 for DBF and 8.56 ± 0.41 μM at pH 5.5 for 5-CF. The OATP2B1 inhibitors, including atorvastatin, bromosulfophthalein, glibenclamide, sulfasalazine, talinolol, and estrone 3-sulfate, inhibited the DBF and the 5-CF transport. Contrastively, testosterone, dehydroepiandrosterone sulfate, and progesterone inhibited the DBF transport but stimulated the 5-CF transport. Natural flavonoid aglycones, such as naringenin and baicalein, also exhibited substrate-dependent effects in this manner.


We found two fluorescein analogs, DBF and 5-CF as the OATP2B1 substrates that exhibited substrate-dependent interactions.

This is a preview of subscription content, access via your institution.

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6
Fig. 7
Fig. 8









Area under the curve










Estrone 3-sulfate








Drug-drug interaction




Dehydroepiandrosterone sulfate


Eosin Y


Food-drug interaction




Oregon Green


Organic anion transporting polypeptide




Rose bengal








  1. Hagenbuch B, Meier PJ. Organic anion transporting polypeptides of the OATP/SLC21 family: phylogenetic classification as OATP/SLCO super-family, new nomenclature and molecular/functional properties. Pflugers Arch Eur J Physiol. 2004;447:653–65.

    Article  CAS  Google Scholar 

  2. Obaidat A, Roth M, Hagenbuch B. The expression and function of organic anion transporting polypeptides in normal tissues and in cancer. Annu Rev Pharmacol Toxicol. 2012;52:135–51.

    Article  CAS  Google Scholar 

  3. Tamai I. Oral drug delivery utilizing intestinal OATP transporters. Adv Drug Deliv Rev. 2012;64:508–14.

    Article  CAS  Google Scholar 

  4. Schulte RR, Ho RH. Organic anion transporting polypeptides: emerging roles in cancer pharmacology. Mol Pharmacol. 2019;95:490–506.

    Article  CAS  Google Scholar 

  5. McFeely SJ, Wu L, Ritchie TK, Unadkat J. Organic anion transporting polypeptide 2B1 – more than a glass-full of drug interactions. Pharmacol Ther. 2019;196:204–15.

    Article  CAS  Google Scholar 

  6. Yu J, Zhou Z, Tay-Sontheimer J, Levy RH, Ragueneau-Majlessi I. Intestinal drug interactions mediated by OATPs: a systematic review of preclinical and clinical findings. J Pharm Sci. 2017;106:2312–25.

    Article  CAS  Google Scholar 

  7. Luo J, Imai H, Ohyama T, Hashimoto S, Hasunuma T, Inoue Y, et al. The pharmacokinetic exposure to fexofenadine is volume-dependently reduced in healthy subjects following Oral administration with apple juice. Clin Transl Sci. 2016;9:201–6.

    Article  CAS  Google Scholar 

  8. Tapaninen T, Neuvonen PJ, Niemi M. Grapefruit juice greatly reduces the plasma concentrations of the OATP2B1 and CYP3A4 substrate aliskiren. Clin Pharmacol Ther. 2010;88:339–42.

    Article  CAS  Google Scholar 

  9. Kashihara Y, Ieiri I, Yoshikado T, Maeda K, Fukae M, Kimura M, et al. Small-dosing clinical study: pharmacokinetic, Pharmacogenomic (SLCO2B1 and ABCG2), and interaction (atorvastatin and grapefruit juice) profiles of 5 probes for OATP2B1 and BCRP. J Pharm Sci. 2017;106:2688–94.

    Article  CAS  Google Scholar 

  10. Shirasaka Y, Shichiri M, Mori T, Nakanishi T, Tamai I. Major active components in grapefruit, orange, and apple juices responsible for OATP2B1-mediated drug interactions. J Pharm Sci. 2013;102:3418–26.

    Article  CAS  Google Scholar 

  11. Fujita D, Saito Y, Nakanishi T, Tamai I. Organic anion transporting polypeptide (OATP) 2B1 contributes to gastrointestinal toxicity of anticancer drug SN-38, active metabolite of irinotecan hydrochloride. Drug Metab Dispos. 2016;44:1–7.

    Article  CAS  Google Scholar 

  12. Takasuna K, Hagiwara T, Watanabe K, Onose S, Yoshida S, Kumazawa E, et al. Optimal antidiarrhea treatment for antitumor agent irinotecan hydrochloride (CPT-11)-induced delayed diarrhea. Cancer Chemother Pharmacol. 2006;58:494–503.

    Article  CAS  Google Scholar 

  13. Ichiki M, Wataya H, Yamada K, Tsuruta N, Takeoka H, Okayama Y, et al. Preventive effect of kampo medicine (hangeshashin-to, TJ-14) plus minocycline against afatinib-induced diarrhea and skin rash in patients with non-small cell lung cancer. Onco Targets Ther. 2017;10:5107–13.

    Article  Google Scholar 

  14. Kawasaki T, Takeichi Y, Tomita M, Uwai Y, Epifano F, Fiorito S, et al. Effects of phenylpropanoids on human organic anion transporters hOAT1 and hOAT3. Biochem Biophys Res Commun. 2017;489:375–80.

    Article  CAS  Google Scholar 

  15. Uwai Y, Kawasaki T, Nabekura T. d-malate decreases renal content of α-ketoglutarate, a driving force of organic anion transporters OAT1 and OAT3, resulting in inhibited tubular secretion of phenolsulfonphthalein, in rats. Biopharm Drug Dispos. 2017;38:479–85.

    Article  CAS  Google Scholar 

  16. Volpe DA, Hamed SS, Zhang LK. Use of different parameters and equations for calculation of IC50 values in efflux assays: potential sources of variability in IC50 determination. AAPS J. 2014;16:172–80.

    Article  CAS  Google Scholar 

  17. Shirasaka Y, Mori T, Shichiri M, Nakanishi T, Tamai I. Functional Pleiotropy of organic anion transporting polypeptide OATP2B1 due to multiple binding sites. Drug Metab Pharmacokinet. 2012;27:360–4.

    Article  CAS  Google Scholar 

  18. Schäfer AM, Bock T, Meyer zu Schwabedissen HE. Establishment and validation of competitive Counterflow as a method to detect substrates of the organic anion transporting polypeptide 2B1. Mol Pharm 2018;15:5501–5513.

  19. Kalliokoski A, Niemi M. Impact of OATP transporters on pharmacokinetics. Br J Pharmacol. 2009;158:693–705.

    Article  CAS  Google Scholar 

  20. U.S. Food and Drug Administration. In Vitro Drug Interaction Studies — Cytochrome P450 Enzyme- and Transporter-Mediated Drug Interactions Guidance for Industry. 2020; Available from

  21. Izumi S, Nozaki Y, Komori T, Takenaka O, Maeda K, Kusuhara H, et al. Investigation of fluorescein derivatives as substrates of organic anion transporting polypeptide (OATP) 1B1 to develop sensitive fluorescence-based OATP1B1 inhibition assays. Mol Pharm. 2016;13:438–48.

    Article  CAS  Google Scholar 

  22. Kullak-Ublick G, Ismair MG, Stieger B, Landmann L, Huber R, Pizzagalli F, et al. Organic anion-transporting polypeptide B (OATP-B) and its functional comparison with three other OATPs of human liver. Gastroenterology. 2001;120:525–33.

    Article  CAS  Google Scholar 

  23. Shirasaka Y, Mori T, Murata Y, Nakanishi T, Tamai I. Substrate- and dose-dependent drug interactions with grapefruit juice caused by multiple binding sites on OATP2B1. Pharm Res. 2014;31:2035–43.

    Article  CAS  Google Scholar 

  24. Grube M, Köck K, Karner S, Reuther S, Ritter C a, Jedlitschky G, et al. Modification of OATP2B1-mediated transport by steroid hormones. Mol Pharmacol. 2006;70:1735–41.

  25. Hoshino Y, Fujita D, Nakanishi T, Tamai I. Molecular localization and characterization of multiple binding sites of organic anion transporting polypeptide 2B1 (OATP2B1) as the mechanism for substrate and modulator dependent drug–drug interaction. Med Chem Commun. 2016;7:1775–82.

    Article  CAS  Google Scholar 

  26. Ojelabi OA, Lloyd KP, De Zutter JK, Carruthers A. Red wine and green tea flavonoids are cis-allosteric activators and competitive inhibitors of glucose transporter 1 (GLUT1)-mediated sugar uptake. J Biol Chem. 2018;293:19823–34.

    Article  CAS  Google Scholar 

  27. Wang X, Wolkoff AW, Morris ME. Flavonoids as a novel class of human organic anion-transporting polypeptide OATP1B1 (OATP-C) modulators. Drug Metab Dispos. 2005;33:1666–72.

    Article  CAS  Google Scholar 

  28. Kawasaki T, Ito H, Omote H. Components of foods inhibit a drug exporter, human multidrug and toxin extrusion transporter 1. Biol Pharm Bull. 2014;37:292–7.

    Article  CAS  Google Scholar 

  29. Kitagawa S, Nabekura T, Takahashi T, Nakamura Y, Sakamoto H, Tano H, et al. Structure–activity relationships of the inhibitory effects of flavonoids on P-glycoprotein-mediated transport in KB-C2 cells. Biol Pharm Bull. 2005;28:2274–8.

    Article  CAS  Google Scholar 

  30. Ogura J, Koizumi T, Segawa M, Yabe K, Kuwayama K, Sasaki S, et al. Quercetin-3-rhamnoglucoside (rutin) stimulates transport of organic anion compounds mediated by organic anion transporting polypeptide 2B1. Biopharm Drug Dispos. 2014;35:173–82.

    Article  CAS  Google Scholar 

Download references

Author information

Authors and Affiliations


Corresponding author

Correspondence to Tomohiro Nabekura.

Ethics declarations

Conflict of Interest

The authors state that they have no conflicts of interest. This article does not contain any studies with human participants or animals performed by any of the authors.

Additional information

Publisher’s Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Electronic supplementary material


(PDF 123 kb)

Rights and permissions

Reprints and Permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Kawasaki, T., Shiozaki, Y., Nomura, N. et al. Investigation of Fluorescent Substrates and Substrate-Dependent Interactions of a Drug Transporter Organic Anion Transporting Polypeptide 2B1 (OATP2B1). Pharm Res 37, 115 (2020).

Download citation

  • Received:

  • Accepted:

  • Published:

  • DOI:

Key Words