Elsevier

Analytical Biochemistry

Volume 405, Issue 1, 1 October 2010, Pages 50-58
Analytical Biochemistry

Fluorescence-based assays for the assessment of drug interaction with the human transporters OATP1B1 and OATP1B3

https://doi.org/10.1016/j.ab.2010.06.012Get rights and content

Abstract

Hepatic disposition plays a significant role in the pharmacokinetics and pharmacodynamics of a variety of drugs. Sinusoidal membrane transporters have been shown to participate in the hepatic disposition of many pharmaceuticals. Two sinusoidal membrane transporters with an established role in hepatic disposition are OATP1B1 and OATP1B3 (organic anion-transporting polypeptides 1B1 and 1B3, respectively). OATP1B1 and OATP1B3 have been implicated in the hepatic uptake of statin drugs, and polymorphisms linked to OATP1B1 have been associated with deleterious patient endpoints. As a result, OATP1B1 and OATP1B3 represent sites for potential drug–drug interactions. Numerous methods exist for identifying potential drug–drug interactions with transporters. However, relatively few offer the convenience and speed of fluorescence-based assays. Here a fluorescence-based assay was developed for measuring the OATP1B1- and OATP1B3-mediated transport of 8-fluorescein-cAMP (8-FcA). The OATP1B1- and OATP1B3-mediated transport of 8-FcA was time dependent and saturable (Km = 2.9 and 1.8 μM, Vmax = 0.20 and 0.33 pmol/min/cm2, respectively). Molecules known to interact with OATPs, including cyclosporin A, rifampicin, and glibenclamide, each demonstrated concentration-dependent inhibition of 8-FcA transport by OATP1B1 and OATP1B3. The in vitro fluorescence-based assays described here using 8-FcA as the substrate are convenient and rapid and have utility in screening drug candidates for potential drug–drug interactions with OATP1B1 and OATP1B3.

Section snippets

Materials

8-FcA was purchased from Biolog Life Science Institute (Bremen, Germany) via its American distributor Axxora (San Diego, CA, USA). Technical information for 8-FcA is available from the manufacturer. All other chemicals were purchased from Sigma–Aldrich (St. Louis, MO, USA). Hank’s balanced salt solution (HBSS) and cell culture reagents were purchased from Mediatech (Herndon, VA, USA). The radiolabeled compounds [3H]p-aminohippurate and [3H]taurocholate were purchased from American Radiolabeled

Transporter specificity of 8-FcA accumulation

To assess the specificity of 8-FcA as a substrate for commonly investigated drug transporters, CHO cells were transiently transfected with a vector control, NTCP, OAT1, OAT3, OATP1B1, OATP1B3, OATP2B1, OCT1, or OCT2, and subsequently exposed to 10 μM 8-FcA for 20 min. A relatively high concentration of 8-FcA and a long exposure time were selected to provide sufficient opportunity for the dye to accumulate in cells even if poorly transported. CHO cells transiently transfected with OATP1B1 and

Discussion

Provided the importance of OATP1B1 and OATP1B3 in the hepatic disposition of drugs, it is essential to have in vitro assays to assess potential drug–drug interactions early in drug development. Fluorescence-based assays offer a straightforward, efficient, and rapid means for evaluating inhibition of transport and, correspondingly, the potential for drug–drug interactions.

The background cellular accumulation of 8-FcA in CHO and CHOvector cells is negligible, consistent with its physicochemical

Acknowledgments

This work was supported by a National Institutes of Health Small Business Innovation Research Grant (R43GM086970). The author thanks Stephen H. Wright (University of Arizona) for his critical review of the manuscript.

References (33)

  • R.H. Ho et al.

    Transporters and drug therapy: implications for drug disposition and disease

    Clin. Pharmacol. Ther.

    (2005)
  • I. Kola et al.

    Can the pharmaceutical industry reduce attrition rates?

    Nat. Rev. Drug Discov.

    (2004)
  • K.M. Giacomini et al.

    Membrane transporters in drug development

    Nat. Rev. Drug. Discov.

    (2010)
  • G. Ahlin et al.

    Structural requirements for drug inhibition of the liver specific human organic cation transport protein

    J. Med. Chem.

    (2008)
  • D. Bednarczyk et al.

    NBD–TMA: a novel fluorescent substrate of the peritubular organic cation transporter of renal proximal tubules

    Pflugers Arch.

    (2000)
  • R.A. Bhasker et al.

    Synthesis and fluorescence of N,N,N-trimethyl-2-[methyl(7-nitrobenzo[c][l,2,5]oxadiazol-4-yl)amino]ethanaminium iodide, a pH-insensitive reporter of organic cation transport

    Synthetic Commun.

    (2006)
  • Cited by (49)

    • Drug Transport—Uptake

      2022, Comprehensive Pharmacology
    • Fluorescent probes for the dual investigation of MRP2 and OATP1B1 function and drug interactions

      2020, European Journal of Pharmaceutical Sciences
      Citation Excerpt :

      Similarly, fluorescent dye substrates of ABCG2 (Hoechst, DyeCycle Violet) have long been used to investigate its function or drug interactions (Mathew et al., 2009; Ozvegy et al., 2002). Whereas the study of the efflux function of ABC transporters requires the use of dyes that can accumulate in the cells (high passive uptake) (Szakacs et al., 2008), ideal test substrates measuring OATP function are cell impermeable (Bednarczyk, 2010; Gui et al., 2010; Kovacsics et al., 2017; Patik et al., 2018; Yamaguchi et al., 2006). To date, fluorescent probes allowing the simultaneous investigation of OATP1B1 and the hepatic ABC transporters have not been identified.

    View all citing articles on Scopus
    View full text