Fluorescence-based assays for the assessment of drug interaction with the human transporters 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)
- et al.
Fluorometric screening for metabolism-based drug–drug interactions
J. Pharmacol. Toxicol. Methods
(2000) - et al.
Fluorescence-based assay for the interaction of small molecules with the human renal organic anion transporter
Anal. Biochem.
(2000) - et al.
Development of a fluorescence-based assay for screening of modulators of human organic anion transporter 1B3 (OATP1B3)
Eur. J. Pharm. Biopharm.
(2006) - et al.
Transport of fluorescent chenodeoxycholic acid via the human organic anion transporters OATP1B1 and OATP1B3
J. Lipid Res.
(2006) - et al.
Role of basolateral efflux transporter MRP4 in the intestinal absorption of the antiviral drug adefovir dipivoxil
Biochem. Pharmacol.
(2010) - et al.
A simple statistical parameter for use in evaluation and validation of high throughput screening assays
J. Biomol. Screen.
(1999) - et al.
Organic anion-transporting polypeptide B (OATP-B) and its functional comparison with three other OATPs of human liver
Gastroenterology
(2001) - et al.
Effect of pregnane X receptor ligands on transport mediated by human OATP1B1 and OATP1B3
Eur. J. Pharmacol.
(2008) - et al.
Interactions of rifamycin SV and rifampicin with organic anion uptake systems of human liver
Hepatology
(2002) - et al.
Identification of a novel gene family encoding human liver-specific organic anion transporter LST-1
J. Biol. Chem.
(1999)
Transporters and drug therapy: implications for drug disposition and disease
Clin. Pharmacol. Ther.
Can the pharmaceutical industry reduce attrition rates?
Nat. Rev. Drug Discov.
Membrane transporters in drug development
Nat. Rev. Drug. Discov.
Structural requirements for drug inhibition of the liver specific human organic cation transport protein
J. Med. Chem.
NBD–TMA: a novel fluorescent substrate of the peritubular organic cation transporter of renal proximal tubules
Pflugers Arch.
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.
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2020, European Journal of Pharmaceutical SciencesCitation 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.