Coproporphyrin I as an in vitro fluorescent probe to measure OATP1B1 transport activity.

The organic anion transporting polypeptide 1B1 (OATP1B1) is a major pharmacologically relevant hepatic uptake transporter that mediates the clearance of many drugs. The OATP1B1 substrate coproporphyrin I (CPI) has been continually shown to be a reliable and specific clinical biomarker of transport activity. These investigations, and others that have characterized OATP1B activity and transport kinetics, have largely relied on expensive, time-consuming, and nonfluorescent methods to detect CPI, such as liquid chromatography-tandem mass spectrometry. In consideration of porphyrin fluorescent properties, we hypothesized that CPI fluorescence can serve as a marker of OATP1B1 transport activity. Cellular accumulation of CPI specifically due to OATP1B1 was measured via fluorescence in the presence or absence of various Food and Drug Administration-approved tyrosine kinase inhibitors (TKIs). Our findings indicate that CPI fluorescence is an appropriate marker of OATP1B1 in vitro activity in overexpressing HEK293 cells. It was also observed that nilotinib, a TKI previously reported as an OATP1B1 inhibitor, could reduce 50% of CPI uptake at a concentration of 1.0 ± 0.5 μM. Using CPI and 8-(2-[Fluoresceinyl]aminoethylthio) adenosine- 3', 5'- cyclic monophosphate, 15 other TKIs were identified as potential OATP1B1 inhibitors, including tivozanib, which was observed to inhibit 50% of OATP1B1 activity at 4.0 ± 2.0 μM. Overall, our findings provide evidence to show that CPI fluorescence can be used as a method to assess OATP1B1-mediated transport in vitro and investigate the potential for drug-drug interactions. SIGNIFICANCE STATEMENT: This paper outlines a methodology for assessing coproporphyrin I accumulation in vitro specific to organic anion transporting polypeptide 1B1 (OATP1B1)-mediated transport. Coproporphyrin I is a reported sensitive clinical biomarker of OATP1B1 activity, and its fluorescent properties serve to provide a substrate with translational relevance in measuring drug-drug interactions in vitro with a low cost and time requirement. This method confirms nilotinib as an effective OATP1B1 inhibitor and identifies new inhibitors that can potentially promote life-threatening drug interactions using a clinically relevant biomarker.