Abstract
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Introduction: The hepatic flux rates of imageable, transportable substrates can potentially be used to measure liver function. Quantifying the time-dependent concentration of substrate directly from PET images may be more straightforward than using MRI. Thus, the purpose of this work was to synthesize and evaluate the hepatobiliary organic anion transport polypeptides (OATPs) transport of a new PET agent, 64Cu-EOB-NOTA (64Cu-6), in wild-type mice and in mice exclusively expressing human hepatic transporters.
Methods: EOB-NOTA (6) was synthesized through multiple-step synthesis and bis-protected NOTA was used to ensure mono-substitution, and enantiomers 6a and 6b were separated by Chiral HPLC. 6a and 6b were separately radiolabeled with 64Cu to form 64Cu-6a/64Cu-6b in high yield. We validated the hepatobiliary transport of each enantiomer using simultaneous dynamic PET/DCE-MRI in wild-type mice and in OATP1B1/1B3 knock-in mice following intravenous injection of a cocktail containing 64Cu-EOB-NOTA and Gd-EOB-DTPA. Mice were then housed in metabolic cages to collect urine and feces for radio-analysis of clearance pathways. In vitro uptake experiments of 64Cu-EOB-NOTA were performed in rat OATP1B2 and human OATP1B3-expressing HEK293 cells.
Results: EOB-NOTA was synthesized as enantiomeric pure compounds, and 6a and 6b were successfully labeled with 64Cu (Fig. 1). In wild-type mice, peak hepatic uptake of 64Cu-EOB-NOTA as measured by PET occurred at ~20 minutes, with slow washout over an hour, with some disparity between the enantiomers. (Fig. 2) This influx and efflux were considerably slower than that of Gd-EOB-DTPA measured by MRI. In mice expressing human OATP1B1/1B3 in the liver, no significant hepatic uptake of either enantiomer of 64Cu-EOB-NOTA was measured by PET, while hepatic uptake of Gd-EOB-DTPA was measured by MRI. Biowastes analysis verified mixed hepatobiliary and renal clearance in wild type mice 64Cu-EOB-NOTA but mostly renal clearance in humanized mice. In vitro cellular experiments measured significantly higher uptake of 64Cu-EOB-NOTA in OATP1B2 cells versus OATP1B3 expressing cells.
Conclusions: 64Cu-EOB-NOTA was transported by native mouse liver following IV injection and in culture by cells expressing rodent hepatic OATPs, but not by transgenic mice or cells expressing human hepatic transporters. This finding was surprising given the efficient transport of a structurally related metal chelate Gd-EOB-DTPA by human hepatic transporters. Alternative approaches to design PET radiotracer for imaging human OATPs are ongoing.
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