Species-Specific Hepatic Uptake of [⁶⁴Cu]Cu-EOB-NOTA, A Newly Designed Hepatospecific PET Agent

Measuring hepatic flux rates of transportable substrates has the potential for assessing liver function. PET imaging of a PET-enabled substrate may provide a more straightforward measurement of time-dependent substrate concentration through the liver than MRI using an MRI contrast agent. Here we synthesized and evaluated the hepatobiliary transport of a new hepatospecific PET agent designed for stable Cu²⁺ chelation and transport by hepatic OATPs, [⁶⁴Cu]Cu-EOB-NOTA.

EOB-NOTA was synthesized, its two enantiomers separated by chiral HPLC, and individually radiolabeled with [⁶⁴Cu]Cu²⁺. Cocktails of each enantiomer of [⁶⁴Cu]Cu-EOB-NOTA and Gd-EOB-DTPA were formulated for simultaneous PET/MRI imaging of hepatic flux by PET and MRI. Two mouse models were evaluated: wild-type mice and mice expressing only human hepatic OATPs.

In wild-type mice, [⁶⁴Cu]Cu-EOB-NOTA hepatic influx and efflux was high, but slower compared to Gd-EOB-DTPA. Neither enantiomer of [⁶⁴Cu]Cu-EOB-NOTA exhibited detectable transport into the liver in mice expressing human OATPs. This was validated by waste clearance studies and in vitro uptake assays in cells engineered to express rodent and human OATPs.

[⁶⁴Cu]Cu-EOB-NOTA exhibited no detectable hepatic uptake by transgenic mice expressing human hepatic transporters. This finding was surprising given the efficient transport of the structurally similar metal chelate Gd-EOB-DTPA, and underscores challenges in the design of imaging molecular probes, including poor predictability for hepatic transport, and the value of validating new agents in mice expressing human hepatic transporters.