Previous studies have investigated the kinetics and affinities of norepinephrine transporter (NET)-targeting radiotracers, including [ ¹²³ I]MIBG, but the role of organic cation transporters (OCTs) remains unclear. This study aimed to evaluate how the structural design of selective NET-targeting tracers affects OCT-mediated non-specific uptake, identifying factors influencing both NET and OCT affinity.

Cellular uptake assays were conducted using SK-N-SH cells expressing human NET, and human OCT1-, OCT2-, and OCT3-expressing cells with [ ³ H]norepinephrine, [ ³ H]MPP ⁺ , and [ ¹³¹ I]MIBG. Competitive uptake assays used non-radioactive reference compounds for several NET-targeting radiopharmaceuticals (MIBG, HED, EPI, PHEN, LMI1195, and PHPG), along with a new PET radiotracer [ ¹⁸ F]AF78, and its two analogs with meta -iodide [ ¹⁸ F]AF78(I) or hydroxyl group [ ¹⁸ F]AF78(OH). Dynamic PET imaging in non-human primates assessed the in vivo uptake of [ ¹⁸ F]AF78 after NET inhibition with desipramine.

Monoamine-based tracers (EPI, PHEN, HED) exhibited high NET selectivity with minimal OCTs interaction, while guanidine-containing tracers (e.g., MIBG, LMI1195) displayed substantial OCTs affinity. Lower lipophilicity in guanidine-containing compounds, influenced by substitutions on the benzene ring (e.g., PHPG, AF78), correlated with weaker OCT interactions. PET imaging confirmed that cardiac uptake of [ ¹⁸ F]AF78 is sensitive to desipramine pretreatment (*** P   <  0.0005), indicating its NET-specificity, while persistent hepatic retention suggests an OCT-mediated transport mechanism.

This study highlights the critical influence of the compounds’ chemical structure on NET and OCT affinities. Structural modifications that reduce OCT-mediated uptake while maintaining high NET affinity could improve the specificity and theranostic potential of NET-targeting ligands. These findings provide insights for designing next-generation radiotracers with enhanced selectivity and clinical utility.