Molecular Imaging-Derived Biomarker of Cardiac Nerve Integrity — Introducing High NET Affinity PET Probe ¹⁸F-AF78

Abstract

Background: Radiolabeled agents that are substrates for the norepinephrine transporter (NET) can be used to quantify cardiac sympathetic nervous conditions and have been demonstrated to identify high-risk congestive heart failure (HF) patients prone to arrhythmic events. We aimed to fully characterize the kinetic profile of the novel 18 F-labeled NET probe AF78 for PET imaging of the cardiac sympathetic nervous system (SNS) among various species. Methods: 18 F-AF78 was compared to norepinephrine (NE) and established SNS radiotracers by employing in vitro cell assays, followed by an in vivo PET imaging approach with healthy rats, rabbits and nonhuman primates (NHPs). Additionally, chase protocols were performed in NHPs with NET inhibitor desipramine (DMI) and the NE releasing stimulator tyramine (TYR) to investigate retention kinetics in cardiac SNS. Results: Relative to other SNS radiotracers, 18 F-AF78 showed higher transport affinity via NET in a cell-based competitive uptake assay (IC 50 0.42 ± 0.14 µM), almost identical to that of NE (IC 50 , 0.50 ± 0.16 µM, n.s.). In rabbits and NHPs, initial cardiac uptake was significantly reduced by NET inhibition. Furthermore, cardiac tracer retention was not affected by a DMI chase protocol but was markedly reduced by intermittent TYR chase, thereby suggesting that 18 F-AF78 is stored and can be released via the synaptic vesicular turnover process. Computational modeling hypothesized the formation of a T-shaped π-π stacking at the binding site, suggesting a rationale for the high affinity of 18 F-AF78. Conclusion: 18 F-AF78 demonstrated high in vitro NET affinity and advantageous in vivo radiotracer kinetics across various species, indicating that 18 F-AF78 is an SNS imaging agent with strong potential to guide specific interventions in cardiovascular medicine.

Rationalizing the Binding Modes of PET Radiotracers Targeting the Norepinephrine Transporter

“…PET/computed tomography (CT) imaging with [ 18 F]AF78(F) was performed using a small animal PET imaging protocol, applied as described previously [ 17 , 18 ]. Systemic distribution of the radiotracer in healthy rats showed homogenous distribution throughout the left ventricular wall.…”

“…The precursors for radiolabeling (with a leaving group to allow for displacement by fluorine-18) with different protecting groups on the guanidine moiety ( N,N′,N″ -tri-Boc protected or non-protected guanidine) as well as different leaving groups (tosylate or brosylate) were used in the radiolabeling procedure for the biodistribution and imaging studies. Radiolabeling was performed analogously according to a previously described protocol [ 17 , 18 ] with variations in terms of a different base and concentration of acid used in deprotection. Radiochemical yield (RCY) is calculated from the isolated final product after HPLC and cartridge purification deducted by radioactivity loaded onto the QMA cartridge with decay correction.…”

“…Evaluation of all synthesized and reference compounds against the uptake of [ 3 H]NE was performed in the human neuroblastoma cell line SK-N-SH stably expressing NET according to a previously described protocol using the NET selective inhibitor desipramine as a control and NE and MIBG as standard references [ 18 ]. A detailed experimental description can be found in the Supplementary Material .…”

“…Preliminary PET imaging of [ 18 F]AF78(F) in rodents (n = 2) and NHPs (n = 2) was obtained according to a previously described protocol [ 18 ]. For the rat biodistribution studies (n = 3–6 for each tracer), radiotracers (1–2 MBq) were administered via the tail vein.…”

“…On the other hand, several drawbacks, including blood flow dependency, low heart-to-liver ratios or challenging radiolabeling protocols with poor labeling yields, have indicated room for improvement. To counteract some of these drawbacks, we recently reported a new NET-targeting radiotracer, AF78, based on a phenethylguanidine core structure with efficient one-step labeling, which also demonstrated high NET affinity and advantageous in vivo kinetics across various species [ 17 , 18 ]. Nevertheless, from a structural point of view, little insight has been obtained for further optimization, even after information has been gathered on the certain similarities and differences between AF78 and other radiotracers ( Figure 1 ).…”