AbstractObjectivesVasopressin 1A (V1A) receptors have been linked to autism spectrum disorder, heart failure, diabetes and renal disease. Currently, there is a lack of validated probes for clinical V1A-targeted imaging and previous PET studies have primarily focused on the brain. To enable non-invasive real-time quantification of V1A receptors in peripheral organs, we sought to develop a suitable PET radioligand that would allow specific and selective V1A receptor imaging in vivo.MethodsThe previously reported triazolobenzodiazepine-based V1A antagonist, PF-184563, served as a structural basis for the development of a suitable V1A-targeted PET probe. Initially, PF-184563 and the respective desmethyl precursor for radiolabeling were synthesized via multistep organic synthesis. Inhibitory constants of PF-184563 for V1A, V1B, V2 and oxytocin (OT) receptors were assessed by competitive radioligand binding or fluorescent-based assays. Molecular docking of PF-184563 to the V1A receptor binding pocket was performed to corroborate the high binding affinity, while carbon-11 labeling was accomplished via radiomethylation. To assess the utility of the resulting PET radioligand, [11C]17, cell uptake studies were performed in a human V1A receptor Chinese hamster ovary (CHO) cell line under baseline and blockade conditions, using a series of V1A, V1B and V2 antagonists in >100-fold excess. Further, to show in vivo specificity, we conducted PET imaging and biodistribution experiments, thereby co-administering the clinical V1A-antagonist, balovaptan (3mg/kg), as a blocking agent.ResultsPF-184563 and the respective desmethyl precursor were synthesized in an overall yield of 49% (over 7 steps) and 40% (over 8 steps), respectively. A subnanomolar inhibitory constant (Ki) of 0.9 nM towards the V1A receptor was observed for PF-184563, while the triazolobenzodiazepine derivative concurrently exhibited excellent selectivity over the related V1B, V2 and OT receptor (IC50 >10,000 nM). [11C]17 was obtained in high radiochemical purity (> 99%), molar activities ranging from 37 - 46 GBq/μmol and a non-decay-corrected radiochemical yield of 8%. Cell uptake studies revealed considerable tracer binding, which was significantly reduced in the presence of V1A antagonists. Conversely, there was no significant blockade in the presence of V1B and V2 antagonists. PET imaging and biodistribution studies in CD-1 mice indicated specific tracer binding in the thyroid, pancreas, spleen and the heart.ConclusionWe report the development of a V1A-targeted PET radioligand that is suitable for subtype-selective in vitro and in vivo receptor imaging. Indeed, [11C]17 proved to specifically visualize V1A receptors in several organs including the heart, pancreas, spleen and thyroid. These results suggest that [11C]17 can be a valuable tool to study the role of V1A receptors in cardiovascular and immune-mediated pathologies.
Development of a Triazolobenzodiazepine-Based PET Probe for Subtype-Selective Vasopressin 1A Receptor Imaging
