Comparison of 4 Radiolabeled Antagonists for Serotonin 5-HT₇ Receptor Neuroimaging: Toward the First PET Radiotracer

Abstract

Brain serotonin 7 (5-hydroxytryptamine 7, or 5-HT₇) is the most recently identified serotonin receptor. It is involved in mood disorders and is studied as a target for antidepressants. Because no radioligand has yet been successfully used to study this receptor by PET neuroimaging, the objective of the present study was to develop a 5-HT₇ ¹⁸F-labeled radiotracer. Methods: Four structural analogs of SB269970, a specific 5-HT₇ receptor antagonist, were synthesized. The nitro precursors of these analogs were radiolabeled by ¹⁸F nucleophilic substitution. Analog antagonist effects were investigated by cellular functional assay. The cerebral distribution of radiolabeled molecules was studied by in vitro autoradiography in rats, and respective selectivity was determined by competition with the 5-HT₇ receptor antagonist SB269970 at different concentrations. Ex vivo small-animal PET studies in rats and in vivo PET studies in cats focused on the 1-(2-{(2R)-1-[(fluorophenyl)sulfonyl]pyrrolidin-2-yl}ethyl)-4-methylpiperidine (FP3) series. Results: Four analogs were synthesized from the SB269970 pharmacophore and divided into an FP3 (¹⁸F-4FP3 and ¹⁸F-2FP3) and an 1-(2-{(2R)-1-[(fluorophenyl)sulfonyl]pyrrolidin-2-yl}ethyl)-4-(2-methoxyphenyl)piperazine (FPMP) (¹⁸F-2FPMP and ¹⁸F-4FPMP) series. The chemical and radiochemical purities of the 4 radiolabeled molecules were greater than 98%. All presented suitable affinity for 5-HT₇ (apparent dissociation constant [K_D] between 1.6 and 14 nM), although the FPMP series showed moderate agonist activity for 5-HT_1A receptors. Lipophilicity values were predictive of good radiotracer blood–brain barrier penetration (logD from 1.4 to 3.9). In vitro competition with a 5-HT₇ antagonist, SB269970, revealed that only radioligands from the FP3 series were displaced by the 5-HT₇–specific antagonist: subsequent in vivo study, therefore, focused on this series. Ex vivo ¹⁸F-4FP3 and ¹⁸F-2FP3 autoradiography was in accordance with the 5-HT₇ brain distribution, with few brain radioactive metabolites. PET scans in cats showed that pretreatment with a 5-HT₇ antagonist significantly reduced ¹⁸F-2FP3 but not ¹⁸F-4FP3 binding. Conclusion: The 4 PET radiotracers had suitable characteristics for 5-HT₇ receptor probing in vitro, although the FP3 series seemed to be more specific for in vivo imaging of 5-HT₇ receptors. In particular, on the basis of the in vivo results, ¹⁸F-2FP3 appears to be the first PET radiotracer to enable in vivo imaging of 5-HT₇ receptors in animal models, possibly leading to neuroimaging studies in humans.