Fluorine-18 radiolabeling of S100/calgranulins: potential probes for molecular imaging of receptor for advanced glycation endproducts

The interaction of S100/calgranulins, a multigenic family of Ca²⁺-modulated proteins, with the receptor for advanced glycation endproducts (RAGE) is hypothesized to be of high relevance in the pathogenesis of various diseases including cardiovascular diseases, inflammatory processes, and cancerogenesis. However, data concerning the role of circulating S100 proteins in these pathologies are scarce. Furthermore, it is currently not known whether RAGE is an universal S100 receptor. One reason for this is the shortage of suitable radiolabeling methods for direct assessment of the metabolic fate of circulating S100 proteins in vivo. We report a novel radiotracer approach using radiolabeling of recombinant human S100A1 with the positron emitter-fluorine-18 (¹⁸F) by conjugation with N-succinimidyl-4-[¹⁸F]fluorobenzoate ([¹⁸F]SFB) and the use of [¹⁸F]fluorobenzoylated S100A1 (¹⁸F-S100A1) in dynamic small animal positron emission tomography (PET) studies in rats. Human S100A1 was cloned as a fusion protein in the bacterial expression vector pGEX-6P-1 and expressed in E. coli strain BL21. Radiolabeling of S100A1 with [¹⁸F]SFB at pH 7.4 resulted in ¹⁸F-S100A1 specifically labeled at the N-terminal glycine residue with radiochemical yields of 2-6% (decay-corrected) and effective specific activities of 0.5-1 GBq/µmol, respectively. In vitro experiments, and biodistribution and metabolite studies in rats in vivo revealed high stability for the ¹⁸F-S100A1. The metabolic fate of ¹⁸F-S100A1 in rats in vivo was delineated by dynamic PET studies using a dedicated small animal PET system. The organ-specific in vivo distribution and kinetics of ¹⁸F-S100A1 correlated well with the anatomical localization of RAGE, e.g., in lungs and in the vascular system. In the presence of molar excess of glycated human low density lipoprotein (glycLDL), a well characterized RAGE ligand, the mean plasma residence time of circulating ¹⁸F-S100A1 increased by 40% from 29.6 ± 1.5 min to 41.3 ± 2.1 min and, vice versa, tissue-associated retention of ¹⁸F-S100A1 decreased by approximately 50% in lungs and 32% in large blood vessels, respectively. These findings indicate first circulating S100A1 to be a specific ligand for RAGE in rats in vivo. In conclusion, radiolabeling of S100/calgranulins with ¹⁸F and the use of small animal PET provide novel probes to delineate functional expression of RAGE under normal and pathophysiological conditions in rodent models of disease in vivo.