Synthesis and evaluation of [18F]-FEAnGA as a PET Tracer for beta-glucuronidase activity

To increase the therapeutic index of chemotherapeutic drugs, prodrugs have been investigated as anticancer agents, as they may present fewer cytotoxic side effects than conventional cytotoxic drugs, while therapeutic efficacy is maintained or even increased. Extracellular beta-glucuronidase (beta-GUS) in the tumors has been investigated as a target enzyme for prodrug therapy, as it can convert nontoxic prodrugs into cytostatic drugs. To optimize beta-GUS-based prodrug therapies, PET imaging could be a useful tool by providing information regarding the localization and quantification of beta-GUS. Here, we describe our first PET tracer for extracellular beta-GUS, [(18)F]-FEAnGA, which consists of a 2-[(18)F]fluoroethylamine ([(18)F]-FEA) group bound to a glucuronic acid via a self-immolative nitrophenyl spacer. [(18)F]-FEAnGA was synthesized by alkylation of its imidazole carbamate precursor with [(18)F]-FEA, followed by deprotection of the sugar moiety with NaOH in 10-20% overall radiochemical yield. [(18)F]-FEAnGA is about 10-fold more hydrophilic than the cleavage product [(18)F]-FEA, and it is stable in PBS and rat plasma for at least 3 h. In the presence of either Escherichia coli beta-GUS or bovine liver beta-GUS, in vitro cleavage of [(18)F]-FEAnGA with complete release of [(18)F]-FEA was observed within 30 min. C6 glioma cells incubated with the tracer and Escherichia coli beta-GUS or bovine liver beta-GUS showed a 4- and 1.5-fold higher uptake of radioactivity, respectively, as compared to control C6 cells without beta-GUS. Incubation of CT26 murine colon adenocarcinoma cells or the genetically engineered CT26mbetaGUS cells, which expressed membrane-anchored GUS on the outer cell membrane, with the tracer, resulted in a 3-fold higher uptake into GUS-expressing cells as compared to control cells. In a preliminary microPET study in mice bearing both CT26 and CT26mbetaGUS tumors, [(18)F]-FEAnGA exhibited a 2-fold higher retention of radioactivity in the tumor expressing beta-GUS than in the control tumor. [(18)F]-FEA did not show any difference in tracer uptake between tumors. These results suggest that [(18)F]-FEAnGA may be a suitable PET tracer for evaluation of beta-GUS activity, since it is specifically cleaved by beta-GUS and the released [(18)F]-FEA remains attached to targeted cells.