Trimeric Radiofluorinated Sulfonamide Derivatives to Achieve In Vivo Selectivity for Carbonic Anhydrase IX-Targeted PET Imaging

Carbonic anhydrase IX (CA-IX), a transmembrane enzyme, mediates cell survival under hypoxic conditions and is overexpressed in solid malignancies. In this study, we synthesized four (18)F sulfonamide derivatives and evaluated their potential for imaging CA-IX expression with PET.

Methods: Azide derivatives of 2 carbonic anhydrase inhibitors, 4-(2-aminoethyl)benzenesulfonamide (AEBS) and 4-aminobenzensulfonamide (ABS), were coupled to radiosynthons with either 1 or 3 alkynes and a pendent ammoniomethyltrifluoroborate (AmBF3) to generate monovalent or trivalent enzyme inhibitors. Binding affinity to CA-IX and other CA isoforms was determined via a stopped-flow, CA-catalyzed CO2 hydration assay. Tracers were radiolabeled via (18)F-(19)F isotope exchange reactions. Imaging/biodistribution studies were performed using HT-29 tumor-bearing immunocompromised mice.

Results: Monomeric AmBF3-AEBS and AmBF3-ABS were obtained in 41% and 40% yields, whereas trimeric AmBF3-(AEBS)3 and AmBF3-(ABS)3 were obtained in 47% and 55% yields, respectively. Derivatives bound CA-I, -II, -IX, and -XII with good affinity (0.49-100.3 nM). (18)F-labeled sulfonamides were obtained in 16.3%-36.8% non-decay-corrected radiochemical yields, with 40-207 GBq/μmol specific activity and greater than 95% radiochemical purity. Biodistribution/imaging studies showed that the tracers were excreted through both renal and hepatobiliary pathways. At 1 h after injection, HT-29 tumor xenografts were clearly visualized in PET images with modest contrast for all 4 tracers. Tumor uptake was 2-fold higher for monovalent tracers (∼0.60 percentage injected dose per gram [%ID/g]) than for trivalent tracers (∼0.30 %ID/g); however, tumor-to-background ratios were significantly better for (18)F-AmBF3-(ABS)3. Preblocking with acetazolamide reduced more than 80% uptake of (18)F-AmBF3-(ABS)3 in HT-29 tumors.

Conclusion: Our data suggest that trimerization of an otherwise nonspecific CA inhibitor greatly enhances the selectivity for CA-IX in vivo and represents a promising strategy for creating multivalent enzyme inhibitors for selectively imaging extracellular enzyme activity by PET.