Novel 68Ga-Labeled Pyridine-Based Fibroblast Activation Protein-Targeted Tracers with High Tumor-to-Background Contrast

Abstract

Compared to quinoline-based fibroblast activation protein (FAP)-targeted radiotracers, pyridine-based FAP-targeted tracers are expected to have faster pharmacokinetics due to their smaller molecular size and higher hydrophilicity, which we hypothesize would improve the tumor-to-background image contrast. We aim to develop 68Ga-labeled pyridine-based FAP-targeted tracers for cancer imaging with positron emission tomography (PET), and compare their imaging potential with the clinically validated [68Ga]Ga-FAPI-04. Two DOTA-conjugated pyridine-based AV02053 and AV02070 were synthesized through multi-step organic synthesis. IC50(FAP) values of Ga-AV02053 and Ga-AV02070 were determined by an enzymatic assay to be 187 ± 52.0 and 17.1 ± 4.60 nM, respectively. PET imaging and biodistribution studies were conducted in HEK293T:hFAP tumor-bearing mice at 1 h post-injection. The HEK293T:hFAP tumor xenografts were clearly visualized with good contrast on PET images by [68Ga]Ga-AV02053 and [68Ga]Ga-AV02070, and both tracers were excreted mainly through the renal pathway. The tumor uptake values of [68Ga]Ga-AV02070 (7.93 ± 1.88%ID/g) and [68Ga]Ga-AV02053 (5.6 ± 1.12%ID/g) were lower than that of previously reported [68Ga]Ga-FAPI-04 (12.5 ± 2.00%ID/g). However, both [68Ga]Ga-AV02070 and [68Ga]Ga-AV02053 showed higher tumor-to-background (blood, muscle, and bone) uptake ratios than [68Ga]Ga-FAPI-04. Our data suggests that pyridine-based pharmacophores are promising for the design of FAP-targeted tracers. Future optimization on the selection of a linker will be explored to increase tumor uptake while maintaining or even further improving the high tumor-to-background contrast.

Synthesis and Preclinical Evaluation of Two Novel 68Ga-Labeled Bispecific PSMA/FAP-Targeted Tracers with 2-Nal-Containing PSMA-Targeted Pharmacophore and Pyridine-Based FAP-Targeted Pharmacophore

“…The enzymatic assay ( Figure 2 B) showed that the FAP-binding affinities of Ga-AV01084 (IC 50 = 10.9 ± 0.67 nM) and Ga-AV01088 (IC 50 = 16.7 ± 1.53 nM) were comparable or even slightly better than that of the previously reported Ga-AV02070 (IC 50 = 17.1 ± 4.60 nM) [ 31 ]. We also measured the FAP-binding affinity of Ga-PSMA-617 to determine whether the PSMA-targeted pharmacophore has any effect on the overall FAP binding of our bispecific ligands or not.…”

“…To synthesize compound 5 ( Scheme 1 ), compound 1 was first coupled with 2,3,5,6-tetrafluorophenol (TFP) to obtain the activated ester 2 in 71% yield. Compound 3, which was synthesized following literature procedures [ 31 ], was first Boc-deprotected using trifluoroacetic acid (TFA), followed by coupling with compound 2 to obtain compound 4 in a 45% yield. The t -Butyl-protecting group of compound 4 was removed using TFA and compound 5 was obtained as a TFA salt.…”

“…The lipophilicity of 68 Ga-labeled tracers was determined by measuring their LogD 7.4 values following literature procedures [ 31 , 36 ]. Briefly, 68 Ga-labeled tracer (50 µL) was mixed with 3 mL n -octanol and 3 mL PBS (pH 7.4) in a 15 mL falcon tube.…”

“…Binding affinities of Ga-labeled ligands to FAP were measured following literature procedures [ 28 , 31 ]. Recombinant human FAP (0.2 µg/mL, 50 µL; BioLegend, San Diego, CA, USA) was added to a costar 96-well plate.…”

“…Many potent pyridine-based FAP inhibitors (FAPIs) have been reported [ 29 , 30 ]; however, there are still few reports on the development of pyridine-based FAP-targeted tracers. Previously, we synthesized and evaluated two novel 68 Ga-labeled pyridine-based FAP-targeted tracers, [ 68 Ga]Ga-AV02053 and [ 68 Ga]Ga-AV02070, and compared their binding affinity and tumor uptake to those of [ 68 Ga]Ga-FAPI-04 [ 31 ]. We discovered that although [ 68 Ga]Ga-FAPI-04 has a higher binding affinity toward FAP and higher tumor uptake, both of our pyridine-based tracers have lower uptake in blood, muscle, and bone, leading to much higher tumor-to-background contrast ratios.…”