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.

Some bispecific radiotracers have been developed to overcome the limitations of monospecific tracers and improve detection sensitivity for heterogeneous tumor lesions. Here, we aim to synthesize two bispecific tracers targeting prostate-specific membrane antigen (PSMA) and fibroblast activation protein (FAP), which are key markers expressed in prostate cancer. A pyridine-based FAP-targeted ligand was synthesized through multi-step organic synthesis and then connected to the 2-Nal-containing PSMA-targeted motif. The Ki(PSMA) values of Ga-complexed bispecific ligands, Ga-AV01084 and Ga-AV01088, were 11.6 ± 3.25 and 28.7 ± 6.05 nM, respectively, and the IC50(FAP) values of Ga-AV01084 and Ga-AV01088 were 10.9 ± 0.67 and 16.7 ± 1.53 nM, respectively. Both [68Ga]Ga-AV01084 and [68Ga]Ga-AV01088 enabled the visualization of PSMA-expressing LNCaP tumor xenografts and FAP-expressing HEK293T:hFAP tumor xenografts in PET images acquired at 1 h post-injection. However, the tumor uptake values from the bispecific tracers were still lower than those obtained from the monospecific tracers, PSMA-targeted [68Ga]Ga-PSMA-617 and FAP-targeted [68Ga]Ga-AV02070. Further investigations are needed to optimize the selection of linkers and targeted pharmacophores to improve the tumor uptake of bispecific PSMA/FAP tracers for prostate cancer imaging.

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

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 Three Novel 68Ga-Labeled Bispecific PSMA/FAP-Targeting Tracers for Prostate Cancer Imaging.

Tumor heterogeneity limits the efficacy and reliability of monospecific radiopharmaceuticals in prostate cancer diagnosis and therapy. To overcome this limitation and improve lesion detection sensitivity, we developed and evaluated three bispecific radiotracers that can target both prostate-specific membrane antigen (PSMA) and fibroblast activation protein (FAP), which are the two key proteins overexpressed in prostate cancer. Three FAP-targeting ligands with various linker lengths were synthesized through multistep organic synthesis, and then connected to the PSMA-targeting motif. IC50(PSMA) and IC50(FAP) values of Ga-complexed bispecific ligands, Ga-AV01017, Ga-AV01030, and Ga-AV01038 were 25.2-71.6 and 1.25-2.74 nM, respectively. The uptake values in PSMA-expressing LNCaP tumor xenografts were 4.38 ± 0.55, 5.17 ± 0.51, and 4.25 ± 0.86 %ID/g for [68Ga]Ga-AV01017, [68Ga]Ga-AV01030, and [68Ga]Ga-AV01038, respectively, which were lower than the monospecific PSMA-targeting tracer [68Ga]Ga-HTK03041 (23.1 ± 6.11 %ID/g). The uptake values in FAP-expressing HEK293T:hFAP tumor xenografts were 2.99 ± 0.37, 3.69 ± 0.81, 3.64 ± 0.83 %ID/g for [68Ga]Ga-AV01017, [68Ga]Ga-AV01030, and [68Ga]Ga-AV01038, respectively, which were also lower than the monospecific FAP-targeting tracer, [68Ga]Ga-FAPI-04 (12.5 ± 2.00 %ID/g). We observed that the bispecific tracers had prolonged blood retention, in which tracers with a longer linker tend to have a higher blood uptake and lower tumor uptake. Further investigations are needed to optimize the linker selection to generate promising bispecific PSMA/FAP-targeting tracers for prostate cancer imaging.