Fibroblast activation protein (FAP) is expressed in the microenvironment of most human epithelial tumors. ⁶⁸Ga-labeled FAP inhibitors based on the cyanopyrrolidine structure (FAPI) are currently used for the detection of the tumor microenvironment by PET imaging. This research aimed to design, synthesize and preclinically evaluate a new FAP inhibitor radiopharmaceutical based on the ^99mTc-((R)-1-((6-hydrazinylnicotinoyl)-D-alanyl) pyrrolidin-2-yl) boronic acid (^99mTc-iFAP) structure for SPECT imaging. Molecular docking for affinity calculations was performed using the AutoDock software. The chemical synthesis was based on a series of coupling reactions of 6-hidrazinylnicotinic acid (HYNIC) and D-alanine to a boronic acid derivative. The iFAP was prepared as a lyophilized formulation based on EDDA/SnCl₂ for labeling with ^99mTc. The radiochemical purity (R.P.) was verified via ITLC-SG and reversed-phase radio-HPLC. The stability in human serum was evaluated by size-exclusion HPLC. In vitro cell uptake was assessed using N30 stromal endometrial cells (FAP positive) and human fibroblasts (FAP negative). Biodistribution and tumor uptake were determined in Hep-G2 tumor-bearing nude mice, from which images were acquired using a micro-SPECT/CT. The iFAP ligand (Ki = 0.536 nm, AutoDock affinity), characterized by UV-Vis, FT-IR, ¹H–NMR and UPLC-mass spectroscopies, was synthesized with a chemical purity of 92%. The ^99mTc-iFAP was obtained with a R.P. >98%. In vitro and in vivo studies indicated high radiotracer stability in human serum (>95% at 24 h), specific recognition for FAP, high tumor uptake (7.05 ± 1.13% ID/g at 30 min) and fast kidney elimination. The results found in this research justify additional dosimetric and clinical studies to establish the sensitivity and specificity of the ^99mTc-iFAP.

Prostate-specific membrane antigen (PSMA) is expressed in a variety of cancer cells, while the fibroblast activation protein (FAP) is expressed in the microenvironment of tumors. Previously, we reported the ability of iPSMA and iFAP ligands to specifically target PSMA and FAP proteins, as well as the preparation of stable ¹⁷⁷Lu₂O₃ nanoparticles (<100 nm) functionalized with target-specific peptides. This research aimed to evaluate the dosimetry and therapeutic response of Lu₂O₃-iPSMA and Lu₂O₃-iFAP nanoparticles activated by neutron irradiation to demonstrate their potential for theranostic applications in nuclear medicine. The biokinetic behavior, radiation absorbed dose, and metabolic activity ([¹⁸F]FDG/micro-PET, SUV) in preclinical tumor tissues (athymic mice), following treatment with ¹⁷⁷Lu₂O₃-iPSMA, ¹⁷⁷Lu₂O₃-iFAP or ¹⁷⁷Lu₂O₃ nanoparticles, were assessed. One patient with multiple colorectal liver metastases (PSMA-positive) received ¹⁷⁷Lu₂O₃-iPSMA under a “compassionate use” protocol. Results indicated no significant difference (p < 0.05) between ¹⁷⁷Lu₂O₃-iPSMA and ¹⁷⁷Lu₂O₃-iFAP, regarding tumor radiation absorbed doses (105 ± 14 Gy, 99 ± 12 Gy and 58 ± 7 Gy for ¹⁷⁷Lu₂O₃-iPSMA, ¹⁷⁷Lu₂O₃-iFAP, and ¹⁷⁷Lu₂O₃, respectively) and tumor metabolic activity (SUV of 0.421 ± 0.092, 0.375 ± 0.104 and 1.821 ± 0.891 for ¹⁷⁷Lu₂O₃-iPSMA, ¹⁷⁷Lu₂O₃-iFAP, and ¹⁷⁷Lu₂O₃, respectively) in mice after treatment, which correlated with the observed therapeutic response. ¹⁷⁷Lu₂O₃-iPSMA and ¹⁷⁷Lu₂O₃-iFAP significantly inhibited tumor progression, due to the prolonged tumor retention and a combination of ¹⁷⁷Lu radiotherapy and iPSMA or iFAP molecular recognition. There were negligible uptake values in non-target tissues and no evidence of liver and renal toxicity. The doses received by the patient’s liver metastases (42–210 Gy) demonstrated the potential of ¹⁷⁷Lu₂O₃-iPSMA for treating colorectal liver metastases.