Synthesis and Evaluation of ^99mTc-Labeled FAP Inhibitors with Different Linkers for Imaging of Fibroblast Activation Proteins in Tumors

Abstract

Fibroblast activation protein (FAP) is a potential target for tumor diagnosis and treatment due to its selective expression on cancer-associated fibroblasts (CAFs) in most solid tumor stroma. Two FAP inhibitor (FAPI) derived ligands (L1 and L2) containing different lengths of D Pro-Gly (PG) repeat units as linkers were designed and synthesized with high affinity for FAP. Two stable hydrophilic 99m Tc-labeled complexes ([ 99m Tc]Tc-L1 and [ 99m Tc]Tc-L2) were obtained. In vitro cellular studies show that the uptake mechanism is correlated with FAP uptake, and [ 99m Tc]Tc-L1 shows a higher cell uptake and specific binding to FAP. A nanomolar K d value for [ 99m Tc]Tc-L1 indicates its significantly high target affinity for FAP. The biodistribution and microSPECT/CT images obtained for U87MG tumor mice show that [ 99m Tc]Tc-L1 has high tumor uptake with specificity to FAP and high tumor-to-nontarget ratios. As an inexpensive, easily made, and widely available tracer, [ 99m Tc]Tc-L1 holds great promise for clinical applications.

Synthesis and Evaluation of [18F]SiFA-Conjugated Ligands for Fibroblast Activation Protein Imaging

“…FAP expression on multipotent bone marrow stromal cells may explain this observation. , Similarly, the bone and blood uptake of [ 18 F]­FGlc-FAPI in HT-1080-FAP tumor-bearing mice 60 min after injection was reduced by 82% with a competitor, which suggests specific binding of the radioligand to bone marrow , The tumor uptake of [ 18 F] 2 was 3.93 ± 0.59% ID/g, which was markedly decreased to 0.33 ± 0.03% ID/g by FAPI-04 (*** P ). The tumor-to-muscle and tumor-to-blood uptake ratios were 9.83 and 4.09 in the control study and 1.73 and 0.61 in the blocking study, respectively.…”

“…13 99m Tc-labeled FAPIs showed reduced bone and blood uptake in the blocking studies. 21,22 The tumor uptake of [ 18 F]2 was 3.93 ± 0.59% ID/g, which was markedly decreased to 0.33 ± 0.03% ID/g by FAPI-04 (***P). The tumor-to-muscle and tumor-to-blood uptake ratios were 9.83 and 4.09 in the control study and 1.73 and 0.61 in the blocking study, respectively.…”

“…Binding assays of 1 and 2 were carried out according to the method reported. , The fluorogenic substrate Gly-Pro-AMC (AAT Bioquest, Pleasanton, CA) was dissolved in DMSO and diluted to 40 μM with the assay buffer containing 25 mM Tris (pH 7.4) and 250 mM NaCl. The ligands were dissolved in DMSO and diluted to various concentrations (4 μM to 40 pM) with the assay buffer.…”

“…CAFs eventually contribute to the migration, invasion, and metastasis of cancer cells by secreting growth factors and cytokines and by modifying the ECM. , FAP, a type II transmembrane serine protease, plays a critical role in ECM remodeling, promoting tumor growth and invasion. ,, Moreover, FAP is highly expressed on the cell surface of activated fibroblasts and is observed in over 90% of epithelial cancers. ,, Therefore, FAP has been recognized as an important target for the diagnosis and therapy of various tumors. , Since ( S )- N -(2-(2-cyano-4,4-difluoropyrrolidin-1-yl)-2-oxoethyl)-quinoline-4-carboxamide (UAMC-1110), a potent and selective FAP inhibitor (FAPI), was reported (Figure ), it has been used as the scaffold for the development of radiopharmaceuticals for the diagnosis and therapy of FAP-expressing tumors . For the last several years, a series of 68 Ga-labeled FAPIs, including [ 68 Ga]­Ga-FAPI-02 and [ 68 Ga]­Ga-FAPI-04 (Figure ), were developed for FAP imaging and showed promise in clinical applications. − Furthermore, these FAPI radioligands showed advantages over [ 18 F]­FDG in the detection of primary tumors and metastases in patients with various cancers, revealing higher tumor uptakes and tumor-to-background contrast ratios for 68 Ga-labeled FAPI than for [ 18 F]­FDG. , More recently, ligands containing the FAPI scaffold were labeled with 18 F, 64 Cu, and 99m Tc for the development of PET and SPECT radioligands for FAP imaging. − …”

Diagnostic and Therapeutic Radiopharmaceuticals: A “Hot” Topic

“…These were radiolabeled with Tc-99m and showed high cell uptake and specific binding to FAP. 33 The review by Mishra et al summarizes publications on 99m Tc-DTPA-bis(Met) evaluated in different clinical studies, e.g., for detection of breast cancer, as a prognostic marker, for detection of recurrent/residual gliomas, and for differentiation of recurrent/residual gliomas from radiation necrosis. 34 The current state and future horizon of SPECT imaging using technetium-99m radiopharmaceuticals are depicted by Riondato and co-workers.…”