Structure Optimization and Their Biodistribution in Tumor-bearing Mice on FAP and αⅤβ3 Dual Targeting Molecular Probes

Fibroblast activation protein (FAP) is a crucial biomarker for the activation of tumor associated fibroblasts, served as an excellent target for both the diagnosis and treatment of cancer. In recent years, a variety of quinoline-based FAP inhibitors (FAPIs) have been developed, such as FAPI-02, FAPI-04 and FAPI-46, which were used to positron emission tomography (PET) imaging for clinical patients. However, high expression of FAP also occurs in chronic inflammation, fibrosis, arthritis, atherosclerotic plaques and cardiac fibrosis, which results in a compromised sensitivity/selectivity in distinguishing cancers from other FAP-positive diseases, such as chronic inflammation and fibrosis. Additionally, integrin receptor αⅤβ3 highly expresses on the surface of various tumor cells and neovascular endothelial cells, including those in lung cancer, glioblastoma, breast cancer, and osteosarcoma, but absent in resting endothelial cells of normal tissues. Integrin receptor αⅤβ3 plays an important role in regulating tumor growth, angiogenesis, local invasiveness, and metastatic potential. However, RGD-based radiotracers, including multimeric RGD peptides with enhanced integrin-targeting efficiency have only moderate tumor uptake. Based on this, in this paper two heterologous dimeric radiotracers, 68Ga-FAPI-RGD-01 and 68Ga-FAPI-RGD-02, were designed and synthesized, which are based on the quinoline-based FAPI-02 for targeting FAP, a cyclic RGD peptide for targeting αⅤβ3, a 1,4,7-triazacyclononanetriacetic acid (NOTA) group for radionuclide labeling, and poly (ethylene glycol) linker. And then, the two radiotracers were used to study the microPET imaging and preliminary biodistribution in U87MG tumor-bearing mice. The excellent in vitro stability of both 68Ga-FAPI-RGD-01 and 68Ga-FAPI-RGD-02 was confirmed through high-performance liquid chromatography analysis, which could maintain radiochemical purity over 95% in PBS buffer solution or in serum medium for at least 4 h. The radiotracers also show favorable binding affinity and specificity through affinity assays of protein and receptor in vitro, and microPET imaging of U87MG tumor-bearing mice in vivo. Subsequently, the pharmacokinetics of the two radiotracers were assessed in healthy ICR mice, microPET imaging and biodistribution were performed in U87MG tumor-bearing mice, and comparing the performance of the 68Ga-FAPI-RGD-01 and 68Ga-FAPI-RGD-02 tracers. Compared to 68Ga-FAPI-RGD-01, 68Ga-FAPI-RGD-02 exhibits higher tumor uptake and lower uptake in the heart, liver, kidneys, and muscle tissues. In addition, compared to FAPI-02, RGD, and FAPI-02+RGD blocking groups, the tumor uptake and retention of 68Ga-FAPI-RGD-02 is very much higher than these blocking groups through microPET imaging studies. It is indicated that superior pharmacokinetic performance and tumor imaging effect for 68Ga-FAPI-RGD-02, demonstrating its enormous potential in clinical disease diagnosis.