Preparation and Bioevaluation of ^99mTc-Labeled FAP Inhibitors as Tumor Radiotracers to Target the Fibroblast Activation Protein

Fibroblast activation protein (FAP) is overexpressed in cancer-associated fibroblasts (CAFs) in a majority of human epithelial cancers. With low expression in normal organs, FAP has become a promising molecular target for tumor theranostics. To develop a lower cost and more widely available alternative to positron emission tomography (PET), two isocyanide-containing FAP inhibitors (CN-C₅-FAPI and CN-PEG₄-FAPI) were synthesized and radiolabeled with ^99mTc to obtain [^99mTc]­[Tc-(CN-C₅-FAPI)₆]⁺ and [^99mTc]­[Tc-(CN-PEG₄-FAPI)₆]⁺ in high yields (>95%). They showed good stability in saline and mouse serum. The partition coefficient (log P) values of [^99mTc]­[Tc-(CN-C₅-FAPI)₆]⁺ and [^99mTc^][Tc-(CN-PEG₄-FAPI)₆]⁺ were −0.86 ± 0.03 and −2.38 ± 0.07, respectively, indicating that they were good hydrophilic complexes. The low nanomolar IC₅₀ values of CN-C₅-FAPI and CN-PEG₄-FAPI indicated that they had specificity to FAP. In vitro cellular uptake and blocking experiments implied a FAP-targeted uptake mechanism. The nanomolar K_d values from the saturation binding assay indicated that they had significantly high target affinity to FAP. The biodistribution and blocking study in BALB/c nude mice bearing U87MG tumors showed that both exhibited specific tumor uptake. [^99mTc]­[Tc-(CN-PEG₄-FAPI)₆]⁺ showed a higher tumor uptake and a higher tumor/nontarget ratio than [^99mTc]­[Tc-(CN-C₅-FAPI)₆]⁺. The results of micro-single-photon emission computed tomography (SPECT) imaging studies of [^99mTc]­[Tc-(CN-C₅-FAPI)₆]⁺ and [^99mTc^][Tc-(CN-PEG₄-FAPI)₆]⁺ were in accordance with the biodistribution results, suggesting that [^99mTc]­[Tc-(CN-PEG₄-FAPI)₆]⁺ is a promising tumor imaging agent for targeting FAP.