Radiochemical synthesis and pre-clinical evaluation of [⁶⁸Ga]Ga-FAPI-4 formulated using in-house developed FAPI-4 kit

The present work describes the formulation of a lyophilized Fibroblast Activation Protein Inhibitor-4 (FAPI-4) kit for preparation of [⁶⁸Ga]Ga-FAPI-4 in high radiochemical yield (RCY) using either generator or cyclotron produced ⁶⁸GaCl₃, suitable for Positron Emission Tomography (PET) based diagnosis of cancers over-expressing Fibroblast Activation Protein (FAP). Experimental parameters such as concentration of FAPI-4, buffer content, storage conditions and shelf-life were optimized to ensure > 95% RCY to utilize the kit for multi-patient dose formulations. A lyophilized FAPI-4 kit containing 50 µg of FAPI-4 ligand could be successfully optimized which consistently yielded [⁶⁸Ga]Ga-FAPI-4 in > 95% RCY on labeling with ~ 1.11 GBq of ⁶⁸GaCl₃. [⁶⁸Ga]Ga-FAPI-4 retained > 95% radiochemical purity (RCP) even after 2 h post labeling when stored at ambient temperature. The kits stored below − 20  °C reproduced the labeling profile up to a period of ~ 3 months (period of study). In the in vitro cell binding experiments performed in HT29 cells, [⁶⁸Ga]Ga-FAPI-4 exhibited specific binding to FAP (16.1 ± 1.8%ID in 10⁶ cells). In the biodistribution studies performed in female SCID mice bearing HT29 colorectal adenocarcinoma, [⁶⁸Ga]Ga-FAPI-4 exhibited tumor uptake of 3.8 ± 0.3% ID/g at 1 h post-injection. The above results demonstrate the utility of developed FAPI-4 kits for formulation of [⁶⁸Ga]Ga-FAPI-4 for PET imaging of FAP over-expressing cancers.