Design, Synthesis, Molecular Docking, and Preclinical Evaluation of a New Radiolabeled PEG3-Linked FAPI Derivative...

Fibroblast activation protein (FAP) is a promising molecular target for cancer theranostic applications. However, current fibroblast activation protein inhibitors (FAPIs) have limitations, including rapid clearance and limited tumor retention.

This study aimed to develop a novel PEG₃-linked FAPI derivative, [⁶⁸Ga]Ga-FAPI-MKG, with enhanced tumor accumulation and retention. FAPI-MKG was prepared by incorporating a PEG₃ linker into the FAPI-04 structure to optimize its pharmacokinetic profile.

The compound was synthesized via an 11-step route starting from quinine sulfate and radiolabeled with gallium-68. In vitro studies included determination of lipophilicity (Log P) and stability assays in saline and human serum albumin. Preclinical evaluation in BALB/c mice bearing CT-26 tumors included biodistribution, blocking studies, and PET/CT imaging. Molecular docking and molecular dynamics simulations were performed to provide mechanistic insights into binding interactions.

[⁶⁸Ga]Ga-FAPI-MKG was successfully synthesized with high radiochemical purity (> 98%) and a molar activity of 414.79 mCi/μmol. It demonstrated moderate hydrophilicity (Log P = -3.26 ± 0.18) compared with the reference radiotracer, [⁶⁸Ga]Ga-FAPI-46 (-3.58 ± 0.29), and high stability (RCP > 90% after 120 minutes). In vivo studies showed significantly higher tumor uptake (7.18 ± 0.56% ID/g at 60 minutes; 3.20 ± 0.11% ID/g at 120 minutes) and prolonged retention compared with the reference radiotracer, along with dual hepatobiliary and renal excretion pathways. Blocking studies confirmed FAP-specific uptake. Computational analyses indicated strong binding energy (-9.8 kcal/mol) and optimized electrostatic interactions with FAP. The strategic incorporation of a PEG₃ linker into [⁶⁸Ga]Ga-FAPI-MKG significantly improved tumor accumulation, extended tumor retention, and increased the tumor-to-background ratio.

These findings suggest that [⁶⁸Ga]Ga-FAPI-MKG may be a promising candidate for clinical translation for imaging and theranostics of FAP-expressing cancers.