The in vitro PSMA-targeting efficiency of PSMA-I&F, the reference PSMA-I&T and their corresponding natGa-/68Ga- and natLu/177Lu-counterparts was determined in LNCaP cells via competitive binding assays (IC50), dual-tracer radioligand- and fluorescence internalization studies. Biodistribution and small-animal PET imaging studies were performed in CB17-SCID and LNCaP xenograft bearing SHO mice, respectively, and complemented by intraoperative far-red fluorescence imaging using a clinical laparoscope. Additionally, fully automated serial cryosectioning and fluorescence imaging of one tumor-bearing animal as well as PSMA immunohistochemistry (IHC) and fluorescence microscopy of organ cryosections (tumor, kidney, spleen) were also performed.
Compared to the parent PSMA-I&T analogs, the PSMA-affinities of PSMA-I&F and its natGa-/natLu-complexes remained high and unaffected by dye conjugation (7.9<IC50<10.5 nM for all ligands). The same was observed for the internalization of 68Ga- and 177Lu-PSMA-I&F. In vivo, blood clearance of 68Ga- and 177Lu-PSMA-I&F was only slightly delayed by high plasma protein binding (94-95%), and very low accumulation in non-target organs was observed already at 1h p.i.. Dynamic PET imaging confirmed PSMA-specific (as demonstrated by coinjection of 2-PMPA) uptake into the LNCaP xenograft (4.5±1.8%iD/g) and the kidneys (106±23%iD/g). Tumor/background ratios of 2.1, 5.2, 9.6 and 9.6 for blood, liver, intestines and muscle, respectively, at 1h p.i. led to excellent imaging contrast in 68Ga-PSMA-I&F PET and in intraoperative fluorescence imaging. Furthermore, fluorescence imaging of tissue cryosections allowed high-resolution visualization of intraorgan PSMA-I&F distribution in vivo and its correlation with PSMA expression as determined by IHC.
Thus, with its high PSMA-targeting efficiency and favorable pharmacokinetic profile, 68Ga/177Lu-PSMA-I&F serves as an excellent proof-of-concept compound for the general feasibility of PSMA-I&T-based hybrid imaging. The PSMA-I&T scaffold represents a versatile PSMA-targeted lead structure, allowing relatively straightforward adaptation to the different structural requirements of dedicated nuclear or hybrid imaging agents.