Evaluation of the Gly-Phe-Lys Linker to Reduce the Renal Radioactivity of a [Cu]Cu-Labeled Multimeric cRGD Peptide.

Radiometal-labeled peptide-based radiopharmaceuticals (RLPB-radiopharmaceuticals) are promising for cancer imaging and targeted radiotherapy; however, their effectiveness is often compromised by the high retention of nonspecific radioactivity in the kidneys due to renal excretion pathways. Current strategies to address this issue have limitations, highlighting the need for innovative approaches to improve targeting specificity and therapeutic efficacy. We aimed to evaluate the applicability of the Gly-Phe-Lys (GFK) tripeptide, a renal brush border (RBB) enzyme-cleavable linkage, to reduce renal radioactivity in RLPB-radiopharmaceuticals using the integrin-targeting radiopeptide [Cu]Cu-cyclam-RAFT-c(-RGDfK-) ([Cu]Cu-cyclam-RaftRGD). We designed and synthesized the model compound [Cu]Cu-cyclam-GFK(benzoyl [Bz]), its predictive metabolites, and GFK-incorporated [Cu]Cu-cyclam-RaftRGD derivatives [Cu]Cu-cyclam-GFK-RaftRGD and [Cu]Cu-cyclam-GFK(beta-alanine [βA])-RaftRGD. studies showed that dual radiometabolites, namely, [Cu]Cu-cyclam-G and [Cu]Cu-cyclam-GF, were simultaneously released from [Cu]Cu-cyclam-GFK(Bz) by different RBB enzymes, whereas both RaftRGD derivatives released only [Cu]Cu-cyclam-GF. When injected into mice, [Cu]Cu-cyclam-GFK(Bz) and the two RaftRGD derivatives led to the urinary excretion of [Cu]Cu-cyclam-G and [Cu]Cu-cyclam-GF, respectively. PET imaging and biodistribution studies showed the increased rates of reduction in renal radioactivity levels for the two RaftRGD derivatives compared to the parental [Cu]Cu-cyclam-RaftRGD (e.g., PET: 1 to 24 h postinjection, 73.0 ± 2.3 and 75.6 ± 1.8 vs 43.0 ± 4.5%, < 0.0001; biodistribution: 3 to 24 h, 61.1 and 74.4 vs 22.8%). Taken together, these results indicate that the designed renal cleavage occurred . We also noted the steric interference of the RaftRGD moiety on enzyme access, the spacer effect of the trimeric βA sequence (reduced steric hindrance), and the altered radiopharmacokinetics (e.g., initially increased renal accumulation) of the RaftRGD compounds upon linker incorporation. These findings provide important insights into the chemical design of RLPB-radiopharmaceuticals with reduced renal retention based on the RBB strategy.