Evaluation of the Gly-Phe-Lys Linker to Reduce the Renal Radioactivity of a [64Cu]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 [⁶⁴Cu]Cu-cyclam-RAFT-c(-RGDfK-)₄ ([⁶⁴Cu]Cu-cyclam-RaftRGD). We designed and synthesized the model compound [⁶⁴Cu]Cu-cyclam-GFK(benzoyl [Bz]), its predictive metabolites, and GFK-incorporated [⁶⁴Cu]Cu-cyclam-RaftRGD derivatives [⁶⁴Cu]Cu-cyclam-GFK-RaftRGD and [⁶⁴Cu]Cu-cyclam-GFK(beta-alanine [βA])₃-RaftRGD. In vitro studies showed that dual radiometabolites, namely, [⁶⁴Cu]Cu-cyclam-G and [⁶⁴Cu]Cu-cyclam-GF, were simultaneously released from [⁶⁴Cu]Cu-cyclam-GFK(Bz) by different RBB enzymes, whereas both RaftRGD derivatives released only [⁶⁴Cu]Cu-cyclam-GF. When injected into mice, [⁶⁴Cu]Cu-cyclam-GFK(Bz) and the two RaftRGD derivatives led to the urinary excretion of [⁶⁴Cu]Cu-cyclam-G and [⁶⁴Cu]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 [⁶⁴Cu]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%, p < 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 in vivo. 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.