Sunflower Trypsin Inhibitor 1 Derivatives as Molecular Scaffolds for the Development of Novel Peptidic Radiopharmaceuticals

Purpose: Peptides with restricted conformation provide increased affinity and stability against degradation as compared to linear peptides. This study investigates the characteristics of derivatives of the sunflower trypsin inhibitor 1 (SFTI-1), a 14 amino acid peptide with high intrinsic stability. Methods: Three SFTI-1 derivatives (cyclic cSFTI, acyclic oSFTI, and DOTA-SFTI) were generated by Fmoc-based automated synthesis. Thereafter, the inhibitory activity for trypsin was determined. After radiolabeling, kinetic and competition studies were done in a variety of tumor cell lines including prostate carcinoma, colon carcinoma, mammary carcinoma, and hepatoma to characterize the binding affinity of the peptides. The stability was determined by incubating the molecules in human serum for increasing time periods. Furthermore, the biodistribution was measured in nude mice bearing human prostate carcinomas. Results: The inhibitory constants for trypsin inhibition were 0.08 nM (cSFTI), 0.15 nM (oSFTI), and 0.3 nM (DOTA-SFTI). Among the different tumor cell lines evaluated, the prostate cancer cell lines PC-3 and DU-145 showed the highest accumulation of the radiolabeled peptides. The open-chain derivatives generally bound better than the cyclic one. Binding was constant during 4 h and could be competed by addition of the cold peptide up to 75%. The stability in serum revealed half-lives of 75.8 h for cSFTI, 34.5 h for oSFTI, and 41.7 h for DOTA-SFTI. The biodistribution showed a rapid renal clearance for all three compounds and tumor uptake values up to 3%ID/g. Conclusions: SFTI derivatives are small stable molecules readily accessible by solid-phase synthesis. The trypsin inhibition was not influenced by the cyclization, and addition of a chelator had no significant influence. The exceptional rigidity and stability allow the use of SFTI derivatives as scaffolds for the introduction of tumor-specific peptide motifs which could be used to increase cell-binding affinities and thus their use as diagnostic and/or therapeutic tools.