Radiopharmacological evaluation of (18)F-labeled phosphatidylserine-binding peptides for molecular imaging of apoptosis

Introduction: Radiolabeled phosphatidylserine (PS)-binding peptides represent an innovative strategy for molecular imaging of apoptosis with positron emission tomography (PET). The goal of this study was the radiopharmacological evaluation of radiolabeled peptides for their binding to PS on apoptotic cancer cells, involving metabolic stability, cellular uptake, biodistribution, and dynamic PET imaging experiments.

Methods: Binding of peptides LIKKPF, PGDLSR, FBz-LIKKPF, FBz-PGDLSR, FBAM-CLIKKPF and FBAM-CPGDLSR to PS was analyzed in a newly developed radiometric binding assay using (64)Cu-labeled wild-type annexin-V as radiotracer. Radiolabeling of most potent peptides with fluorine-18 was carried out with thiol-selective prosthetic group [(18)F]FBAM to give [(18)F]FBAM-CLIKKPF and [(18)F]FBAM-CPGDLSR. [(18)F]FBAM-labeled peptides were studied in camptothecin-induced apoptotic human T lymphocyte Jurkat cells, and in a murine EL4 tumor model of apoptosis using dynamic PET imaging and biodistribution.

Results: Peptides LIKKPF and PGDLSR inhibited binding of (64)Cu-labeled annexin-V to immobilized PS in the millimolar range (IC50 10-15 mM) compared to annexin-V (45 nM). Introduction of FBAM prosthetic group slightly increased inhibitory potencies (FBAM-CLIKKPF: IC50 = 1 mM; FBAM-CPGDLSR: IC50 = 6 mM). Radiolabeling succeeded in good radiochemical yields of 50-54% using a chemoselective alkylation reaction of peptides CLIKKPF and CPGDLSR with [(18)F]FBAM. In vivo metabolic stability studies in mice revealed 40-60% of intact peptides at 5 min p.i. decreasing to 25% for [(18)F]FBAM-CLIKKPF and less than 5% for [(18)F]FBAM-CPGDLSR at 15 min p.i.. Cell binding of [(18)F]FBAM-CLIKKPF in drug-treated Jurkat cells was significantly higher compared to untreated cells, but this was not observed for [(18)F]FBAM-CPGDLSR. Dynamic PET imaging experiments showed that baseline uptake of [(18)F]FBAM-CLIKKPF in EL4 tumors was higher (SUV(5min) 0.46, SUV(60min) 0.13) compared to [(18)F]FBAM-CPGDLSR (SUV(5min) 0.16, SUV(60min) 0.10). Drug-treated EL4 tumors did not show an increased uptake for both [(18)F]FBAM-labeled peptides.

Conclusion: Although both (18)F-labeled peptides [(18)F]FBAM-CLIKKPF and [(18)F]FBAM-CPGDLSR showed higher binding to apoptotic Jurkat cells in vitro, their in vivo uptake profiles were not different in apoptotic EL4 tumors. This may explained by the relatively low potency of both compounds to compete with binding of (64)Cu-labeled annexin-V to PS. Overall the novel competitive radiometric PS-binding assay with (64)Cu-labeled annexin-V represents a versatile and very robust screening platform to analyze potential PS-binding compounds in vitro. Further studies will be necessary to evaluate alternative peptide structures toward their use as PET radiotracers imaging apoptosis in vivo.

Advances in knowledge and implications for patient care: Development of peptide-based radiotracers for imaging apoptosis in vivo remains a significant challenge.