title: Radiopharmacological evaluation of (18)F-labeled phosphatidylserine-binding peptides for molecular imaging of apoptosis.
abstract: BACKGROUND
  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.
  
  
  CONCLUSIONS
  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.
  
  
  CONCLUSIONS
  Development of peptide-based radiotracers for imaging apoptosis in vivo remains a significant challenge.
authors.affiliation: Department of Oncology, University of Alberta, Cross Cancer Institute, 11560 University Avenue, Edmonton AB, T6G 1Z2, Canada. Electronic address: mwuest@ualberta.ca.
authors.forename: Melinda
authors.initials: M
authors.lastname: Wuest
authors.affiliation: Department of Oncology, University of Alberta, Cross Cancer Institute, 11560 University Avenue, Edmonton AB, T6G 1Z2, Canada.
authors.forename: Amanda
authors.initials: A
authors.lastname: Perreault
authors.affiliation: Department of Oncology, University of Alberta, Cross Cancer Institute, 11560 University Avenue, Edmonton AB, T6G 1Z2, Canada.
authors.forename: Janice
authors.initials: J
authors.lastname: Kapty
authors.affiliation: Department of Oncology, University of Alberta, Cross Cancer Institute, 11560 University Avenue, Edmonton AB, T6G 1Z2, Canada.
authors.forename: Susan
authors.initials: S
authors.lastname: Richter
authors.affiliation: Department of Oncology, University of Alberta, Cross Cancer Institute, 11560 University Avenue, Edmonton AB, T6G 1Z2, Canada.
authors.forename: Christian
authors.initials: C
authors.lastname: Foerster
authors.affiliation: Department of Oncology, University of Alberta, Cross Cancer Institute, 11560 University Avenue, Edmonton AB, T6G 1Z2, Canada.
authors.forename: Cody
authors.initials: C
authors.lastname: Bergman
authors.affiliation: Department of Oncology, University of Alberta, Cross Cancer Institute, 11560 University Avenue, Edmonton AB, T6G 1Z2, Canada.
authors.forename: Jenilee
authors.initials: J
authors.lastname: Way
authors.affiliation: Department of Oncology, University of Alberta, Cross Cancer Institute, 11560 University Avenue, Edmonton AB, T6G 1Z2, Canada.
authors.forename: John
authors.initials: J
authors.lastname: Mercer
authors.affiliation: Department of Oncology, University of Alberta, Cross Cancer Institute, 11560 University Avenue, Edmonton AB, T6G 1Z2, Canada.
authors.forename: Frank
authors.initials: F
authors.lastname: Wuest
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D017209:Apoptosis; D005260:Female; D005462:Fluorine Radioisotopes; D006571:Heterocyclic Compounds / Q000737:chemistry; D006573:Heterocyclic Compounds, 1-Ring; D006801:Humans; D019169:Jurkat Cells; D051379:Mice; D008958:Models, Molecular; D057054:Molecular Imaging / Q000379:methods*; D009842:Oligopeptides / Q000737:chemistry*; D010718:Phosphatidylserines / Q000737:chemistry*; D011487:Protein Conformation
chemical_list: D005462:Fluorine Radioisotopes; D006571:Heterocyclic Compounds; D006573:Heterocyclic Compounds, 1-Ring; D009842:Oligopeptides; D010718:Phosphatidylserines; C048993:1,4,7-triazacyclononane-N,N',N''-triacetic acid
keywords: (18)F; Apoptosis; Peptide; Phosphatidylserine; Positron emission tomography (PET)