title: In vitro and in vivo evaluation of 64Cu-labeled SarAr-bombesin analogs in gastrin-releasing peptide receptor-expressing prostate cancer.
abstract: Bombesin is a 14-amino-acid amphibian peptide that binds with high affinity to the gastrin-releasing peptide receptor (GRPR), which is overexpressed on a variety of solid tumors. It has been demonstrated that bombesin analogs can be radiolabeled with a variety of radiometals for potential diagnosis and treatment of GRPR-positive tumors. In this regard, several studies have used different chelators conjugated to the 8 C-terminal amino acids of bombesin(7-14) for radiolabeling with (64)Cu. These analogs have demonstrated GRPR-specific small-animal PET of tumors but have various advantages and disadvantages. The objective of this study was to conjugate the previously described (1-N-(4-aminobenzyl)-3,6,10,13,16,19-hexaazabicyclo[6.6.6]-eicosane-1,8-diamine) (SarAr) chelator to bombesin(7-14), radiolabel the conjugate with (64)Cu, and evaluate in vitro and in vivo.
  
  
  METHODS
  SarAr was synthesized as previously published and conjugated to bombesin(7-14) by solid-phase peptide synthesis using standard Fmoc chemistry. Succinic acid (SA), 8-aminooctanoic acid (Aoc), and Gly-Ser-Gly (GSG) were used as linkers between SarAr and bombesin(7-14) to yield the resulting SarAr-SA-Aoc-bombesin(7-14) and SarAr-SA-Aoc-GSG-bombesin(7-14) peptides. The unlabeled peptides were evaluated in a competitive binding assay using PC-3 prostate cancer cells and (125)I-Tyr(4)-bombesin to determine the inhibitory concentration of 50%. The peptides were radiolabeled with (64)Cu and evaluated for internalization into PC-3 cells in vitro and for in vivo tumor uptake in mice bearing PC-3 xenografts using biodistribution and small-animal PET/CT studies.
  
  
  RESULTS
  The competitive binding assay demonstrated that both SarAr-SA-Aoc-bombesin(7-14) and SarAr-SA-Aoc-GSG-bombesin(7-14) bound with high affinity to GRPR with an inhibitory concentration of 50% of 3.5 and 4.5 nM, respectively. Both peptides were radiolabeled with (64)Cu at room temperature without further purification and demonstrated similar internalization into PC-3 cells. In vivo, the radiolabeled peptides demonstrated tumor-specific uptake (13.0 and 8.5 percentage injected dose per gram for (64)Cu-SarAr-SA-Aoc-bombesin(7-14) and (64)Cu-SarAr-SA-Aoc-GSG-bombesin(7-14), respectively, at 1 h) and imaging that was comparable to, or better than, that of the previously reported (64)Cu-labeled bombesin analogs. The (64)Cu-SarAr-SA-Aoc-GSG-bombesin(7-14) had more rapid blood clearance and lower tumor and normal-tissue uptake than (64)Cu-SarAr-SA-Aoc-bombesin(7-14), resulting in similar tumor-to-blood ratios for each analog (15.1 vs. 11.3 for (64)Cu-SarAr-SA-Aoc-bombesin(7-14) and (64)Cu-SarAr-SA-Aoc-GSG-bombesin(7-14), respectively, at 1 h).
  
  
  CONCLUSIONS
  These studies demonstrate that (64)Cu-SarAr-SA-Aoc-bombesin(7-14) and (64)Cu-SarAr-SA-Aoc-GSG-bombesin(7-14) bound with high affinity to GRPR-expressing cells and that these peptides can be used for PET of GRPR-expressing prostate cancer.
authors.affiliation: Department of Radiation Oncology, Washington University School of Medicine, St. Louis, MO, USA.
authors.forename: Kimberly A
authors.initials: KA
authors.lastname: Lears
authors.forename: Riccardo
authors.initials: R
authors.lastname: Ferdani
authors.forename: Kexian
authors.initials: K
authors.lastname: Liang
authors.forename: Alexander
authors.initials: A
authors.lastname: Zheleznyak
authors.forename: Rebecca
authors.initials: R
authors.lastname: Andrews
authors.forename: Christopher D
authors.initials: CD
authors.lastname: Sherman
authors.forename: Samuel
authors.initials: S
authors.lastname: Achilefu
authors.forename: Carolyn J
authors.initials: CJ
authors.lastname: Anderson
authors.forename: Buck E
authors.initials: BE
authors.lastname: Rogers
mesh_terms: D000814:Aniline Compounds / Q000737:chemistry / Q000493:pharmacokinetics; D000818:Animals; D001839:Bombesin / Q000031:analogs & derivatives / Q000493:pharmacokinetics*; D019086:Bridged Bicyclo Compounds, Heterocyclic / Q000737:chemistry / Q000493:pharmacokinetics; D003301:Copper Radioisotopes / Q000493:pharmacokinetics*; D005260:Female; D019886:Gastrin-Releasing Peptide / Q000378:metabolism*; D006801:Humans; D007553:Isotope Labeling / Q000379:methods; D008297:Male; D008657:Metabolic Clearance Rate; D051379:Mice; D009928:Organ Specificity; D011471:Prostatic Neoplasms / Q000000981:diagnostic imaging* / Q000378:metabolism*; D011877:Radionuclide Imaging; D019275:Radiopharmaceuticals / Q000138:chemical synthesis / Q000493:pharmacokinetics; D014018:Tissue Distribution
chemical_list: C525132:1-N-(4-aminobenzyl)-3,6,10,13,16,19-hexaazabicyclo(6.6.6)eicosane-1,8-diamine; D000814:Aniline Compounds; D019086:Bridged Bicyclo Compounds, Heterocyclic; D003301:Copper Radioisotopes; D019275:Radiopharmaceuticals; D019886:Gastrin-Releasing Peptide; D001839:Bombesin