title: Preparation of a promising angiogenesis PET imaging agent: 68Ga-labeled c(RGDyK)-isothiocyanatobenzyl-1,4,7-triazacyclononane-1,4,7-triacetic acid and feasibility studies in mice.
abstract: Arg-Gly-Asp (RGD) derivatives have been labeled with various radioisotopes for the imaging of angiogenesis in ischemic tissue, in which alpha(v)beta(3) integrin plays an important role. In this study, cyclic Arg-Gly-Asp-D-Tyr-Lys [c(RGDyK)] was conjugated with 2-(p-isothiocyanatobenzyl)-1,4,7-triazacyclononane-1,4,7-triacetic acid (SCN-Bz-NOTA) and then labeled with (68)Ga. The labeled RGD so produced was subjected to an in vitro binding assay and in vivo biodistribution and PET studies.
  
  
  METHODS
  A mixture of SCN-Bz-NOTA (660 nmol) and c(RGDyK) (600 nmol) in 0.1 M sodium carbonate buffer (pH 9.5) was allowed to react for 20 h at room temperature in the dark for thiourea bond formation. The conjugate obtained was purified by semipreparative high-performance liquid chromatography (HPLC). The purified c(RGDyK)-SCN-Bz-NOTA (NOTA-RGD) was then labeled with (68)Ga from a (68)Ge/(68)Ga generator and purified by semipreparative HPLC. A competitive binding assay for c(RGDyK) and NOTA-RGD was performed with (125)I-c(RGDyK) as a radioligand and alpha(v)beta(3) integrin-coated plates as a solid phase. (68)Ga-NOTA-RGD (0.222 MBq/100 microL) was injected, through a tail vein, into mice with hind limb ischemia and into mice bearing human colon cancer SNU-C4 xenografts. Biodistribution and imaging studies were performed at 1 and 2 h after injection.
  
  
  RESULTS
  The labeling of NOTA-RGD with (68)Ga was straightforward. The K(i) values of c(RGDyK) and NOTA-RGD were 1.3 and 1.9 nM, respectively. In the biodistribution study, the mean +/- SD uptake of (68)Ga-NOTA-RGD by ischemic muscles was 1.6+/-0.2 percentage injected dose per gram (%ID/g); this uptake was significantly blocked by cold c(RGDyK) to 0.6+/-0.3 %ID/g (P<0.01). Tumor uptake was 5.1+/-1.0 %ID/g, and the tumor-to-blood ratio was 10.3+/-4.8. Small-animal PET revealed rapid excretion through the urine and high levels of tumor and kidney uptake.
  
  
  CONCLUSIONS
  Stable (68)Ga-NOTA-RGD was obtained in a straightforward manner at a high yield and showed a high affinity for alpha(v)beta(3) integrin, specific uptake by angiogenic muscles, a high level of uptake by tumors, and rapid renal excretion. (68)Ga-NOTA-RGD was found to be a promising radioligand for the imaging of angiogenesis.
authors.affiliation: Department of Nuclear Medicine, Cancer Research Institute College of Medicine, Seoul National University, Jongro-gu, Seoul, Korea. jmjng@snu.ac.kr
authors.forename: Jae Min
authors.initials: JM
authors.lastname: Jeong
authors.forename: Mee Kyung
authors.initials: MK
authors.lastname: Hong
authors.forename: Young Soo
authors.initials: YS
authors.lastname: Chang
authors.forename: Yun-Sang
authors.initials: YS
authors.lastname: Lee
authors.forename: Young Joo
authors.initials: YJ
authors.lastname: Kim
authors.forename: Gi Jeong
authors.initials: GJ
authors.lastname: Cheon
authors.forename: Dong Soo
authors.initials: DS
authors.lastname: Lee
authors.forename: June-Key
authors.initials: JK
authors.lastname: Chung
authors.forename: Myung Chul
authors.initials: MC
authors.lastname: Lee
mesh_terms: D000818:Animals; D056831:Coordination Complexes / Q000138:chemical synthesis / Q000737:chemistry*; D005240:Feasibility Studies; D005710:Gallium Radioisotopes; D006571:Heterocyclic Compounds / Q000138:chemical synthesis / Q000737:chemistry*; D006614:Hindlimb / Q000098:blood supply / Q000473:pathology; D006801:Humans; D000860:Hypoxia / Q000000981:diagnostic imaging; D007511:Ischemia / Q000000981:diagnostic imaging; D017879:Isothiocyanates / Q000138:chemical synthesis / Q000737:chemistry*; D008297:Male; D051379:Mice; D009389:Neovascularization, Pathologic / Q000000981:diagnostic imaging*; D009842:Oligopeptides / Q000138:chemical synthesis / Q000737:chemistry*; D049268:Positron-Emission Tomography / Q000379:methods*; D013194:Staining and Labeling; D014183:Transplantation, Heterologous
chemical_list: C584295:68Ga-1,4,7-triazacyclononane-1,4-7-triacetic acid-isothiocyanatobenzyl-c(Arg-Gly-Asp-Tyr-Lys); D056831:Coordination Complexes; D005710:Gallium Radioisotopes; D006571:Heterocyclic Compounds; D017879:Isothiocyanates; D009842:Oligopeptides; C082060:2-(4-isothiocyanatobenzyl)-1,4,7-triazacyclononane-1,4,7-triacetic acid; C047981:arginyl-glycyl-aspartic acid