Synthesis and evaluation of (68)Ga-labeled dimeric cNGR peptide for PET imaging of CD13 expression with ovarian cancer xenograft

Introduction: Previous studies have shown that peptides containing the asparagine-glycine-arginine (NGR) sequence can specifically bind to CD13 (aminopeptidase N) receptor, a tumor neovascular biomarker that is over-expressed on the surface of angiogenic blood vessels and various tumor cells, and it plays an important role in angiogenesis and tumor progression. In the present study, we aimed to evaluate the efficacy of a gallium-68 ((68)Ga)-labeled dimeric cyclic NGR (cNGR) peptide as a new molecular probe that binds to CD13 in vitro and in vivo. Materials and Methods: A dimeric cNGR peptide conjugated with 1,4,7,10-tetraazacyclododecane-N,N',N'',N'''-tetraacetic acid (DOTA) was synthesized and labeled with (68)Ga. In vitro uptake and binding analyses of the (68)Ga- DOTA-c(NGR)(2) were performed in two ovarian tumor cell lines, ES2 and SKOV3, which had different CD13 expression patterns. An in vivo biodistribution study was performed in normal mice, and micro positron emission tomography (PET) imaging was conducted in nude mice bearing ES2 and SKOV3 tumors. Results: (68)Ga-DOTA-c(NGR)(2) was prepared with high radiochemical purity (>95%), and it was stable both in saline at room temperature and in bovine serum at 37°C for 3 h. In vitro studies showed that the uptake of (68)Ga-DOTA-c(NGR)(2) in ES2 cells was higher compared with SKOV3 cells, and such uptake could be blocked by the cold DOTA-c(NGR)(2). Biodistribution studies demonstrated that (68)Ga-DOTA-c(NGR)(2) was rapidly cleared from blood and mainly excreted from the kidney. MicroPET imaging of ES2 tumor xenografts showed the focal uptake of (68)Ga-DOTA-c(NGR)(2) in tumors from 1 to 1.5 h post-injection. The high-contrast tumor visualization occurred at 1 h, corresponding to the highest tumor/background ratio of 10.30±0.26. The CD13-specific tumor targeting of the (68)Ga-DOTA-c(NGR)(2) was further supported by the reduced uptake of the probe in ES2 tumors by co-injection of the unlabeled cold peptide. In SKOV3 tumor models, the tumor was not obviously visible under the same imaging conditions. Conclusions: (68)Ga-DOTA-c(NGR)(2) was easily synthesized, and it showed favorable CD13-specific targeting ability by in vitro data and microPET imaging with ovarian cancer xenografts. Collectively, (68)Ga-DOTA-c(NGR)(2) might be a potential PET imaging probe for non-invasive evaluation of the CD13 receptor expression in tumors.