In Vitro and In Vivo Evaluation of 111In- DTPAGlu-G-CCK8 for Cholecystokinin-B Receptor Imaging

Regulatory peptides and their analogs are being extensively investigated as radiopharmaceuticals for cancer imaging and therapy. Receptors of the cholecystokinin family have been shown to be overexpressed in different types of neuroendocrine tumors. The purposes of this study were to evaluate the chole- cystokinin octapeptide amide (CCK8) peptide tagged with a diethylenetriaminepentaacetic acid derivative (DTPAGlu) and to test whether a 111In-labeled conjugate (111In-DTPAGlu-G-CCK8, a derivative containing the chelating agent DTPAGlu bound through a glycine linker at the N-terminal end of the bioactive peptide CCK8) is suitable for cholecystokinin-B receptor (CCKBR) imaging. Methods: CCK8 was synthesized by solid- phase techniques and covalently coupled to DTPAGlu through a glycine linker at its amino terminus. The compound was labeled with 111In. The radiochemical purity and stability of the com- pound were assessed by chromatographic methods. NIH-3T3 and A431 cells overexpressing CCKBR were used to charac- terize the in vitro properties of the compound. Nude mice bear- ing control and CCKBR-overexpressing A431 xenografts were used as an in vivo model. Results: DTPAGlu-G-CCK8 showed rapid and efficient labeling with 111In. The radiolabeled conju- gate showed specific binding to both cell lines overexpressing CCKBR. Binding was saturable, with a dissociation constant of ?20 nmol/L in both cell systems. Both cell lines showed inter- nalization of the ligand after interaction with the receptor. Bio- distribution studies showed rapid localization of 111In-DTPAGlu- G-CCK8 on CCKBR-overexpressing A431 xenografts that was severalfold higher than that on control tumors at all time points tested. Unbound activity showed rapid clearance of over 80% through the kidneys by 30 min after injection. The labeled pep- tide conjugate was very stable in serum but showed a rapid breakdown after injection. Incubation with kidney homogenates suggested that most breakdown occurred in the kidneys, favor- ing the clearance of unbound activity. Conclusion: Our findings indicate that the in vitro and in vivo characteristics of 111In- DTPAGlu-G-CCK8 are favorable for CCKBR imaging, as the peptide shows high-affinity binding to the receptor, is internal- ized in CCKBR-expressing cells, and shows avid uptake in CCKBR-overexpressing xenografts, with rapid clearance of un- bound radioactivity through the kidneys. Furthermore, the ease of synthesis, high labeling efficiency, and chemical stability of DTPAGlu make this chelating moiety an ideal candidate for widespread use in peptide radiolabeling for nuclear medicine applications.