AbstractRegulatory peptides and their analogs are being extensively investigated as radiopharmaceuticals for cancer imaging. In particular, cholecystokinin (CCK) receptors of the subtype B (CCK‐BR) have been shown to be overexpressed in certain neuroendocrine tumors including medullary thyroid cancer. Our recent work has focused on new methods to radiolabel the CCK8 peptide with 111In or 99mTc for the purpose of developing radiopharmaceuticals for in vivo CCK‐B receptor imaging. Labeling of CCK8 with 111In was achieved at the N‐terminus of the peptide by adding, in solid phase, a glutamate coupled diethylenetriaminepentaacetic acid (DTPA) moiety through a glycine linker, yielding DTPA–Glu–G–CCK8. For labeling with 99mTc, the CCK8 peptide was modified at its N‐terminus by introducing, in the following order—cysteine, glycine, and a diphenylphosphinopropionyl moiety—giving a 10‐residue peptide derivative, Phos–GC–CCK8. A cell culture model was developed for the purpose of evaluating the binding properties of these two ligands. The human epidermoid carcinoma cell line, A431, was transfected with a plasmid containing the full coding sequence of the human CCK‐BR under a strong viral promoter, obtaining a number of receptors in the range of 2–5 × 106 per cell. Control cells were transfected with vector alone. An animal tumor model utilizing these two cell lines was developed to evaluate the specificity of interaction with the CCK‐BR and biodistribution properties of the compounds. CCK‐BR positive and control cells were subcutaneously injected in opposite flanks of CD1 female nude mice in order to obtain xenografts differing only in their ability to express CCK‐B receptors. High performance liquid chromatography (HPLC) and other chromatographic methods were utilized to assess stability of the radiolabeled compounds after injection. Both 111In–DTPA–Glu–G–CCK8 and 99mTc–Phos–GC–CCK8 showed similar binding affinities for cultured CCK‐BR expressing cells, with dissociation constants in the range of 20–40 nM. With the two xenograft approach, we were able to demonstrate specific interaction with the receptor of both CCK analogs in our animal model. The data obtained shows rapid specific localization of both compounds on the CCK‐BR overexpressing xenografts. Both tracers show rapid plasma clearance of unbound peptide. Clearance of 111In–DTPA–Glu–G–CCK8 appears to be preferentially through the kidneys, whereas 99mTc–Phos–GC–CCK8 clearance occurs both through kidneys and the hepatobiliary system. Both our labeling approaches appear adequate for clinical use of peptide based radiopharmaceuticals, although 99mTc–Phos–GC–CCK8 shows elevated accumulation in the gastrointestinal tract, which causes high background activity. © 2003 Wiley Periodicals, Inc. Biopolymers (Pept Sci) 66: 370–380, 2002