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

(A) Radioactive trace of RP HPLC analysis after labeling for 60 min at room temperature. ¹¹¹In-DTPAGlu-G-CCK8 shows a retention time of approximately 22 min on the CH₃CN:H₂O gradient. Unincorporated ¹¹¹In, which has a retention time of approximately 3 min, is barely detectable. (B) Specificity of interactions of ¹¹¹In-DTPAGlu-G-CCK8 with NIH-3T3-CCKBR cells and control cells. There is a progressive increase of cell-associated activity in receptor-positive cells incubated with the radioactive peptide alone (▴). Receptor-negative cells (•) and receptor-positive cells incubated with excess unlabeled peptide (▪) show very little interaction with the radiolabeled peptide. There were 3 samples per time point; error bars indicate SDs.

Binding of ¹¹¹In-DTPAGlu-G-CCK8 on NIH-3T3-CCKBR cells (A) and A431-CCKBR cells (B) at 4°C. Both cell lines showed saturable binding of the peptide, with equivalent K_ds (22 ± 11 nmol/L [mean ± SD] for NIH-3T3-CCKBR cells; 23 ± 4 nmol/L for A431-CCKBR cells). A431-CCKBR cells had higher B_max values (1.6 × 10⁶ ± 0.4 × 10⁶ sites per cell [mean ± SD] for NIH-3T3-CCKBR cells; 4.7 × 10⁶ ± 0.4 × 10⁶ sites per cell for A431-CCKBR cells). There were 2 samples per concentration; error bars indicate SDs.

Cellular internalization and release of ¹¹¹In-DTPAGlu-G-CCK8 for A431-CCKBR cells. (A) Internalization. Cells were incubated at 4°C (□) and 37°C (▪) for 60 and 120 min with radiolabeled peptide (20 nmol/L). Excess unlabeled peptide was added during the 2-h incubation (third set of bars from left) or after the 2-h incubation and after washes in PBS to displace label on the cell surface (fourth set of bars from left). There is a progressive increase of cell-bound activity at 37°C, whereas a constant level is observed at 4°C. Coincubation with cold ligand produced a very low level of binding under both conditions. The addition of cold ligand after the 2-h incubation displaced most of the radioactivity from cells incubated at 4°C, consistent with the ligand being on the cell surface, whereas very little displacement was observed for cells incubated at 37°C, indicating that the label was in an intracellular compartment in these cells. (B) Release. Cells incubated at 37°C for 2 h were rinsed with PBS. Fresh medium was added, and cell-associated radioactivity was determined at the indicated times. There were 3 samples per time point; error bars indicate SDs.

Biodistribution of ¹¹¹In-DTPAGlu-G-CCK8 after intravenous injection. Organ-associated radioactivity is expressed as the %ID per gram of tissue normalized for a 20-g mouse. There were at least 5 samples per time point; error bars indicate SDs. GI = gastrointestinal.

Pinhole γ-camera image obtained at 2 h after injection of ¹¹¹In-DTPAGlu-G-CCK8. Avid accumulation of the peptide was seen in a CCKBR-positive xenograft (right thigh) but not in a control tumor (left thigh). Hot spots in the abdomen are consistent with kidney accumulation.