Two Technetium-99m-Labeled Cholecystokinin-8 (CCK8) Peptides for Scintigraphic Imaging of CCK Receptors

A broad spectrum of radiolabeled peptides with high affinity for receptors expressed on tumor cells is currently under preclinical and clinical investigation for scintigraphic imaging and radionuclide therapy. The present paper evaluates two 99mTc-labeled forms of the C-terminal octapeptide of cholecystokinin (CCK8): sulfated (s)CCK8, with high affinity for CCK1 and CCK2 receptors, and nonsulfated (ns)CCK8, with high affinity for CCK2 receptors but low affinity for CCK1 receptors. Peptides were conjugated with the bifunctional chelator N-hydroxysuccinimidyl hydrazino niconitate (s-HYNIC). 99mTc-labeling, performed in the presence of nicotinic acid and tricine, was highly efficient (~95%) and yielded products with a high specific activity (~700 Ci/mmol) and good stability (~5% release of radiolabel during 16 h incubation in phosphate buffered saline at 37 (degree)C). Chinese hamster ovary cells stably expressing the CCK1 receptor (CHO-CCK1 cells) internalized ~3% of added 99mTc-sCCK8 per confluent well during 2 h at 37 (degree)C. Internalization was effectively blocked by excess unlabeled sCCK8. CHO-CCK1 cells did not internalize 99mTc-nsCCK8. Displacement of 99mTc-sCCK8 and -nsCCK8 by unlabeled CCK-8 (performed at 0 (degree)C to prevent internalization) revealed 50% inhibitory concentrations (IC50) of 8 nM and >1 (mu)M, respectively. CHO-CCK2 cells internalized ~25% and ~5% of added 99mTcsCCK8 and -nsCCK8, respectively. In both cases internalization was blocked by excess unlabeled peptide. IC50 values for the displacement of 99mTc-sCCK8 and -nsCCK8 were 3 nM and 10 nM, respectively. CHO-CCK1 cell-derived tumors present in one flank of athymic mice accumulated 2.0% of injected 99mTc-sCCK8 per gram tissue at 1 h postinjection. This value decreased to 0.6% following coinjection with excess unlabeled peptide. Uptake of 99mTc-nsCCK8 was low (0.2%) and not did change by excess unlabeled peptide (0.3%). Accumulation of 99mTc-sCCK8 and -nsCCK8 by CHOCCK2 cell-derived tumors (present in the other flank) amounted to 4.2% and 0.6%, respectively. In both cases uptake was significantly reduced by excess unlabeled peptide to 1.0% and 0.4% for sCCK8 and nsCCK8, respectively. Accumulation of 99mTc-sCCK8 was also high in pancreas (11.7%), stomach (2.0%), and kidney (2.1%), whereas uptake of 99mTc-nsCCK8 was high in stomach (0.7%) and kidney (1.4%). Both radiolabeled peptides showed a rapid blood clearance. In conclusion, these data show that CCK8 analogues can be efficiently labeled with 99mTc using s-HYNIC as chelator and nicotinic acid/tricine as coligand system without compromising receptor binding. Furthermore, the present study demonstrates that CCK1 tumors hardly accumulate 99mTc-nsCCK8, CCK2 tumors accumulate 2 times more 99mTc-sCCK8 than CCK1 tumors, and CCK2 tumors accumulate 15 times more 99mTc-sCCK8 than 99mTc-nsCCK8. Although accumulation in some nontarget organs was also higher with 99mTcsCCK8, this may not reflect the human situation due to a different receptor expression pattern in humans as compared to mice. Therefore, further studies are warranted to investigate the possible use of 99mTc-sCCK8 for scintigraphic imaging of CCK receptor-positive tumors in humans.