99mTc-HYNIC-Gastrin Peptides: Assisted Coordination of 99mTc by Amino Acid Side Chains Results in Improved Performance Both In Vitro and In Vivo

The aim of this study was to determine the effects of assisted coordination by amino acids such as histidine and glutamic acid on the function of 99mTc-labeled gastrin peptide–hydrazinonicotinamide (HYNIC) conjugates and their ability to target cholecystokinin-R in small-animal models. Methods: Three peptide–HYNIC conjugates containing the -AYGWMDF-NH2 C-terminal sequence and combinations of histidine, glutamic acid, and glycine were synthesized, radiolabeled with 99mTc/99Tc using either tricine or ethylenediaminediacetic acid as a coligand, and analyzed by the high-performance liquid chromatography and liquid chromatography–mass spectrometric techniques. Stability, receptor binding, and internalization and in vivo targeting in AR42J-bearing mice were assessed. Results: When radiolabeling was performed using tricine as a coligand, the insertion of a histidine residue near the HYNIC residue resulted in the displacement of one molecule of tricine from the coordination sphere, a reduction in the number of radiolabeled species formed, an improvement in the in vitro stability, an increase in the rate of radiopeptide internalization, and a significant improvement in tumor uptake in vivo. When radiolabeling was performed using ethylenediaminediacetic acid as a coligand, no effect on coligand binding, homogeneity, or in vitro stability was observed but a significant improvement in the internalization in vitro and tumor uptake in vivo was again found. All of the complexes formed showed similar receptor affinity in competitive radioligand binding assays. Conclusion: The insertion of histidine into the sequence of peptide–HYNIC conjugates can result in more stable, more homogeneous complexes that show improvements in tumor-targeting performance both in vitro and in vivo.