TrifluoroacetylHYNIC-peptides have recently been shown to label directly with 99mTc as efficiently as their non-trifluoroacetylated analogs. In this work, the trifluoroacetyl (Tfa) moiety has been evaluated as a protecting group for HYNIC against reaction with strong electrophiles. Fmoc-(trifluoroacetylHYNIC)-lysine, the chosen model starting material, was found to be resistant against acetaldehyde and benzylchloroformate challenges, at 1molequiv and a 1000M excess, respectively. In contrast, the Fmoc-(HYNIC)-lysine derivative, with a free hydrazine group, was quantitatively converted to the corresponding hydrazone after a 1h incubation with acetaldehyde. Fmoc-(trifluoroacetylHYNIC)-lysine was also found to be stable over a wide pH range (3.6–10) to the acetaldehyde challenge. High efficiency 99mTc-radiolabelling (99%) was achieved in the presence of acetaldehyde using Fmoc-(trifluoroacetylHYNIC)-lysine, as compared to a poor radiolabelling yield (34%) obtained with the non-trifluoroacetylated analog. These findings firmly establish the trifluoroacetyl group as a convenient and effective protecting group for HYNIC, and as a promising alternative to currently available labelling strategies.

Two cyclized minigastrin analogues for gastrin receptor scintigraphy were synthesized and derivatized with HYNIC at the N-terminus for labeling with 99mTc. Radiolabeling efficiency, stability, cell internalization, and receptor binding on CCK-2 receptor expressing AR42J cells were studied and the biodistribution evaluated in tumor bearing nude mice, including NanoSPECT/CT imaging. Metabolites in urine, liver, and kidneys were analyzed by radio-HPLC. Radiolabeled cyclic MG showed high stability in vitro and receptor mediated uptake in AR42J cells. In the animal tumor model, fast renal clearance and low nonspecific uptake in most organs were observed. A tumor uptake >3% was calculated ex vivo 1 h p.i. for both 99mTc-EDDA-HYNIC-cyclo-MG1 and 99mTc-EDDA-HYNIC-cyclo-MG2. In an imaging study with 99mTc-EDDA-HYNIC-cyclo-MG1, the tumor was clearly visualized. The metabolite analysis indicated rapid enzymatic degradation 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 Tc-99m-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 Tc-99m/Tc-99 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.

The aim of this study was to determine the effects of assisted coor- dination by amino acids such as histidine and glutamic acid on the function of 99m Tc-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 com- binations of histidine, glutamic acid, and glycine were synthesized, radiolabeled with 99mTc/99Tc using either tricine or ethylenediamine- diacetic acid as a coligand, and analyzed by the high-performance liquid chromatography and liquid chromatography-mass spectro- metric techniques. Stability, receptor binding, and internalization and in vivo targeting in AR42J-bearing mice were assessed. Re- sults: When radiolabeling was performed using tricine as a coli- gand, the insertion of a histidine residue near the HYNIC residue resulted in the displacement of one molecule of tricine from the co- ordination 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 signif- icant 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. Conclu- sion: The insertion of histidine into the sequence of peptide-HYNIC conjugates can result in more stable, more homogeneous com- plexes that show improvements in tumor-targeting performance both in vitro and in vivo.

Fmoc-lys(HYNIC-Boc)-OH, a precursor for solid-phase synthesis of 99mTc-labeled peptides, was synthesized efficiently without HPLC purification. HPLC-ESMS showed that deprotection and decoupling of peptide from the resin with trifluoroacetic acid gave initially HYNIC-peptide, which was trifluoroacetylated upon prolonged incubation. The trifluoroacetyl-HYNIC group was hydrolyzed during 99mTc labeling, rendering deprotection unnecessary. Trifluoroacetyl-HYNIC peptide was 99mTc-labeled as efficiently, producing the same product, as HYNIC-peptide. These modifications enhance the versatility of HYNIC for 99mTc peptide labeling.

Hydrazinonicotinamide (HYNIC) is an established bifunctional complexing agent for technetium-99m ((99m)Tc) but the structure of the technetium coordination sphere remains uncertain. To gain further insight into this, we have prepared conjugates of HYNIC and hydrazinobenzoic acid (HYBA) with a model peptide, and radiolabelled them with (99m)Tc using three well-established co-ligand systems: EDDA, tricine and tricine-nicotinic acid. The labelled peptides were studied by LC-MS and by subjecting them to serum stability and protein binding assays. For each co-ligand system, HYNIC conjugates formed fewer and more stable labelled species than the corresponding HYBA conjugates. LC-MS analysis showed that all conjugates contained one hydrazine moiety bound to Tc, that binding of Tc to HYNIC-peptide and co-ligand occurs with displacement of 5H(+) indicating a Tc formal oxidation state of +5, and that the Tc has no oxo- or halide ligands. LC-MS also shows that complexes formed with the HYNIC conjugate contain fewer coordinating co-ligand molecules than the HYBA conjugate indicating that HYNIC is able to more effectively satisfy the coordination requirement of technetium, perhaps by binding in chelating mode.