Technetium Complexes of a Hydrazinonicotinamide-Conjugated Cyclic Peptide and 2-Hydrazinopyridine:  Synthesis and Characterization

Abstract

Ternary ligand technetium complexes of a hydrazinonicotinamide-conjugated cyclic peptide (HYNICtide: cyclo(D-Val-NMeArg-Gly-Asp-Mamb(5-(6-(6-hydrazinonicotinamido)hexanamide)))) and 2-hydrazinopyridine (HYPY) were prepared and characterized by various spectroscopic methods. The HPLC concordance experiments for (99m)Tc and (99)Tc analogues show clearly that the same complexes are prepared on the no-carrier-added ((99m)Tc) and the carrier-added ((99)Tc) levels. Using a chirality experiment, it was demonstrated that the presence of two radiometric peaks in the HPLC chromatograms of RP444, RP445, and RP446 is due to the resolution of diastereomers, which result from the presence of chiral cyclic peptide and the formation of two enantiomers of the technetium chelate. In a ligand challenge experiment, we found that the high solution stability of these ternary ligand [(99m)Tc]HYNICtide complexes is due to their kinetic inertness. The 1:1:1:1 composition for Tc:HYNICtide:L:tricine (L = TPPTS, TPPDS, and TPPMS) in these ternary ligand [(99)Tc]HYNICtide complexes is confirmed by (1)H NMR and FAB mass spectral data and is completely consistent with that determined on the tracer ((99m)Tc) level. In addition, the IC(50) values of RP444, RP445, and RP446 and the two isomeric forms of RP444 were determined using a platelet IIb/IIIa binding assay. Both isomeric forms of RP444 were found to have the same binding affinity (IC(50) = 13 +/- 2 nM). Complexes [(99)Tc(HYPY)(PPh(3))(2)Cl(2)] and [(99)Tc(HYPY)(PPh(3))(tricine)] were isolated from the reaction of HYPY with [n-Bu(4)N][TcOCl(4)(-)] in the presence of excess tricine and triphenylphosphine. [(99)Tc(HYPY)(PPh(3))(tricine)] serves as a model for ternary ligand [(99m)Tc]HYNICtide complexes. Both complexes have been characterized by HPLC, spectroscopic (IR, NMR, and FAB-MS) methods, and elemental analysis. The HPLC concordance for complexes [(99m)Tc(HYPY)(PPh(3))(tricine)] and [(99)Tc(HYPY)(PPh(3))(tricine)] shows that the two complexes are identical. The NMR ((1)H and (13)C) data suggests that the complex [(99)Tc(HYPY)(PPh(3))(tricine)] have an octahedral coordination geometry with a monodentate diazenido HYPY, a tetradentate tricine, and a monodentate triphenylphosphine coligand.

6-Hydrazinonicotinamide Derivatives as Bifunctional Coupling Agents for 99mTc-Labeling of Small Biomolecules

“…Using a chirality experiment, it has been demonstrated that the presence of two isomeric forms is due to the resolution of diastereomers resulting from a combination of the chiral technetium chelate and chiral centers in the cyclic peptide. It was also found that the two diastereomers of RP444 possess equal biological activity [46]. The chemical composition of these ternary ligand 99m Tc complexes has been determined to be 1:1:1:1 for Tc:HYNIC:L:tricine through a series of mixed-ligand experiments [42], and was confirmed by LC-MS at both the tracer level and the macroscopic level [46,54].…”

“…Since then, the HYNIC technology has successfully been used for the 99m Tc-labeling of antibodies [3][4][5][6][7][8] and small biomolecules (BMs), including chemotactic peptides [9][10][11][12][13][14][15][16][17][18], somatostatin analogs [19][20][21][22][23][24][25],"stealth" liposomes [26,27], antisense oligonucleotides [28][29][30][31], a folate receptor ligand [32], and polypeptides [32][33][34]. A ternary ligand system (HYNIC, tricine, and trisodium triphenylphosphine-3,3¢,3≤-trisulfonate, TPPTS) has been used for the 99m Tc-labeling of chemotactic peptides [35] and leukotriene B 4 (LTB 4 ) receptor antagonists [36][37][38][39] for imaging infection and inflammation, integrin a v b 3 receptor antagonists for tumor imaging [40], and a glycoprotein IIb/IIIa (GPIIb/IIIa) receptor antagonist for imaging thrombosis [41][42][43][44][45][46]…”

“…A ternary ligand system (HYNIC, tricine, and trisodium triphenylphosphine-3,3¢,3≤-trisulfonate, TPPTS) has been used for the 99m Tc-labeling of chemotactic peptides [35] and leukotriene B 4 (LTB 4 ) receptor antagonists [36][37][38][39] for imaging infection and inflammation, integrin a v b 3 receptor antagonists for tumor imaging [40], and a glycoprotein IIb/IIIa (GPIIb/IIIa) receptor antagonist for imaging thrombosis [41][42][43][44][45][46][47][48]. The advantage of HYNIC is its high labeling efficiency (rapid and high yield radiolabeling) and the high in vivo stability of the resulting complexes [35][36][37][38][39][40][41][42][43][44][45][46][49][50][51][52]. Since HYNIC can only occupy one or two coordination sites, a coligand such as tricine is needed to complete the coordination sphere of technetium.…”

“…Since HYNIC can only occupy one or two coordination sites, a coligand such as tricine is needed to complete the coordination sphere of technetium. The choice of coligands allows easy modification of hydrophilicity and pharmacokinetics of 99m Tc-labeled small BMs [35][36][37][38][39][40][41][42][43][44][45][46][49][50][51][52]]. …”

Coligand effects on the solution stability, biodistribution and metabolism of the 99mTc-labeled cyclic RGDfK tetramer

“…For the last decade, we have been using ternary ligand systems (HYNIC, tricine, water soluble phosphine or pyridine analog) for 99m Tc-labeling of small biomolecules, including chemotactic peptides [40] and LTB 4 antagonists [41,42] for imaging infection/inflammation, integrin α v β 3 antagonists for tumor imaging [27][28][29][30], and a GPIIb/IIIa antagonist for imaging thrombosis [43][44][45][46]. Regardless of the biomolecule, the Tc:HYNIC:L:tricine ratio in [ 99m Tc(HYNIC-biomolecule)(tricine)(L)] always remains 1:1:1:1 as demonstrated via mixed ligand experiments at the tracer level [43,44], and has been further confirmed by the LC-MS data of [ 99m Tc(HYNIC-biomolecule)(tricine)(L)] (L = TPPTS and ISONIC) at both macroscopic and tracer levels [47,48]. Based these results, it is reasonable to believe that ternary ligand complexes [ 99m Tc(HYNIC-tetramer)(tricine)(L)] (L = ISONIC and PDA) contain one HYNIC-tetramer, one tricine, and one monodentate ISONIC or PDA.…”