Evaluation of K(HYNIC)₂ as a Bifunctional Chelator for ^99mTc-Labeling of Small Biomolecules

Abstract

This study sought to evaluate K(HYNIC)2 (K = lysine and HYNIC = 6-hydrazinonicotinyl) as a bifunctional chelator for 99mTc-labeling of biomolecule. In this study, four K(HYNIC)2–conjugated cyclic RGD peptides, K(HYNIC)2-RGD2 (RGD2 = E[c(RGDfK)]2), K(HYNIC)2-3G-RGD2 (3G-RGD2 = Gly-Gly-Gly-E[Gly-Gly-Gly-c(RGDfK)]2), K(HYNIC)2-2P-RGD2 (2P-RGD2 = E[PEG4-c(RGDfK)]2, and PEG4 = 15-amino-4,7,10,13-tetraoxapentadecanoic acid), and K(HYNIC)2-3P-RGD2 (3P-RGD2 = PEG4-E[PEG4-c(RGDfK)]2) were prepared, and evaluated for their integrin αvβ3 binding affinity. IC50 values were determined to be 47 ± 2, 35 ± 2, 37 ± 2, 85 ± 2 and 422 ± 15 nM for K(HYNIC)2-2P-RGD2, K(HYNIC)2-3P-RGD2, K(HYNIC)2-3G-RGD2, K(HYNIC)2-RGD2 and c(RGDyK), respectively, against 125I-echistatin bound to U87MG cells. Macrocyclic complexes [99mTc(K(HYNIC)2-RGD2)(tricine)] (1), [99mTc(K(HYNIC)2-3G-RGD2)(tricine)] (2), [99mTc(K(HYNIC)2-2P-RGD2)(tricine)] (3), and [99mTc(K(HYNIC)2-3P-RGD2)(tricine)] (4) were prepared, and evaluated in athymic nude mice bearing U87MG glioma xenografts for their tumor targeting capability and biodistribution. It was found that 1 – 4 all had high solution stability and more than two isomers, as evidenced by the presence of multiple radiometric peaks in their radio-HPLC chromatograms. The tumor uptake of 1 – 4 was 3.78 ± 0.81, 7.46 ± 1.68, 9.74 ± 1.65 and 8.59 ± 1.52 %ID/g, respectively, which was completely consistent with trend of integrin αvβ3 binding affinity for cyclic RGD peptides. Replacing [99mTc(HYNIC)(tricine)(TPPTS)] (TPPTS = trisodium triphenylphosphine-3,3′,3″-trisulfonate) with [99mTc(K(HYNIC)2)(tricine)] had little impact on radiotracer tumor uptake; but it had significant effect on the uptake of radiotracer in kidneys, lungs and spleen. The tumor was clearly visualized by SPECT/CT with excellent contrast in a glioma-bearing mouse administered with 4. K(HYNIC)2 would be particularly useful for 99mTc-labeling of small biomolecules with one or more disulfide linkages.

Radiolabeled Cyclic RGD Peptide Bioconjugates as Radiotracers Targeting Multiple Integrins

“…107–124 Studies show that the α v β 3 is over-expressed on both tumor cells and activated endothelial cells of tumor neovasculature. 44,47,115 It is believed that the α v β 3 on endothelial cells modulate the cell adhesion and migration during tumor angiogenesis, while the α v β 3 on carcinoma cells potentiate metastasis by facilitating the invasion and movement of tumor cells across blood vessels. 119–124 It has also been shown that the α v β 3 expression levels correlate well with the metastatic potential and aggressiveness of solid tumors.…”

“…46,47 Since [ 99m Tc(HYNIC-K(NIC))(tricine)] is structurally similar to [ 99m Tc(K(HYNIC) 2 )(tricine)] (Figure 10), it was not surprising that 99m Tc-3P-RGD 2 -A and 99m Tc-3P-RGD 2 -B shared almost identical biodistribution properties. 47 A major advantage of using K(HYNIC) 2 and HYNIC-K(NIC) as BFCs is the use of SnCl 2 as a reducing agent during 99m Tc-labeling because TPPTS can easily reduce the S-S disulfide bond, which is often important for small biomolecules to maintain their biological activity and tumor-targeting capability. The disadvantage of K(HYNIC) 2 and HYNIC-K(NIC) is that there are more than two isomers in [ 99m Tc(HYNIC-K(NIC))(tricine)] and [ 99m Tc(K(HYNIC) 2 )(tricine)].…”

“…The disadvantage of K(HYNIC) 2 and HYNIC-K(NIC) is that there are more than two isomers in [ 99m Tc(HYNIC-K(NIC))(tricine)] and [ 99m Tc(K(HYNIC) 2 )(tricine)]. 46,47 …”

“…It is also unknown about which receptor binding unit actually contributes to the tumor uptake of “bifunctional radiotracers”. Since the PC-3 tumors have low α v β 3 expression and the MDA-MB-435 tumors have little or no expression of GRPR, 40,47,181,182 the xenografted PC-3 and MDA-MB-435 tumor-bearing models are not appropriate to prove the concept of “bivalent heterodimers” because the α v β 3 and GRPR receptors must be co-localized and the distance between them must be short in order for the bifunctional radiotracer to achieve bivalency. Otherwise, it is very difficult to observe the “synergetic effect” even if they are able to target individual receptors.…”

“…200 This is consistent with the α v β 3 expression levels on three xenografted tumors. 44,47 Highly vascularized U87MG tumors with high levels of α v β 3 and CD31 have better response to linifanib therapy than poorly vascularized PC-3 tumors with low levels of α v β 3 and CD31 (Figure 15). Thus, 99m Tc-3P-RGD 2 has the potential as the pre-treatment screening tool to select appropriate patients who will benefits most the antiangiogenic treatment.…”