Impact of Multiple Negative Charges on Blood Clearance and Biodistribution Characteristics of (99m)Tc-Labeled Dimeric Cyclic RGD Peptides

[Image: see text] This study sought to evaluate the impact of multiple negative charges on blood clearance kinetics and biodistribution properties of (99m)Tc-labeled RGD peptide dimers. Bioconjugates HYNIC-P6G-RGD(2) and HYNIC-P6D-RGD(2) were prepared by reacting P6G-RGD(2) and P6D-RGD(2), respectively, with excess HYNIC-OSu in the presence of diisopropylethylamine. Their IC(50) values were determined to be 31 ± 5 and 41 ± 6 nM, respectively, against (125)I-echistatin bound to U87MG glioma cells in a whole-cell displacement assay. Complexes [(99m)Tc(HYNIC-P6G-RGD(2))(tricine)(TPPTS)] ((99m)Tc-P6G-RGD(2)) and [(99m)Tc(HYNIC-P6D-RGD(2))(tricine)(TPPTS)] ((99m)Tc-P6D-RGD(2)) were prepared in high radiochemical purity (RCP > 95%) and specific activity (37–110 GBq/μmol). They were evaluated in athymic nude mice bearing U87MG glioma xenografts for their biodistribution. The most significant difference between (99m)Tc-P6D-RGD(2) and (99m)Tc-P6G-RGD(2) was their blood radioactivity levels and tumor uptake. The initial blood radioactivity level for (99m)Tc-P6D-RGD(2) (4.71 ± 1.00%ID/g) was ∼5× higher than that of (99m)Tc-P6G-RGD(2) (0.88 ± 0.05%ID/g), but this difference disappeared at 60 min p.i. (99m)Tc-P6D-RGD(2) had much lower tumor uptake (2.20–3.11%ID/g) than (99m)Tc-P6G-RGD(2) (7.82–9.27%ID/g) over a 2 h period. Since HYNIC-P6D-RGD(2) and HYNIC-P6G-RGD(2) shared a similar integrin α(v)β(3) binding affinity (41 ± 6 nM versus 31 ± 5 nM), the difference in their blood activity and tumor uptake is most likely related to the nine negative charges and high protein binding of (99m)Tc-P6D-RGD(2). Despite its low uptake in U87MG tumors, the tumor uptake of (99m)Tc-P6D-RGD(2) was integrin α(v)β(3)-specific. SPECT/CT studies were performed using (99m)Tc-P6G-RGD(2) in athymic nude mice bearing U87MG glioma and MDA-MB-231 breast cancer xenografts. The SPECT/CT data demonstrated the tumor-targeting capability of (99m)Tc-P6G-RGD(2), and its tumor uptake depends on the integrin α(v)β(3) expression levels on tumor cells and neovasculature. It was concluded that the multiple negative charges have a significant impact on the blood clearance kinetics and tumor uptake of (99m)Tc-labeled dimeric cyclic RGD peptides.