Evaluation of In-111-Labeled Cyclic RGD Peptides: Effects of Peptide and Linker Multiplicity on Their Tumor Uptake, Excretion Kinetics and Metabolic Stability

Purpose: The purpose of this study was to demonstrate the valence of cyclic RGD peptides, P-RGD (PEG(4)-c(RGDfK): PEG(4) = 15-amino-4,710,13-tetraoxapentadecanoic acid), P-RGD(2) (PEG(4)-E[c(RGDfK)](2), 2P-RGD(4) (E{PEG(4)-E[c(RGDfK)](2)}(2), 2P(4)G-RGD(4) (E{PEG(4)-E[G(3)-c(RGDfK)](2)}(2): G(3) = Gly-Gly-Gly) and 6P-RGD(4) (E{PEG(4)-E[PEG(4)-c(RGDfK)](2)}(2)) in binding to integrin alpha(v)beta(3), and to assess the impact of peptide and linker multiplicity on biodistribution properties, excretion kinetics and metabolic stability of their corresponding In-111 radiotracers. Methods: Five new RGD peptide conjugates (DOTA-P-RGD (DOTA = 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetracetic acid), DOTA-P-RGD(2), DOTA-2P-RGD(4), DOTA-2P4G-RGD(4), DOTA-6P-RGD(4)), and their In-111 complexes were prepared. The integrin alpha(v)beta(3) binding affinity of cyclic RGD conjugates were determined by a competitive displacement assay against I-125-c(RGDyK) bound to U87MG human glioma cells. Biodistribution, planar imaging and metabolism studies were performed in athymic nude mice bearing U87MG human glioma xenografts. Results: The integrin alpha(v)beta(3) binding affinity of RGD conjugates follows the order of: DOTA-6P-RGD(4) (IC50 = 0.3 +/- 0.1 nM) similar to DOTA-2P4G-RGD(4) (IC50 = 0.2 +/- 0.1 nM) similar to DOTA-2P-RGD(4) (IC50 = 0.5 +/- 0.1 nM) > DOTA-3P-RGD(2) (DOTA-PEG(4)-E[PEG(4)-c(RGDfK)](2): IC50 = 1.5 +/- 0.2 nM) > DOTA-P-RGD(2) (IC50 = 5.0 +/- 1.0 nM) >> DOTA-P-RGD (IC50 = 44.3 +/- 3.5 nM) similar to c(RGDfK) (IC50 = 49.9 +/- 5.5 nM) >> DOTA-6P-RGK(4) (IC50 = 437 +/- 35 nM). The fact that DOTA-6P-RGK(4) had much lower integrin alpha(v)beta(3) binding affinity than DOTA-6P-RGD(4) suggests that the binding of DOTA-6P-RGD(4) to integrin alpha(v)beta(3) is RGD-specific. This conclusion is consistent with the lower tumor uptake for In-111(DOTA-6P-RGK(4)) than that for In-111(DOTA-6P-RGD(4)). It was also found that the G(3) and PEG(4) linkers between RGD motifs have a significant impact on the integrin alpha(v)beta(3)-targeting capability, biodistribution characteristics, excretion kinetics and metabolic stability of In-111-labeled cyclic RGD peptides. Conclusion: On the basis of their integrin alpha(v)beta(3) binding affinity and tumor uptake of their corresponding In-111 radiotracers, it was conclude that 2P-RGD(4), 2P4G-RGD(4) and 6P-RGD(4) are most likely bivalent in binding to integrin alpha(v)beta(3), and extra RGD motifs might contribute to the long tumor retention times of In-111(DOTA-2P-RGD(4)), In-111(DOTA-2P4G-RGD(4)) and In-111(DOTA-6P-RGD(4)) than that of In-111(DOTA-3P-RGD3) at 72 h p.i. Among the In-111-labeled cyclic RGD tetramers evaluated in the glioma model, In-111(DOTA-2P4G-RGD(4)) has very high tumor uptake with the best tumor/kidney and tumor/liver ratios, suggesting that Y-90(DOTA-2P4G-RGD(4)) and Lu-177(DOTA-2P4G-RGD(4)) might have the potential for targeted radiotherapy of integrin alpha(v)beta(3)-positive tumors.Medicine, Research & ExperimentalSCI(E)16ARTICLE322-340