Evaluation of 111In-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), 2P4G-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 α(v)β(3), and to assess the impact of peptide and linker multiplicity on biodistribution properties, excretion kinetics and metabolic stability of their corresponding (111)In 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 (111)In complexes were prepared. The integrin α(v)β(3) binding affinity of cyclic RGD conjugates were determined by a competitive displacement assay against (125)I-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 α(v)β(3) binding affinity of RGD conjugates follows the order of: DOTA-6P-RGD(4) (IC(50) = 0.3 ± 0.1 nM) ~ DOTA-2P4G-RGD(4) (IC(50) = 0.2 ± 0.1 nM) ~ DOTA-2P-RGD(4) (IC(50) = 0.5 ± 0.1 nM) > DOTA-3P-RGD(2) (DOTA-PEG(4)-E[PEG(4)-c(RGDfK)](2): IC(50) = 1.5 ± 0.2 nM) > DOTA-P-RGD(2) (IC(50) = 5.0 ± 1.0 nM) >> DOTA-P-RGD (IC(50) = 44.3 ± 3.5 nM) ~ c(RGDfK) (IC(50) = 49.9 ± 5.5 nM) >> DOTA-6P-RGK(4) (IC(50) = 437 ± 35 nM). The fact that DOTA-6P-RGK(4) had much lower integrin α(v)β(3) binding affinity than DOTA-6P-RGD(4) suggests that the binding of DOTA-6P-RGD(4) to integrin α(v)β(3) is RGD-specific. This conclusion is consistent with the lower tumor uptake for (111)In(DOTA-6P-RGK(4)) than that for (111)In(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 α(v)β(3)-targeting capability, biodistribution characteristics, excretion kinetics and metabolic stability of (111)In-labeled cyclic RGD peptides. CONCLUSION On the basis of their integrin α(v)β(3) binding affinity and tumor uptake of their corresponding (111)In radiotracers, it was conclude that 2P-RGD(4), 2P4G-RGD(4) and 6P-RGD(4) are most likely bivalent in binding to integrin α(v)β(3), and extra RGD motifs might contribute to the long tumor retention times of (111)In(DOTA-2P-RGD(4)),( 111)In(DOTA-2P4G-RGD(4)) and (111)In(DOTA-6P-RGD(4)) than that of (111)In(DOTA-3P-RGD(3)) at 72 h p.i. Among the (111)In-labeled cyclic RGD tetramers evaluated in the glioma model, (111)In(DOTA-2P4G-RGD(4)) has very high tumor uptake with the best tumor/kidney and tumor/liver ratios, suggesting that (90)Y(DOTA-2P4G-RGD(4)) and (177)Lu(DOTA-2P4G-RGD(4)) might have the potential for targeted radiotherapy of integrin α(v)β(3)-positive tumors.