| Summary: | <p><b>Purpose: </b>The purpose of this study was to demonstrate the valence of cyclic RGD peptides, P-RGD (PEG<sub>4</sub>-c(RGDfK): PEG<sub>4</sub> = 15-amino-4,710,13-tetraoxapentadecanoic acid), P-RGD<sub>2</sub> (PEG<sub>4</sub>-E[c(RGDfK)]<sub>2</sub>, 2P-RGD<sub>4</sub> (E{PEG<sub>4</sub>-E[c(RGDfK)]<sub>2</sub>}<sub>2</sub>, 2P4G-RGD<sub>4</sub> (E{PEG<sub>4</sub>-E[G<sub>3</sub>-c(RGDfK)]<sub>2</sub>}<sub>2</sub>: G<sub>3</sub> = Gly-Gly-Gly) and 6P-RGD<sub>4</sub> (E{PEG<sub>4</sub>-E[PEG<sub>4</sub>-c(RGDfK)]<sub>2</sub>}<sub>2</sub>) in binding to integrin α<sub>v</sub>β<sub>3</sub>, and to assess the impact of peptide and linker multiplicity on biodistribution properties, excretion kinetics and metabolic stability of their corresponding <sup>111</sup>In radiotracers.</p><p><b>Methods: </b>Five new RGD peptide conjugates (DOTA-P-RGD (DOTA =1,4,7,10-tetraazacyclododecane-1,4,7,10-tetracetic acid), DOTA-P-RGD<sub>2</sub>, DOTA-2P-RGD<sub>4</sub>, DOTA-2P4G-RGD<sub>4</sub>, DOTA-6P-RGD<sub>4</sub>), and their <sup>111</sup>In complexes were prepared. The integrin α<sub>v</sub>β<sub>3</sub> binding affinity of cyclic RGD conjugates were determined by a competitive displacement assay against <sup>125</sup>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.</p><p><b>Results:</b> The integrin α<sub>v</sub>β<sub>3</sub> binding affinity of RGD conjugates follows the order of: DOTA-6P-RGD<sub>4</sub> (IC<sub>50</sub> = 0.3 ± 0.1 nM) <b>~ </b>DOTA-2P4G-RGD<sub>4</sub> (IC<sub>50</sub> = 0.2 ± 0.1 nM) ~ DOTA-2P-RGD<sub>4</sub> (IC<sub>50</sub> = 0.5 ± 0.1 nM) > DOTA-3P-RGD<sub>2</sub> (DOTA-PEG<sub>4</sub>-E[PEG<sub>4</sub>-c(RGDfK)]<sub>2</sub>: IC<sub>50</sub> = 1.5 ± 0.2 nM) <b>> </b>DOTA-P-RGD<sub>2</sub> (IC<sub>50</sub> = 5.0 ± 1.0 nM) >> DOTA-P-RGD (IC<sub>50</sub> = 44.3 ± 3.5 nM) ~ c(RGDfK) (IC<sub>50</sub> = 49.9 ± 5.5 nM) >> DOTA-6P-RGK<sub>4</sub> (IC<sub>50</sub> = 437 ± 35 nM). The fact that DOTA-6P-RGK<sub>4</sub> had much lower integrin α<sub>v</sub>β<sub>3</sub> binding affinity than DOTA-6P-RGD<sub>4</sub> suggests that the binding of DOTA-6P-RGD<sub>4</sub> to integrin α<sub>v</sub>β<sub>3</sub> is RGD-specific. This conclusion is consistent with the lower tumor uptake for <sup>111</sup>In(DOTA-6P-RGK<sub>4</sub>) than that for <sup>111</sup>In(DOTA-6P-RGD<sub>4</sub>). It was also found that the G<sub>3</sub> and PEG<sub>4</sub> linkers between RGD motifs have a significant impact on the integrin α<sub>v</sub>β<sub>3</sub>-targeting capability, biodistribution characteristics, excretion kinetics and metabolic stability of <sup>111</sup>In-labeled cyclic RGD peptides.</p><p><b>Conclusion: </b>On the basis of their integrin α<sub>v</sub>β<sub>3</sub> binding affinity and tumor uptake of their corresponding <sup>111</sup>In radiotracers, it was conclude that 2P-RGD<sub>4</sub>, 2P4G-RGD<sub>4</sub> and 6P-RGD<sub>4</sub> are most likely bivalent in binding to integrin α<sub>v</sub>β<sub>3</sub>, and extra RGD motifs might contribute to the long tumor retention times of <sup>111</sup>In(DOTA-2P-RGD<sub>4</sub>),<sup> 111</sup>In(DOTA-2P4G-RGD<sub>4</sub>) and <sup>111</sup>In(DOTA-6P-RGD<sub>4</sub>) than that of <sup>111</sup>In(DOTA-3P-RGD<sub>3</sub>) at 72 h p.i. Among the <sup>111</sup>In-labeled cyclic RGD tetramers evaluated in the glioma model, <sup>111</sup>In(DOTA-2P4G-RGD<sub>4</sub>) has very high tumor uptake with the best tumor/kidney and tumor/liver ratios, suggesting that <sup>90</sup>Y(DOTA-2P4G-RGD<sub>4</sub>) and <sup>177</sup>Lu(DOTA-2P4G-RGD<sub>4</sub>) might have the potential for targeted radiotherapy of integrin α<sub>v</sub>β<sub>3</sub>-positive tumors.</p>
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