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

Abstract

Purpose: The purpose of this study was to demonstrate the valence of cyclic RGD peptides, P-RGD (PEG4-c(RGDfK): PEG4 = 15-amino-4,710,13-tetraoxapentadecanoic acid), P-RGD2 (PEG4-E[c(RGDfK)]2, 2P-RGD4 (E{PEG4-E[c(RGDfK)]2}2, 2P4G-RGD4 (E{PEG4-E[G3-c(RGDfK)]2}2: G3 = Gly-Gly-Gly) and 6P-RGD4 (E{PEG4-E[PEG4-c(RGDfK)]2}2) in binding to integrin αvβ3, and to assess the impact of peptide and linker multiplicity on biodistribution properties, excretion kinetics and metabolic stability of their corresponding 111In radiotracers.Methods: Five new RGD peptide conjugates (DOTA-P-RGD (DOTA =1,4,7,10-tetraazacyclododecane-1,4,7,10-tetracetic acid), DOTA-P-RGD2, DOTA-2P-RGD4, DOTA-2P4G-RGD4, DOTA-6P-RGD4), and their 111In complexes were prepared. The integrin αvβ3 binding affinity of cyclic RGD conjugates were determined by a competitive displacement assay against 125I-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 αvβ3 binding affinity of RGD conjugates follows the order of: DOTA-6P-RGD4 (IC50 = 0.3 ± 0.1 nM) ~ DOTA-2P4G-RGD4 (IC50 = 0.2 ± 0.1 nM) ~ DOTA-2P-RGD4 (IC50 = 0.5 ± 0.1 nM) > DOTA-3P-RGD2 (DOTA-PEG4-E[PEG4-c(RGDfK)]2: IC50 = 1.5 ± 0.2 nM) > DOTA-P-RGD2 (IC50 = 5.0 ± 1.0 nM) >> DOTA-P-RGD (IC50 = 44.3 ± 3.5 nM) ~ c(RGDfK) (IC50 = 49.9 ± 5.5 nM) >> DOTA-6P-RGK4 (IC50 = 437 ± 35 nM). The fact that DOTA-6P-RGK4 had much lower integrin αvβ3 binding affinity than DOTA-6P-RGD4 suggests that the binding of DOTA-6P-RGD4 to integrin αvβ3 is RGD-specific. This conclusion is consistent with the lower tumor uptake for 111In(DOTA-6P-RGK4) than that for 111In(DOTA-6P-RGD4). It was also found that the G3 and PEG4 linkers between RGD motifs have a significant impact on the integrin αvβ3-targeting capability, biodistribution characteristics, excretion kinetics and metabolic stability of 111In-labeled cyclic RGD peptides.Conclusion: On the basis of their integrin αvβ3 binding affinity and tumor uptake of their corresponding 111In radiotracers, it was conclude that 2P-RGD4, 2P4G-RGD4 and 6P-RGD4 are most likely bivalent in binding to integrin αvβ3, and extra RGD motifs might contribute to the long tumor retention times of 111In(DOTA-2P-RGD4), 111In(DOTA-2P4G-RGD4) and 111In(DOTA-6P-RGD4) than that of 111In(DOTA-3P-RGD3) at 72 h p.i. Among the 111In-labeled cyclic RGD tetramers evaluated in the glioma model, 111In(DOTA-2P4G-RGD4) has very high tumor uptake with the best tumor/kidney and tumor/liver ratios, suggesting that 90Y(DOTA-2P4G-RGD4) and 177Lu(DOTA-2P4G-RGD4) might have the potential for targeted radiotherapy of integrin αvβ3-positive tumors.

FITC-Conjugated Cyclic RGD Peptides as Fluorescent Probes for Staining Integrin αvβ3/αvβ5 in Tumor Tissues

“…64−66 The integrin α v β 3 /α v β 5 binding affinity followed a general trend: FITC-3P-RGD 2 ∼ FITC-Galacto-RGD 2 > FITC-RGD 2 ≫ c(RGDfK) > FITC-3P-RGK 2 , which was consistent with the results for their DOTA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetracetic acid) and HYNIC (6-(2-(2-sulfonatobenzaldehyde)hydrazono)nicotinyl) derivatives. 44,60−71 The lower binding affinity of FITC-3P-RGK 2 (IC 50 = 589 ± 73 nM) than that of FITC-3P-RGD 2 (IC 50 = 32 ± 7 nM) clearly demonstrated the RGD-specificity of the FITC-conjugated cyclic RGD peptides.…”

Radiolabeled Cyclic RGD Peptide Bioconjugates as Radiotracers Targeting Multiple Integrins

“…The fact that the tumor uptake of 99m Tc-3G-RGD 2 and 99m Tc-3P-RGD 2 is well-comparable to that of 99m Tc-RGD 4 (Figure 7) within experimental errors suggests that multimeric cyclic RGD peptides are not necessarily multivalent in binding to integrins, and the contribution from the concentration factor is not as much as that from the bivalency factor. 39,41 In addition, the capability of a multimeric cyclic RGD peptide to achieve bivalency also depends on the α v β 3 density in tumor tissues. If the tumor α v β 3 density is high, the distance between two neighboring α v β 3 sites will be short.…”

“…39,41,42 It was found that the glioma uptake (% ID/g at < 24 h p.i.) follow the general ranking order of 111 In-6G-RGD 4 ~ 111 In-6P-RGD 4 ~ 111 In-3P-RGD 2 ≫ 111 In-P-RGD 2 > 111 In-P-RGD (Figure 9).…”

The Practicality of Nanoceria-PAN-Based 68Ge/68Ga Generator Toward Preparation of 68Ga-Labeled Cyclic RGD Dimer as a Potential PET Radiotracer for Tumor Imaging

“…7,[16][17][18][19][32][33][34][35][36][37][38][39][40][41][42] This trend is observed from derivatives having monomer-to-tetramer RGD derivatives. [16][17][18][19]32,33,[36][37][38][40][41][42] However, a steady increase of uptake in other nontarget organs, for example, the liver, intestine, and kidneys, is also reported as the number of RGD molecule increase. A comparison of biological behavior of radiolabeled RGD monomer, dimer, and tetramer revealed that dimers exhibit rapid tumor localization and significant tumor retention with excellent target/nontarget ratios and is the most suitable candidate for tumor imaging.…”