Evaluation of In-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 (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.

Keywords: 111In-labeled cyclic RGD peptides; integrin αvβ3; tumor imaging.

Figure 1

Cyclic RGD peptide tetramer conjugates: DOTA-6G-RGD₄, DOTA-6P-RGD₄, DOTA-2P-RGD₄ and DOTA-2P4G-RGD₄.

Figure 2

Competitive inhibition curves of ¹²⁵I-c(RGDyK) bound to the U87MG human glioma cells by c(RGDyK), DOTA-P-RGD, DOTA-P-RGD₂, DOTA-3P-RGD₂, DOTA-2P-RGD₄, DOTA-2P4G-RGD₄, DOTA-6P-RGD₄ and DOTA-6P-RGK₄. Their IC₅₀ values were calculated to be 49.9 ± 5.5, 44.3 ± 3.5, 5.0 ± 1.0, 1.5 ± 0.2, 0.5 ± 0.1, 0.2 ± 0.1, 0.3 ± 0.1, and 437 ± 35 nM, respectively. The integrin α_vβ₃ binding affinity follows the order: DOTA-6P-RGD₄ ~ DOTA-2P4G-RGD₄ ~ DOTA-2P-RGD₄ > DOTA-3P-RGD₂ > DOTA-P-RGD₂ > DOTA-P-RGD >> DOTA-6P-RGK₄.

Figure 3

Comparison of the uptake (%ID/g) in tumor and intestine, as well as the tumor/kidney and tumor/liver ratios for ¹¹¹In(L) (L = DOTA-P-RGD, DOTA-P-RGD₂, DOTA-3P-RGD₂, DOTA-2P-RGD₄, DOTA-2P4G-RGD₄, DOTA-6P-RGD₄ and DOTA-6G-RGD₄) in the athymic nude mice (n = 5) bearing U87MG glioma xenografts. Biodistribution data and T/B ratios for ¹¹¹In(DOTA-3P-RGD₃) and ¹¹¹In(DOTA-6G-RGD₄) were obtained from our previous reports , .

Figure 4

A: Comparison of the 60-min biodistribution data of ¹¹¹In(DOTA-6P-RGD₄) in the athymic nude mice (n = 5) bearing U87MG human glioma xenografts in the absence/presence of excess RGD₂ to demonstrate its integrin α_vβ₃-specificity. B: Comparison of the 60-min biodistribution data of ¹¹¹In(DOTA-6P-RGD₄) and ¹¹¹In(DOTA-6P-RGK₄) in the athymic nude mice (n = 5) bearing U87MG glioma xenografts to demonstrate the RGD-specificity.

Figure 5

A: Representative microscopic fluorescence images of the organ tissues (U87MG glioma, lung, liver and kidney) obtained from the U87MG tumor-bearing mice. For β3 staining (red), frozen tissue slices were incubated with a β3 primary antibody followed by a DyLight 594-conjugated secondary antibody. For CD31 staining (green), slices were incubated with a CD31 primary antibody followed by a FITC-conjugated secondary antibody (×100). Yellow color indicates the overlay of integrin β3 and CD31. Phase contrast pictures were shown as histological reference. B: Western blot results showing expression of integrin β₃ in U87MG glioma, lungs, liver and kidneys obtained from the U87MG tumor-bearing mice.

Figure 6

The whole-body planar images of the tumor-bearing mice (bearing U87MG human glioma xenografts) administered with ~100 μCi of ¹¹¹In(DOTA-3P-RGD₂) (top), ¹¹¹In(DOTA-2P-RGD₄) (middle) and ¹¹¹In(DOTA-6P-RGD₄) (bottom) to illustrate the long tumor retention times of ¹¹¹In-labeled cyclic RGD tetramers. Arrows indicate the presence of tumors.

Figure 7

Representative radio-HPLC chromatograms of ¹¹¹In(DOTA-2P-RGD₄) (left) and ¹¹¹In(DOTA-6P-RGD₄) (right) in saline before injection, in urine at 30 min and 120 min p.i., and in feces at 120 min p.i. Each mouse was administered with ~100 μCi radiotracer. ¹¹¹In(DOTA-2P4G-RGD₄) has the same metabolic stability in the same animal model.