Improving Tumor-Targeting Capability and Pharmacokinetics of ^99mTc-Labeled Cyclic RGD Dimers with PEG₄ Linkers

Abstract

This report describes the synthesis of two cyclic RGD (Arg-Gly-Asp) conjugates, HYNIC-2PEG 4 -dimer (HYNIC = 6-hydrazinonicotinyl; 2PEG 4 -dimer = E[PEG 4 -c(RGDfK)] 2 ; and PEG 4 = 15-amino-4,7,10,13-tetraoxapentadecanoic acid) and HYNIC-3PEG 4 -dimer (3PEG 4 -dimer = PEG 4 -E [PEG 4 -c(RGDfK)] 2 ), and evaluation of their 99m Tc complexes [ 99m Tc(HYNIC-2PEG 4 -dimer) (tricine)(TPPTS)] ( 99m Tc-2PEG 4 -dimer: TPPTS = trisodium triphenylphosphine-3,3′,3″-trisulfonate) and [ 99m Tc(HYNIC-3PEG 4 -dimer)(tricine)(TPPTS)] ( 99m Tc-3PEG 4 -dimer) as novel radiotracers for imaging integrin α v β 3 expression in athymic nude mice bearing U87MG glioma and MDA-MB-435 breast cancer xenografts. The integrin α v β 3 binding affinities of RGD peptides were determined by competitive displacement of 125 I-c(RGDyK) on U87MG glioma cells. It was found that the two PEG 4 linkers between RGD motifs in HYNIC-2PEG 4 -dimer (IC 50 = 2.8 ± 0.5 nM) and HYNIC-3PEG 4 -dimer (IC 50 = 2.4 ± 0.7 nM) are responsible for their higher integrin α v β 3 binding affinity than that of HYNIC-PEG 4 -dimer (PEG 4 -dimer = PEG 4 -E[c(RGDfK)] 2 ; IC 50 = 7.5 ± 2.3 nM). Addition of extra PEG 4 linker in HYNIC-3PEG 4 -dimer has little impact on integrin α v β 3 binding affinity. 99m Tc-2PEG 4 -dimer and 99m Tc-3PEG 4 -dimer were prepared in high yield with >95% radiochemical purity and the specific activity of > 10 Ci/μmol. Biodistribution studies clearly demonstrated that PEG 4 linkers are particularly useful for improving the tumor uptake and clearance kinetics of 99m Tc-2PEG 4 -dimer and 99m Tc-3PEG 4 -dimer from non-cancerous organs. It was also found that there was a linear relationship between the tumor size and radiotracer tumor uptake expressed as %ID (percentage of the injected dose) in U87MG glioma and MDA-MB-435 breast tumor models. The blocking experiment showed that the tumor uptake of 99m Tc-2PEG 4 -dimer is integrin α v β 3 -mediated. In the metabolism study, 99m Tc-2PEG 4 -dimer had high metabolic stability during its excretion from renal and hepatobiliary routes. 99m Tc-3PEG 4 -dimer also remained intact during thee excretion from the renal route, but, had ~30% metabolism during the excretion from the hepatobiliary route. Planar imaging studies in U87MG glioma and MDA-MB-435 breast tumor models showed that the tumors of ~5 mm in diameter could be readily visualized with excellent contrast. Thus, 99m Tc-3PEG 4 -dimer is a very promising radiotracer for the early detection of integrin α v β 3 -positive tumors, and may have the potential for non-invasive monitoring of tumor growth or treatment efficacy.

Imaging Integrin αvβ3 on Blood Vessels with 111In-RGD2 in Head and Neck Tumor Xenografts

“…The development of multimeric RGD peptides to enhance specific uptake in integrin a v b 3 -expressing tumors through multidentate binding sites (36)(37)(38) was thought to reduce specific uptake in nontumor tissues. Despite the multimeric nature of 111 In-RGD 2 , uptake of the tracer was still visualized in these nontumor tissues, as seen in the biodistribution studies described here.…”

Integrin αvβ3 Imaging of Radioactive Iodine–Refractory Thyroid Cancer Using 99mTc-3PRGD2

“…The adhesion of endothelial cells and extracellular matrix, mediated by integrin a v b 3 , is vital for vascular endothelial growth factor receptor-2 activation (5). Ligands bearing RGD (Arg-GlyAsp) peptide have a high affinity and specificity for integrin a v b 3 , especially cyclic RGD dimmers with PEG4 linkers (6). Unlike 18 F-FDG PET/CT as a diagnosis-only modality, integrin a v b 3 imaging by radiolabeled RGD peptide provides a specific method for visualizing tumor angiogenesis and a therapeutic target for antiangiogenetic and antiintegrin drugs (7).…”

“…Moreover, the product can be kit-formulated and ready for routine clinical use with a simple, convenient labeling procedure and easy quality control. 99m Tc-PEG 4 -E [PEG 4 -c(RGDfK)] 2 ( 99m Tc-3PRGD2) is a new 99m Tc-labeled cyclic RGD dimer peptide with increased receptor binding affinity and improved kinetics for the in vivo imaging of integrin a v b 3 expression in xenografted tumor-bearing models (6,15,16). The blood clearance kinetics, biodistribution, and radiation dosimetry of a kit-formulated 99m Tc-3PRGD2 were also evaluated in nonhuman primates in our department, and no adverse reactions were observed to date (17).…”

Evaluation of ανβ3-Mediated Tumor Expression with a 99mTc-Labeled Ornithine-Modified RGD Derivative During Glioblastoma Growth In Vivo

“…99m Tc], which even if it disposes lower tumor accumulation in relation to multimeric 99m Tc-RGD peptides [39][40][41][42][43] was able to clearly detect integrin expression variations during glioblastoma tumor growth. Nevertheless, apart from dimerization that contributes to a higher integrin a v b 3 binding affinity, a direct comparison of tumor uptake among 99m Tc-labeled RGD derivatives is somehow difficult due to the wide variety of (1) the linkers and chelating groups [44][45][46] and (2) the tumor cell lines with different a m b 3 receptor density levels that are used.…”