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-2PEG₄-dimer (HYNIC = 6-hydrazinonicotinyl; 2PEG₄-dimer = E[PEG₄-c(RGDfK)]₂; and PEG₄ = 15-amino-4,7,10,13-tetraoxapentadecanoic acid) and HYNIC-3PEG₄-dimer (3PEG₄-dimer = PEG₄-E[PEG₄-c(RGDfK)]₂), and evaluation of their ^99mTc complexes [^99mTc(HYNIC-2PEG₄-dimer)(tricine)(TPPTS)] (^99mTc-2PEG₄-dimer: TPPTS = trisodium triphenylphosphine-3,3′,3′′-trisulfonate) and [^99mTc(HYNIC-3PEG₄-dimer)(tricine)(TPPTS)] (^99mTc-3PEG₄-dimer) as novel radiotracers for imaging integrin α_vβ₃ expression in athymic nude mice bearing U87MG glioma and MDA-MB-435 breast cancer xenografts. The integrin α_vβ₃ binding affinities of RGD peptides were determined by competitive displacement of ¹²⁵I-c(RGDyK) on U87MG glioma cells. It was found that the two PEG₄ linkers between RGD motifs in HYNIC-2PEG₄-dimer (IC₅₀ = 2.8 ± 0.5 nM) and HYNIC-3PEG₄-dimer (IC₅₀ = 2.4 ± 0.7 nM) are responsible for their higher integrin α_vβ₃ binding affinity than that of HYNIC-PEG₄-dimer (PEG₄-dimer = PEG₄-E[c(RGDfK)]₂; IC₅₀ = 7.5 ± 2.3 nM). Addition of extra PEG₄ linker in HYNIC-3PEG₄-dimer has little impact on integrin α_vβ₃ binding affinity. ^99mTc-2PEG₄-dimer and ^99mTc-3PEG₄-dimer were prepared in high yield with >95% radiochemical purity and the specific activity of >10 Ci/μmol. Biodistribution studies clearly demonstrated that PEG₄ linkers are particularly useful for improving the tumor uptake and clearance kinetics of ^99mTc-2PEG₄-dimer and ^99mTc-3PEG₄-dimer from noncancerous 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 ^99mTc-2PEG₄-dimer is integrin α_vβ₃-mediated. In the metabolism study, ^99mTc-2PEG₄-dimer had high metabolic stability during its excretion from renal and hepatobiliary routes. ^99mTc-3PEG₄-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, ^99mTc-3PEG₄-dimer is a very promising radiotracer for the early detection of integrin α_vβ₃-positive tumors, and may have the potential for noninvasive monitoring of tumor growth or treatment efficacy.