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.

Integrin alphavbeta3 plays a critical role in tumor angiogenesis and metastasis. Radiolabeled RGD peptides that are integrin alphavbeta3-specific are very useful for noninvasive imaging of integrin expression in rapidly growing and metastatic tumors. In this study, we determined the binding affinity of E{E[c(RGDfK)]2}2 (tetramer) and its 6-hydrazinonicotinamide conjugate (HYNIC-tetramer) against the binding of 125I-echistatin to the integrin alphavbeta3-positive MDA-MB-435 breast cancer cells. The athymic nude mice bearing MDA-MB-435 xenografts were used to evaluate the potential of ternary ligand complex [99mTc(HYNIC-tetramer)(tricine)(TPPTS)] (TPPTS = trisodium triphenylphosphine-3,3',3' '-trisulfonate) as a new radiotracer for imaging breast cancer integrin alphavbeta3 expression by single photon emission computed tomography (SPECT). It was found that the binding affinity of tetramer (IC50 = 51 +/- 11 nM) was slightly higher than that of its dimeric analogue (IC50 = 78 +/- 27 nM) and is comparable to that of the HYNIC-tetramer conjugate (IC50 = 55 +/- 11 nM) within the experimental error. Biodistribution data showed that [99mTc(HYNIC-tetramer)(tricine)(TPPTS)] had a rapid blood clearance (4.61 +/- 0.81 %ID/g at 5 min postinjection (p.i.) and 0.56 +/- 0.12 %ID/g at 120 min p.i.) and was excreted mainly via the renal route. [99mTc(HYNIC-tetramer)(tricine)(TPPTS)] had high tumor uptake with a long tumor retention (5.60 +/- 0.87 %ID/g and 7.30 +/- 1.32 %ID/g at 5 and 120 min p.i., respectively). The integrin alphavbeta3-specificity was demonstrated by co-injection of excess E[c(RGDfK)]2, which resulted in a significant reduction in tumor uptake of the radiotracer. The metabolic stability of [99mTc(HYNIC-tetramer)(tricine)(TPPTS)] was determined by analyzing urine and feces samples from the tumor-bearing mice at 120 min p.i. In the urine, about 20% of [99mTc(HYNIC-tetramer)(tricine)(TPPTS)] remained intact while only approximately 15% metabolized species was detected in feces. SPECT images displayed significant radiotracer localization in tumor with good contrast as early as 1 h p.i. The high tumor uptake and fast renal excretion make [99mTc(HYNIC-tetramer)(tricine)(TPPTS)] a promising radiotracer for noninvasive imaging of the integrin alphavbeta3-positive tumors by SPECT.