Glycosylated RGD-containing peptides, tracer for tumor targeting and angiogenesis imaging with improved biokinetics

The alpha (V)beta (3) integrin plays an important role in metastasis and tu mor-induced angiogenesis, Targeting with radiolabeled ligands of the alpha (V)beta (3) integrin may provide information about the receptor status and enable specific therapeutic planning. Previous studies from our group resul ted in tracers that showed alpha (V)beta (3)-selective tumor uptake. Howeve r, these first-generation compounds predominantly revealed hepatobiliary ex cretion with high radioactivity found in the liver. In this report, the syn thesis and biological evaluation of the first glycosylated RGD-containing p eptide (RGD-peptide) for the noninvasive imaging of alpha (V)beta (3) expre ssion are described. Methods: Peptides were assembled on a solid support us ing fluorenylmethoxycarbonyl-coupling protocols. The precursor cyclo(-Arg-G ly-Asp-D-Tyr-Lys(SAA)-) GP1 was synthesized by coupling 3-acetamido-2,6-anh ydro-4,5,7-tri-O-benzyl-3-deoxy-beta -o-glycero-D-gulo-heptonic acid (SAA(B n-3)) with cyclo(-Arg(Mtr)-Gly-Asp(OtBu)-D-Tyr(tBu)-Lys-) and subsequent re moval of the protection groups, iodine labeling was performed by the lodo-G en method (radiochemical yield > 50%). The in vitro binding assays were per formed using purified immobilized alpha (IIb)beta (3), alpha (V)beta (5), a nd alpha (V)beta (3) integrins, For in vivo experiments, nude mice bearing xenotransplanted melanomas and mice with osteosarcomas were used. Results: The glycosylated peptide 3-iodo-Tyr(4)-cyclo(-Arg-Gly-Asp-D-Tyr-Lys(SAA)-) ly-Asp-D-Tyr-Lys(SAA)-)GP2 showed high affinity and selectivity for alpha ( V)beta (3) in vitro (50% inhibitory concentration = 40 nmol/L), Pretreatmen t studies indicate specific binding of [I-125]GP2 on alpha (V)beta (3)-expr essing tumors in vivo. Comparison of the pharmacokinetics of [I-125]GP2 and [I-125]-3-iodo-Tyr(4)-cyclo(-Arg-Gly-Asp-D-Tyr-Val-) [I-125]P2 revealed fo r [I-125]GP2 an increased activity concentration in the blood (e.g., 3.59 /- 0.35 percentage injected dose [%ID]/g vs. 1.72 +/- 0.44 %ID/g at 10 min postinjection) and a significantly reduced uptake in the liver (e.g., 2.59 +/- 0.24 %ID/g vs. 21.96 +/- 2.78 %ID/g at 10 min postinjection). Furthermo re, a clearly increased activity accumulation in the tumor was found (e.g., 3.05 +/- 0.31 %ID/g vs. 0.92 +/- 0.16 %ID/g at 240 min postinjection), whi ch remained almost constant between 60 and 240 min postinjection, This resu lted in good tumor-to-organ ratios for the glycosylated tracer (e.g., 240-m in postinjection osteosarcoma model: tumor-to-blood = 16; tumor-to-muscle = 7; tumor-to-liver = 2.5), which were confirmed by the first gamma-camera i mages of osteosarcoma-bearing mice at 240 min postinjection. Conclusion: Th is study demonstrates that the introduction of a sugar moiety improves the pharmakokinetic behavior of a hydrophobic peptide-based tracer. Additionall y, this alpha (V)beta (3)-selective glycosylated radioiodinated second-gene ration tracer GP2 shows high tumor uptake and good tumor-to-organ ratios th at allow noninvasive visualization of alpha (V)beta (3)-expressing tumors a nd monitoring therapy with alpha (V)beta (3) antagonists. Finally, the favo rable biokinetics make the glycosylated RGD-peptide a promising lead struct ure for tracers to quantify the alpha (V)beta (3) expression using PET.