In vitro and in vivo evaluation of a technetium-99m-labeled cyclic RGD peptide as a specific marker of αVβ3 integrin for tumor imaging

Three amino acids residues, Arg-Gly-Asp (RGD), in vitronectin and fibronectin show affinity for alpha(v)beta(3) integrins expressed in vascular endothelial cells. That tumor growth can upregulate the expression of these integrins on tumor cells for invasion and metastasis and in tissue neovasculature suggests the potential of developing radiolabeled RGD peptides as antagonists of alpha(v)beta(3) integrins for broad spectrum tumor specific imaging. The polypeptide RGD-4C, which contains four cysteine residues for cyclization, has shown preferential localization on integrins at sites of tumor angiogenesis. Both RGD-4C and RGE (Arg-Gly-Glu)-4C (as control) were purchased and conjugated with 6-hydrazinopyridine-3-carboxylic acid (HYNIC) for Tc-99m radiolabeling. After purification of the conjugated peptides by a C18 Sep-Pak cartridge with 20% methanol, both peptides were radiolabeled using tricine. For cell binding studies, both Tc-99m peptides were further purified by SE HPLC. High specific radioactivity of labeled cyclized RGD/E (cyclized RGD/E will be simplified as RGD/E through out the text) of about 20 Ci/mumol was achieved. Both Tc-99m complexes were stable in the labeling solution for over 24 h at room temperature. In the human umbilical vein endothelial (HUVE) cell studies, the binding at 1 h of radiolabeled RGD/E was determined at 4 degreesC and at concentrations in the picomolar to nanomolar range. Under these conditions, cell accumulation of 99mTc in the case of RGD was as much as 16 times greater than the control RGE. As a check on specificity, 7 nM of native cyclized RGD blocked 50% of the binding of Tc-99m-labeled RGD to cells. The binding percentage of Tc-99m-labeled RGD to purified alpha(v)beta(3) integrin protein, as determined by SE HPLC, increased with the concentration of the integrin while Tc-99m-labeled RGE showed no binding. The association constant for Tc-99m-RGD was modest at 7 x 10(6) M-1. In both human renal adenocarcinoma (ACHN) and human colon cancer cell line (LS174T) nude mouse tumor models, the accumulation of Tc-99m-labeled RGD/E exhibited no statistical difference. In conclusion, possibly because of limited numbers Of alpha(v)beta(3) integrin receptors per tumor cell and low binding affinity, radiolabeled RGD peptides may have limitations as tumor imaging agents.