Direct targeting of tumor cell F(1)F(0) ATP-synthase by radioiodine angiostatin in vitro and in vivo.

UNLABELLED: Recent discoveries have identified endothelial cell-surface F(1)F(0) adenosine triphosphate (ATP) synthase as the key target for angiostatin (AST) action. As this enzyme is also present on tumor cells, we investigated whether radiolabeled AST may directly target cancer cell-surface ATP synthase in vitro and in vivo. METHODS: Cell-binding characteristics of (125)I-AST was evaluated on human umbilical vein endothelial (HUVE) and SNU-C5 colon carcinoma cells. The molecular target for binding was investigated with anti-ATP synthase antibodies and RGDyV. Biodistribution and imaging experiments were performed in mice xenografted with carcinoma and sarcoma tumors. Tumor localization of ATP synthase and exogenous AST was compared via double immunostaining. RESULTS: Both HUVE and SNU-C5 cells showed specific (125)I-AST binding that was dose-dependently inhibited by excess AST, with IC(50) values of 3.5 and 0.2 microM, respectively. Both cell types stained positive for ATP synthase and demonstrated an approximately 50% reduction in AST binding by antibodies against the alpha- and beta-subunit of the enzyme. (123)I-AST injected in mice allowed for the clear tumor visualization with significant tumor accumulation and uptake ratios. Immunostaining revealed a localization of exogenous AST to closely correlate to that of tumor-cell ATP synthase. CONCLUSIONS: AST can directly target tumor-cell ATP synthase, and AST imaging may thus be useful for monitoring tumor ATP synthase expression.

Distribution and pharmacokinetic analysis of angiostatin radioiodine labeled with high stability.

OBJECTIVE: Radiotracers of anticancer agents provide important information on its in vivo handling. Angiostatin (AST) is a promising anticancer drug with potent antiangiogenic effects, but reported AST radiotracers suffer from poor in vivo stability. In this study, we synthesized an AST probe radioiodinated via the Bolton-Hunter reagent (125I-BH-AST) and investigated its stability and biokinetics in mice. METHODS: 125I-BH-AST and conventional direct radioiodinated 125I-AST were evaluated for human endothelial cell binding characteristics. In vivo stability of the radiotracers was compared by biodistribution studies in normal ICR mice. Angiostatin pharmacokinetics was analyzed by serial blood sampling after intravenous injection of 125I-BH-AST with varying AST concentrations in mice. RESULTS: Both 125I-AST and 125I-BH-AST retained selective endothelial binding as demonstrated by dose-dependent inhibition by nonradiolabeled AST. 125I-BH-AST was substantially more stable in mice than 125I-AST, with 28- and 7-fold lower 24-h thyroid and blood activities, respectively (15.5+/-1.5 vs. 430.9+/-32.2 and 0.1+/-0.0 vs. 0.8+/-0.0 %ID/g; both P<.005). Using (125)I-BH-AST, we found that 24-h AST accumulation was highest in the kidneys, followed by the liver and lungs. Kinetic analysis of 125I-BH-AST revealed AST to have linear pharmacokinetics with a T(1/2) of 5.8+/-2.6 h, volume of distribution (V(d)) of 6.8+/-1.3 ml and clearance of 0.8+/-0.1 ml/h. CONCLUSION: Radioiodine-labeled AST prepared by the BH method provides a radioprobe with superior stability and improved in vivo biokinetics that is useful for distribution and pharmacokinetic studies.