Evaluation of 64Cu- and 125I-Radiolabeled Bitistatin as Potential Agents for Targeting (alpha)v(beta)3 Integrins in Tumor Angiogenesis

The formation of new blood vessels (angiogenesis) is a feature common to all solid tumors. The integrin receptor (alpha)V (beta)3, which is found on endothelial cells lining newly growing blood vessels at a higher density than on mature blood vessels, is being explored as a marker for tumor angiogenesis. Bitistatin, a member of the disintegrin family of polypeptides, has affinity for (alpha)V(beta)3 integrins. To determine whether radiolabeled bitistatin could target tumors, its biodistribution was tested in tumor-bearing mice. For initial validation studies, 125I-bitistatin was injected into BALB/c mice bearing EMT-6 mouse mammary carcinoma tumors, a model that is highly vascular but which lacks (alpha)V(beta)3 directly on tumor cells. Tumor uptake reached maximal values (11.7 +- 4.6 %ID/g) at 2 h. Co-injection of 200 (mu)g of unlabeled bitistatin reduced tumor uptake 62%, suggesting that the binding of 125I-bitistatin is receptor-mediated. This work was extended to include the (beta) +-emitting radionuclide 64Cu, which was attached to bitistatin via 1,4,7,10-tetraazacyclododecane-N,Nʹ,Nʹ ʹ,Nʹ ʹʹ-tetraacetic acid (DOTA). This modification did not significantly alter receptor binding in vitro. MicroPET images obtained with 64CuDOTA-bitistatin showed that the tumor could easily be identified 4 h after administering the radiopharmaceutical. The biodistribution of 64Cu-DOTA-bitistatin differed from the 125I analogue, in that maximum tumor uptake was nearly 8-fold lower and took at least 6 h to reach maximal binding (1.6 +- 0.2 %ID/g). As with 125I-labeled bitistatin, the 64Cu conjugate showed a 50% reduction in tumor uptake with the co-injection of 200 (mu)g of unlabeled bitistatin (0.8 +- 0.2 %ID/g). Competition studies with integrin-specific peptides indicated that the tumor uptake was related to both (alpha)v(beta)3 and (alpha)IIb(beta)3 integrin binding. To see if tumor uptake could be improved upon, 64Cu was tethered to bitistatin using bromoacetamidobenzyl-1,4,7,10-tetraazacyclododecane-N,Nʹ,Nʹ ʹ,Nʹ ʹʹ-tetraacetic acid (BAD). Tumor uptake for 64CuBAD-2IT-bitistatin was higher than the DOTA conjugate at all time points, reaching a maximum at least 6 h postinjection (5.2 +- 0.6 %ID/g); however, this was accompanied by higher uptake in nontarget organs at all time points. Radiolabeled ligands of this type may be useful in the targeting of tumor angiogenesis, but the choice of radiolabeling approach has a significant impact on the in vivo properties of the radioligand.