Molecular, colloidal and cellular approaches to attack tumor vasculature

Abstract

This thesis is focused on improvement of blood vessel disruption. New systems have been developed that effectuate tumor blood vessel disruption at the molecular, colloidal and cellular level. Vascular disrupting agents (VDAs) are generally small molecular weight drugs that bind to cytoskeletal alfa-tubulin and thereby temporary inhibit or destabilize microtubule formation. Interruption of tubulin polymerization can initiate arrest of cell dynamics, cell shape change and ultimately leads to apoptosis. These changes induce thrombus formation and consequent blood vessel congestion, leading to local oxygen and nutrient deprivation and cell death. A large quantity of tumor tissue could be killed by VDAs, however, always a typical viable rim remains at the tumor periphery inducing rapid repopulation and tumor progression. In the molecular approach, we synthesized PEGylated prodrug derivatives of colchicine, the parent VDA. In chapter 2 the synthesis, in vitro efficacy and the in vivo behaviour of the PEGylated prodrugs is discussed. Two biodegradable prodrugs were designed (one degrading fast, the other slow) and characterized to enhance vascular disruption in tumors and to reduce systemic adverse effects normally seen for colchicine. Although both in vitro and in vivo results were promising further research is needed to optimize the prodrugs. The second vascular disruption system we investigated is colloidal delivery of VDAs or combination of VDAs with colloidal therapeutics. In chapter 3, a small molecular VDA developed by AstraZeneca, called ZD6126, was encapsulated into (targeted) liposomes. Single and multiple doses of both liposomal ZD6126 formulations showed significant prolonged therapeutic efficacy for established tumors when compared to the same dose of free ZD6126. Endothelial targeting proved not to be beneficial and unfortunately, multiple 100 mg/kg liposomal injections appeared toxic. In chapter 4, a combination therapy strategy was tested to attack tumor core and tumor periphery simultaneously. Free ZD6126 was exploited to kill the tumor core and additionally radionuclide 177Lu decorated (targeted) liposomes were designed to attack the remaining tumor rim. No additional or synergistic efficacy compared to single agent therapy was seen. Moreover, massive liver and spleen accumulation was seen and 177Lu-liposome doses of 20 MBq appeared to be very toxic. The combination therapy strategy using radionuclides in combination with VDAs is promising but further optimalization and fine-tuning is crucial. The third way we explored to target the tumor vasculature and consequently induce disruption is by specific targeting of (red blood) cells. In chapter 5, we describe tumor targeting by erythrocytes modified with RGD-peptides. RGD-modified erythrocytes furthermore represent a model for opsonized apoptotic and/or aged erythrocytes. Angiogenic endothelial cells, incubated with apoptotic cell opsonin (lactadherin) bound particles, apoptotic cells and RGD-modified erythrocytes, showed massive binding and consequent internalization. Injection of RGD-modified erythrocytes into tumor-bearing mice, showed tumor endothelial cell phagocytosis, damage and ultimately massive cell death. Further optimization of the model, was described and discussed in chapter 6. We treated the RGD-modified erythrocytes by glutaraldehyde to make the erythrocytes more rigid, a feature of erythrocytes in apoptosis. Incubation with HUVECs showed a significant increase in association compared to RGD-modified erythrocytes without glutaraldehyde treatment.