It has long been known that blood vessels, supplying tumors and sites of inflammation, are hyperpermeable to plasma and plasma proteins. Angiogenesis inhibitors, such as TNP-470 and angiostatin are emerging as a new class of anticancer drugs. Although TNP-470 has shown promise in clinical trials, higher doses of this angiognesis inhibitor necessary for tumor regression, showed signs of neurotoxicity. We have previously described the synthesis and characterization of a novel non-toxic HPMA copolymer-TNP-470 conjugate (Satchi-Fainaro et al., Nature Medicine 10, 255-261, 2004). It has enhanced and prolonged antitumor activity and no neurotoxicity compared to TNP-470. This conjugate accumulates selectively in tumor tissue by the enhanced permeability and retention (EPR) effect and has a significantly longer half-life than free TNP-470 due to its increased size. The conjugate bears a tetrapeptide linker designed to be cleaved in lysosmes of tumor endothelial cells where cathepsin B is known to be overexpressed. Here we investigated the effects of several inhibitors of angiogenesis on vascular permeability. Our study shows that, TNP-470, its polymeric conjugate HPMA copolymer-TNP-470 and angiostatin, reduce plasma macromolecule extravasation from the pathologically hyperpermeable vasculature supplying tumors and inflammatory sites, and also from blood vessels rendered hyperpermeable by three vascular permeabilizing mediators, VEGF, PAF and histamine. Treatment for three days with these inhibitors also reduced edema in tumors and pulmonary edema induced by IL-2 therapy and so could be useful as adjuvant therapy for tumors, inflammatory conditions such as delayed-type hypersensitivity, or complications of chemotherapy or immunotherapy. TNP-470 or HPMA copolymer-TNP-470 conjugate inhibited VEGF-induced phosphorylation of VEGFR-2, calcium influx and Rho A activation in cultured endothelial cells. Our results have identified an important new activity of TNP-470, that of inhibiting vessel hyperpermeability. We describe a novel mechanism of action for TNP-470 and possibly other endogenous proteins with antiangiogenic activity. This activity likely contributes to TNP-470’s antiangiogenic effect and suggests that HPMA copolymer-TNP-470 can be used in the treatment of cancer and inflammation. We also tested the effect of combination therapy on tumor growth. We selected two angiogenesis inhibitors that block VEGF-induced vessel permeability. The combination of HPMA-TNP-470 and Avastin showed synergistic effect on COLO-205 human colon carcinoma, causing complete tumor regression following 155 days of treatment.

[Proc Amer Assoc Cancer Res, Volume 46, 2005]