NPI-2358, a novel diketopiperazine, induces tubulin depolymerization in vitro and tumor vascular collapse in vivo

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NPI-2358 is a novel diketopiperazine that was selected for further study following a SAR analysis of over 110 analogs. NPI-2358 displays high cytotoxic activity (IC₅₀ values of 10-15nM) against various human tumor cell lines and maintains full activity in multi-drug resistant (MDR) human tumor cell lines and is not susceptible to the P-glycoprotein (P-gp) mediated mechanism of Taxol resistance. In vivo studies using human tumor xenografts demonstrated that NPI-2358 has significant anti-tumor activity when used in combination with chemotherapeutic agents. In this study we show that NPI-2358 is able to induce tubulin depolymerization in human umbilical vascular endothelial cells (HuVECs) at a concentration of 20nM and within 30 min. Other tubulin depolymerizing agents such as Combretastatin A-4-phosphate (CA4P) and ZD6126 have been shown in vivo to cause a rapid collapse of the tumor vasculature. NPI-2358 was therefore evaluated in an in vitro model that mimics in vivo vascular collapse. Results from these experiments demonstrated that NPI-2358 increased monolayer permeability in HuVEC cells in a dose dependent manner. Colchicine and Vincristine are two microtubule destabilizing agents that display in vivo an effect on the tumor vasculature only when used at their maximum tolerated dose. When tested in comparison with NPI-2358, Colchicine and Vincristine induced monolayer permeability in HuVECs at 2μM, while NPI-2358 induced permeability in HuVECs at both 20nM and 2μM. To evaluate the effect of NPI-2358 on tumor vasculature and tumor growth in vivo, intravital microscopy of tumor spheroids with fluorescently labeled DNA implanted in a dorsal skin fold chamber was used. Preliminary data indicate that NPI-2358 induces a rapid vascular collapse resulting in a necrotic center in established breast tumors. Additional in vivo experiments are on-going and will be presented.