Solid tumors rely on a functional tumor vasculature for their survival and continuous growth. Specific targeting of the vascular network in growing tumors with vascular disrupting agents results in a rapid decrease in blood flow followed by central tumor anoxia and cell death. Proliferating tumor endothelium has an increased reliance on the microtubule cytoskeleton for structural integrity. As such, microtubule depolymerizing agents represent one class of molecules with vascular disrupting activity. We studied the biological activity of the novel diketopiperazine, NPI-2358. In vitro experiments revealed that NPI-2358 inhibits the polymerization of MAP-rich bovine brain tubulin by a mechanism distinct from that of colchicine even though both compounds share overlapping binding sites on tubulin. The inhibition of microtubule formation was confirmed by the observation that the mean microtubule length was reduced by ∼70% in the presence of NPI-2358. Further, at nM concentrations NPI-2358 blocks mitotic spindle formation and chromosome alignment in MCF7 human breast cancer cells resulting in cell cycle arrest at prometaphase. At similar concentrations, NPI-2358 disrupts the microtubule network in human umbilical vein endothelial cells (HuVECs). When tested in vitro, NPI-2358 exhibited direct anti-tumor activity against human tumor cell lines with IC50 values ranging from 4-18nM and maintained equivalent cytotoxic activity when tested against parental or multidrug resistant tumor cell lines. To evaluate the effect of NPI-2358 on tumor vasculature and tumor growth in vivo, intravital microscopy was performed using the murine transgenic breast tumor N202 transfected with GFP-H2B. The N202 spheroids were co-implanted with mammary fat pad in a dorsal skin fold chamber in nu/nu mice. Data from two experiments indicated that NPI-2358 selectively induced a rapid tumor vascular collapse resulting in central tumor necrosis and tumor regression in the established breast tumors. In several xenograft models, NPI-2358 demonstrates activity as a single agent and/or markedly potentiates the anti-tumor effects of CPT-11, paclitaxel and taxotere, without adversely affecting the body weight of the animals. Taken together, these data demonstrate that NPI-2358 is a tubulin binding agent that disrupts tumor vasculature in vivo resulting in tumor cell death. NPI-2358 is currently advancing towards clinical trials.

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