The alpha(v)
beta(3) integrin is overexpressed on proliferating endothelial cells such as those present in growing
tumors as well as on
tumor cells of various origins.
Tumor-induced angiogenesis can be inhibited in vivo by antagonizing the alpha(v)
beta(3) integrin with small
peptides containing the
arginyl-glycyl-aspartic acid (RGD) amino acid sequence. The divalent
cyclic peptide E-[c(RGDfK)(2)] is a novel
ligand-based vascular-targeting agent that binds
integrin alpha(v)beta(3) and demonstrated high uptake in OVCAR-3 xenograft
tumors. In this work, we coupled the 2'-OH-group of
paclitaxel through an aliphatic
ester to the amino group of E-[c(RGDfK)(2)] or the control
peptide c(RADfK), thus obtaining the derivatives E-[c(RGDfK)(2)]-
paclitaxel and c(RADfK)-
paclitaxel. Subsequently, we investigated the activity of the
paclitaxel derivatives using several well-established in vitro angiogenesis assays: using a standard 72 h endothelial cell proliferation assay, we showed that both E-[c(RGDfK)(2)]-
paclitaxel and c(RADfK)-
paclitaxel inhibit the proliferation of human umbilical vein endothelial cells (HUVEC) in a similar manner as free
paclitaxel (IC(50) value approximately 0.4 nM), an observation that can be explained by the half-life of the
paclitaxel ester bond in the conjugates of approximately 2h at pH 7. In contrast, a 30-min exposure of the cells to the three drugs showed a clear difference between free
paclitaxel, E-[c(RGDfK)(2)]-
paclitaxel and c(RADfK)-
paclitaxel with IC(50) values of 10nM, 25 nM, and 60 nM, respectively. These differences are very likely due to the different routes of cellular entry of these three molecules. While the hydrophobic
paclitaxel diffuses rapidly through the cell membrane, the charged
peptide-containing derivative E-[c(RGDfK)(2)]-
paclitaxel binds to the overexpressed alpha(v)
beta(3) integrin in order to enter the cells via receptor-mediated endocytosis. The differences between the derivatives were further demonstrated using an endothelial cell adhesion assay. Inhibition of cell attachment was observed only with the E-[c(RGDfK)(2)]-
paclitaxel derivative indicating its specificity to the growing endothelial cells. Furthermore, E-[c(RGDfK)(2)]-
paclitaxel inhibited both endothelial cells migration and capillary-like tube formation. These results further demonstrate their antiangiogenic properties. In vivo studies in an OVCAR-3 xenograft model demonstrated no antitumor efficacy for either E-[c(RGDfK)(2)] or E-[c(RGDfK)(2)]-
paclitaxel compared to moderate efficacy for
paclitaxel.