Synthesis and Biological Evaluation of Paclitaxel Conjugates Involving Linkers Cleavable by Lysosomal Enzymes and alvhaVbeta3-Integrin Ligands for Tumor Targeting

Two cyclo[DKP-RGD]-PTX (PTX = paclitaxel) and two cyclo[RGDfK]-PTX conjugates containing the Gly-Phe-Leu-Gly (GFLG) linker, which is cleavable by lysosomal enzymes, were synthesized and compared to two cyclo[DKP-RGD]-Val-Ala-PTX conjugates. The conjugates were evaluated for their ability to inhibit biotinylated vitronectin binding to the isolated (v3) receptor, retaining good binding affinity, in the same nanomolar range of the free ligands. Cell viability assays were performed for the six conjugates in the (v3)+ U87 and in the (v3)- HT29 cell lines. Loss of potency was observed for all the conjugates, attenuated by the presence of a tetraethylene glycol (PEG-4) spacer. A good Targeting Index (TI = Relative Potency in the (v3)+ U87/Relative Potency in the (v3)- HT29) was displayed by the conjugates, in particular by cyclo[DKP-RGD]-PEG-4-Val-Ala-PTX 9 (TI = 533). This conjugate was tested in the (v3)+ U87 cell line in the presence of 50-fold excess free cyclo[DKP-RGD] ligand 2. In this competition experiment, a fivefold decrease of the conjugate cytotoxicity was calculated, suggesting that the conjugate is possibly internalized by an (v3) integrin-mediated process.