Novel peptide conjugates for tumor-specific chemotherapy

One of the major problems in cancer chemotherapy are the severe side effect s that limit the dose of the anticancer drugs because of their unselectivit y for tumor versus normal cells. In the present work, we show that coupling of anthracyclines to peptides is a promising approach to obtain selectivit y. The peptide-drug conjugate was designed to bind to specific receptors ex pressed on the tumor cells with subsequent internalization of the ligand-re ceptor complex. Neuropeptide Y (NPY), a 36-amino acid peptide of the pancre atic polypeptide family, was chosen as model peptide because NPY receptors are overexpressed in a number of neuroblastoma tumors and the thereof deriv ed cell lines. Daunorubicin and doxorubicin, two widely used antineoplastic agents in tumor therapy, were covalently linked to NPY via two spacers tha t differ in stability: an acid-sensitive hydrazone bond at the 13-keto posi tion of daunorubicin and a stable amide bond at the 3 ' -amino position of daunorubicin and doxorubicin. Receptor binding of these three conjugates ([ C-15]-NPY-Dauno-HYD, [C-15]-NPY-Dauno-MBS, and [C-15]-NPY-Doxo-MBS) was det ermined at the human neuroblastoma cell line SK-N-MC, which selectively exp resses the NPY Y-1 receptor subtype, and cytotoxic activity was evaluated u sing a XTT-based colorimetric cellular cytotoxicity assay. The different co njugates were able to bind to the receptor with affinities ranging from 25 to 51 nM, but only the compound containing the acid-sensitive bond ([C-15] -NPY-Dauno-HYD) showed cytotoxic activity comparable to the free daunorubic in. This cytotoxicity is Y1 receptor-mediated as shown in blocking studies with BIBP 3226, because tumor cells that do not express NPY receptors were sensitive to free daunorubicin, but not to the peptide-drug conjugate. The intracellular distribution was investigated by confocal laser scanning micr oscopy. We found evidence that the active conjugate [C-15]-NPY-Dauno-HYD re leases daunorubicin, which is localized close to the nucleus, whereas the i nactive conjugate [C-15]-NPY-Dauno-MBS is distributed distantly from the nu cleus and does not seem to release the drug within the cell.