One of the major problems in
cancer chemotherapy are the severe side effects that limit the dose of the anticancer drugs because of their unselectivity for
tumor versus normal cells. In the present work, we show that coupling of
anthracyclines to
peptides is a promising approach to obtain selectivity. The
peptide-
drug conjugate was designed to bind to specific receptors expressed on the
tumor cells with subsequent internalization of the
ligand-receptor complex.
Neuropeptide Y (NPY), a 36-amino
acid peptide of the
pancreatic polypeptide family, was chosen as model
peptide because NPY receptors are overexpressed in a number of
neuroblastoma tumors and the thereof derived cell lines.
Daunorubicin and
doxorubicin, two widely used
antineoplastic agents in
tumor therapy, were covalently linked to NPY via two spacers that differ in stability: an
acid-sensitive
hydrazone bond at the 13-keto position 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 determined at the human
neuroblastoma cell line SK-N-MC, which selectively expresses the NPY Y(1) receptor subtype, and cytotoxic activity was evaluated using a XTT-based colorimetric cellular cytotoxicity assay. The different conjugates 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
daunorubicin. This cytotoxicity is Y(1) 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 microscopy. We found evidence that the active conjugate [C(15)]-NPY-Dauno-HYD releases
daunorubicin, which is localized close to the nucleus, whereas the inactive conjugate [C(15)]-NPY-Dauno-MBS is distributed distantly from the nucleus and does not seem to release the
drug within the cell.