Synthesis of a covalent epirubicin-(C(3)-amide)-anti-HER2/neu immunochemotherapeutic utilizing a UV-photoactivated anthracycline intermediate.

The C(3)-monoamine on the carbohydrate moiety (daunosamine -NH(2)-3') of epirubicin was reacted under anhydrous conditions with succinimidyl 4,4-azipentanoate to create a covalent UV-photoactivated epirubicin-(C(3)-amide) intermediate with primary amine-reactive properties. A synthetic covalent bond between the UV-photoactivated epirubicin-(C(3)-amide) intermediate and the ɛ-amine of lysine residues within the amino acid sequence of anti-HER2/neu monoclonal immunoglobulin was subsequently created by exposure to UV light (354 nm) for 15 minutes. Size-separation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis combined with immunodetection analysis and chemiluminescent autoradiographic imaging revealed a lack of IgG-IgG polymerization or degradative protein fragmentation of the covalent epirubicin-(C(3)-amide)-[anti-HER2/neu] immunochemotherapeutic. Retained binding-avidity of epirubicin-(C(3)-amide)-[anti-HER2/neu] was validated by cell-ELISA utilizing monolayer populations of chemotherapeutic-resistant mammary adenocarcinoma SKBr-3 which highly overexpress membrane-associated HER2/neu complexes. Between epirubicin-equivalent concentrations of 10(-10) to 10(-6) M the covalent epirubicin-(C(3)-amide)-[anti-HER2/neu] immunochemotherapeutic consistently evoked levels of cytotoxic anti-neoplastic potency that were highly analogous to chemotherapeutic-equivalent concentrations of epirubicin. Cytotoxic anti-neoplastic potency of epirubicin-(C(3)-amide)-[anti-HER2/neu] against chemotherapeutic-resistant mammary adenocarcinoma SKBr-3 challenged with epirubicin-(C(3)-amide)-[anti-HER2/neu] at an epirubicin-equivalent concentration of 10(-6) M was 88.5% (e.g., 11.5% residual survival). Between final epirubicin-equivalent concentrations of 10(-8) and 10(-7) M there was a marked threshold increase in the mean cytotoxic anti-neoplastic activity for epirubicin-(C(3)-amide)-[anti-HER2/neu] from 9.9% to 66.9% (90.2% to 33.1% residual survival).