title: Synthesis of a covalent epirubicin-(C(3)-amide)-anti-HER2/neu immunochemotherapeutic utilizing a UV-photoactivated anthracycline intermediate.
abstract: 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).
authors.affiliation: Department of Basic Sciences, College of Veterinary Medicine, Mississippi State University, Mississippi State, Mississippi, USA. coyne@cvm.msstate.edu
authors.forename: Cody P
authors.initials: CP
authors.lastname: Coyne
authors.forename: Toni
authors.initials: T
authors.lastname: Jones
authors.forename: Ryan
authors.initials: R
authors.lastname: Bear
mesh_terms: D000230:Adenocarcinoma / Q000188:drug therapy / Q000201:enzymology / Q000235:genetics / Q000473:pathology; D000903:Antibiotics, Antineoplastic / Q000138:chemical synthesis* / Q000737:chemistry / Q000494:pharmacology; D001943:Breast Neoplasms / Q000188:drug therapy* / Q000201:enzymology / Q000276:immunology / Q000473:pathology; D045744:Cell Line, Tumor; D004797:Enzyme-Linked Immunosorbent Assay; D015251:Epirubicin / Q000031:analogs & derivatives* / Q000138:chemical synthesis / Q000494:pharmacology; D005260:Female; D006801:Humans; D007074:Immunoglobulin G / Q000276:immunology; D055668:Photochemical Processes; D018719:Erb-b2 Receptor Tyrosine Kinases / Q000037:antagonists & inhibitors*; D014407:Tumor Cells, Cultured; D014466:Ultraviolet Rays
chemical_list: D000903:Antibiotics, Antineoplastic; D015251:Epirubicin; D007074:Immunoglobulin G; D018719:Erb-b2 Receptor Tyrosine Kinases