Synthesis of a Covalent Epirubicin-(C₃-amide)-Anti-HER2/neuImmunochemotherapeutic 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 e-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 chemotherapeuticequivalent 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).

Synthesis of Gemcitabine-(C<sub>4</sub>-<i>amide</i>)-[anti-HER2/<i>neu</i>] Utilizing a UV-Photoactivated Gemcitabine Intermediate: Cytotoxic Anti-Neoplastic Activity against Chemotherapeutic-Resistant Mammary Adenocarcinoma SKBr-3

“…The observed molecular weight of 150-kDa for both gemcitabine-(C 4 - amide )-[anti-HER2/ neu ] and gemcitabine-(C 5 - methylcarbamate )-[anti-HER2/ neu ] directly corresponds with the known molecular weight/mass of reference control anti-HER2/ neu monoclonal immunoglobulin fractions (Figure 3). Analogous results have been reported for similar covalent immunochemotherapeutics [2,7,36,71,72,74]. …”

“…Size-separation of covalent immunochemotherapeutics like gemcitabine-(C 4 - amide )-[anti-HER2/ neu ] and gemcitabine-(C 5 - methylcarbamate )-[anti-HER2/ neu ] by micro-scale exchange column chromatography consistently yields preparations that contain <4.0% of residual chemotherapeutic that is not covalently bound to the immunoglobulin fraction [7,36,71,72]. Small residual amounts of non-covalently bound chemotherapeutic remaining within covalent immunochemotherapeutic preparations is generally considered to not be available for further removal through any additional sequential column chromatography separations [73].…”

“…In contrast to the anthracyclines, [7,71,72] gemcitabine can not be measured directly within covalent immunochemotherapeutic preparations by spectrophotometric absorption [36]. Alternatively it is possible to calculate the amount of gemcitabine that has been covalent incurporated into immunochemotherapeutics by measuring residual unbound gemcitabine before and after the Phase II reaction or by measuring the difference in non-chemotherapeutic-occupied sites associated with either amine or reduced sulfhydryl groups within anti-HER2/ neu monoclonal immunoglobulin compared to gemcitabine-immunochemotherapeutics [36,51,52].…”

Design andIn VivoCharacterization of Immunoconjugates Targeting HIV gp160

“…Epirubicin-C 3 -amide-7B2 was produced by employing a 2-phase synthesis regimen with succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate and established protocols (57)(58)(59). Residual unreacted reagents were removed by microscale column chromatography, and the percentage of non-immunoglobulin-bound epirubicin was measured in a chloroform-propanol supernatant extract following centrifugation of the resulting protein precipitate.…”

“…The proportion of covalently bound epirubicin was above 97%, and the molar incorporation index was 57%. Gemcitabine-C 2 -amide-SS-7B2 was produced by employing a 2-phase synthesis regimen with succinimidyl 2-[(4,4=-azipentanamido)ethyl]-1,3=-dithioproprionate and established protocols (57)(58)(59). Residual unreacted reagents were removed by microscale column chromatography.…”