Department of Basic Sciences, College of Veterinary Medicine, Mississippi State University, Mississippi State, Mississippi, USA.
Cancer Biother Radiopharm. 2012 Feb;27(1):41-55. doi: 10.1089/cbr.2011.1097. Epub 2011 Dec 22.
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).
阿霉素糖基部分（道诺霉素-NH(2)-3'）上的 C(3)-单胺在无水条件下与琥珀酰亚胺 4,4-氮杂戊酸反应，生成具有伯胺反应性的共价光活化阿霉素-(C(3)-酰胺)中间物。随后，通过暴露于 354nm 的紫外线光照射 15 分钟，在 UV 光活化的阿霉素-(C(3)-酰胺)中间物和抗 HER2/neu 单克隆免疫球蛋白的氨基酸序列内赖氨酸残基的ε-胺之间创建了一个合成的共价键。通过十二烷基硫酸钠-聚丙烯酰胺凝胶电泳的大小分离，结合免疫检测分析和化学发光放射自显影成像，显示出共价阿霉素-(C(3)-酰胺)-[抗 HER2/neu]免疫化疗药物缺乏 IgG-IgG 聚合或蛋白降解片段。通过利用高表达膜相关 HER2/neu 复合物的化疗耐药乳腺腺癌 SKBr-3 的单层群体进行细胞酶联免疫吸附试验（cell-ELISA），验证了阿霉素-(C(3)-酰胺)-[抗 HER2/neu]的保留结合亲和力。在 10(-10)至 10(-6)M 的阿霉素等效浓度范围内，共价阿霉素-(C(3)-酰胺)-[抗 HER2/neu]免疫化疗药物始终引起与阿霉素等效浓度相当的细胞毒性抗肿瘤效力水平。阿霉素-(C(3)-酰胺)-[抗 HER2/neu]对化疗耐药乳腺腺癌 SKBr-3 的细胞毒性抗肿瘤效力与阿霉素-(C(3)-酰胺)-[抗 HER2/neu]在 10(-6)M 的阿霉素等效浓度下对 SKBr-3 的挑战相当，为 88.5%（即 11.5%的残余存活率）。在最终的阿霉素等效浓度为 10(-8)至 10(-7)M 之间，阿霉素-(C(3)-酰胺)-[抗 HER2/neu]的平均细胞毒性抗肿瘤活性有明显的阈值增加，从 9.9%增加到 66.9%（90.2%到 33.1%的残余存活率）。
