Synthesis of a covalent gemcitabine-(carbamate)-[anti-HER2/neu] immunochemotherapeutic and its cytotoxic anti-neoplastic activity against chemotherapeutic-resistant SKBr-3 mammary carcinoma

Gemcitabine is a potent chemotherapeutic that exerts cytotoxic activity against several leukemias and a wide spectrum of carcinomas. A brief plasma half-life in part due to rapid deamination and chemotherapeutic-resistance frequently limit the utility of gemcitabine in clinical oncology. Selective ‘targeted’ delivery of gemcitabine represents a potential molecular strategy for simultaneously prolonging its plasma half-life and minimizing exposure of innocent tissues and organ systems.

Materials and methods

Gemcitabine was combined in molar excess with N-[p-maleimidophenyl]-isocyanate (PMPI) so that the isocyanate moiety of PMPI which exclusively reacts with hydroxyl groups preferentially created a carbamate covalent bond at the terminal C₅-methylhydroxy group of gemcitabine. Monoclonal immunoglobulin with binding-avidity specifically for HER2/neu was thiolated with 2-iminothiolane at the terminal ??-amine group of lysine amino acid residues. The gemcitabine-(carbamate)-PMPI intermediate with a maleimide moiety that exclusively reacts with reduced sulfhydryl groups was then combined with thiolated anti-HER2/neu monoclonal immunoglobulin. Western-blot analysis was utilized to delineate the molecular weight profile for gemcitabine-(carbamate)-[anti-HER2/neu] while cell binding characteristics were determined by cell-ELISA utilizing SKBr-3 mammary carcinoma which highly over-expresses HER2/neu receptors. Cytotoxic anti-neoplastic potency of gemcitabine-(carbamate)-[anti-HER2/neu] between the gemcitabine-equivalent concentrations of 10⁻¹² and 10⁻⁶ M was determined utilizing vitality staining analysis of chemotherapeutic-resistant SKBr-3 mammary carcinoma.

Results

Gemcitabine-(carbamate)-[anti-HER2/neu] was synthesized at a molar incorporation index of 1:1.1 (110%) and had a molecular weight of 150 kDa that was indistinguishable from reference control immunoglobulin fractions. Cell-ELISA detected progressive increases in SKBr-3 mammary carcinoma associated immunoglobulin with corresponding increases in covalent gemcitabine immunochemotherapeutic concentrations. The in vitro cytotoxic anti-neoplastic potency of gemcitabine-(carbamate)-[anti-HER2/neu] was approximately 20% and 32% at 10⁻⁷ and 10⁻⁶ M (gemcitabine-equivalent concentrations) after a 182-h incubation period.

Discussion

The investigations describes for the first time a methodology for synthesizing a gemcitabine anti-HER2/neu immunochemotherapeutic by creating a covalent bond structure between the C₅-methylhydroxy group of gemcitabine and thiolated lysine amino acid residues of monoclonal antibody or other biologically active protein fractions. Gemcitabine-(carbamate)-[anti-HER2/neu] possessed binding-avidity at HER2/neu receptors highly over-expressed by chemotherapeutic-resistant SKBr-3 mammary carcinoma. Alternatively, gemcitabine can be covalently linked at its C₅-methylhydroxy group to monoclonal immunoglobulin fractions that possess binding-avidity for other receptors and membrane complexes uniquely highly over-expressed by a variety of neoplastic cell types. Compared to chemotherapeutic-resistant SKBr-3 mammary carcinoma, gemcitabine-(carbamate)-[anti-HER2/neu] immunochemotherapeutic is anticipated to exert higher levels of cytotoxic anti-neoplastic potency against other neoplastic cell types like pancreatic carcinoma, small-cell lung carcinoma, neuroblastoma, glioblastoma, oral squamous cell carcinoma, cervical epithelioid carcinoma, or leukemia/lymphoid neoplastic cell types based on their reportedly greater sensitivity to gemcitabine and gemcitabine covalent conjugates.