General Molecular Structure of Covalent Gemcitabine and Epirubicin Immunochemotherapeutics

Figure 1 Molecular design and chemical composition of covalent gemcitabine and epirubicin immunochemotherapeutics. Legends: (Panel 1) gemcitabine-(C4-amide)-[anti-EGFR]; and (Panel 2) epirubicin-(C3-amide)-[anti-HER2/neu]. Both covalent immunochemotherapeutics were synthesized utilizing a 2-stage organic chemistry reaction scheme that initially produces a chemotherapeutic analog that is a UV-photoactivated intermediate. Covalent bonds are formed at the monoamine groups of gemcitabine or epirubicin and the side chains of amino acid residues within the sequence of immunoglobulin fractions.

SDS/PAGE MolecularWeight Separation and Chemiluminescent AutoradiographyAnalysis of Covalent Gemcitabine and Epirubicin Immunochemotherapeutics

Figure 2 Characterization of the molecular weight profile for the covalent immunochemotherapeutics, epirubicin-(C3-amide)-[anti-HER2/neu] and gemcitabine-(C4-amide)-[anti-EGFR] relative to reference control anti- EGFR and anti-HER2/neu monoclonal immunoglobulin fractions. Legends: (Lane-1) murine anti-human EGFR monoclonal immunoglobulin; (Lane-2) gemcitabine-(C4-amide)-[anti-EGFR]; (Lane-3) murine antihuman HER2/neu monoclonal immunoglobulin; and (Lane-4) epirubicin- (C3-amide)-[anti-HER2/neu]; Covalent immunochemotherapeutics and monoclonal immunoglobulin fractions were size-separated by non-reducing SDS-PAGE followed by lateral transfer onto sheets of nitrocellulose membrane to facilitate detection with biotinylated goat anti-mouse IgG immunoglobulin. Subsequent analysis entailed incubation of membranes with strepavidin-HRPO in combination with the use of a HRPO chemiluminescent substrate and the acquisition of autoradiography images.

Detection of Retained Seletive BindingAvidityof Covalent- Gemcitabine and Epirubicin Immunochemotherapeutics

Figure 3Detection of total immunoglobulin in the form of gemcitabine- (C4-amide)-[anti-EGFR] or epirubicin-(C3-amide)-[anti-HER2/neu] selectively bound to the exterior surface membrane of chemotherapeuticresistant mammary adenocarcinoma.

Legends: (◆) gemcitabine-(C4-amide)-[anti-EGFR]; and (■) epirubicin- (C3-amide)-[anti-HER2/neu]. Covalent gemcitabine-(C4-amide)-[anti- EGFR] or epirubicin-(C3-amide)-[anti-HER2/neu] immunochemotherapeutics formulated at gradient concentrations were incubated with triplicate monolayer populations of chemotherapeutic-resistant mammary adenocarcinoma (SKBr-3) over a 4-hour period and total immunoglobulin bound to the exterior surface membrane was then measured by cell- ELISA.

Influence of ContactIncubation Period onAnti-Neoplastic Cytotoxicityof Selectively"Targeted" Covalent Gemcitabine and Epirubicin Immunochemotherapeutics

Figure 4Relative anti-neoplastic cytotoxicity for the covalent immunochemotherapeutic dual-combination of gemcitabine-(C4-amide)-[anti- EGFR] and epirubicin-(C3-amide)-[anti-HER2/neu] compared to gemcitabine-( C4-amide)-[anti-EGFR] alone against chemotherapeutic-resistant human mammary adenocarcinoma.

Legends: (■) gemcitabine-(C4-amide)-[anti-EGFR] in dual-combination with epirubicin-(C3-amide)-[anti-HER2/neu] at 182-hours; (▲) gemcitabine-( C4-amide)-[anti-EGFR] at 182-hours; and (◆) epirubicin-(C3- amide)-[anti-HER2/neu] at 96-hours; (●) epirubicin-(C3-amide)-[anti- HER2/neu] at 182-hours. Individual covalent immunochemotherapeutics or the dual 50/50 combination of gemcitabine-(C4-amide)-[anti-EGFR] with epirubicin-(C3-amide)-[anti-HER2/neu] were formulated at gradient chemotherapeutic-equivalent concentrations and incubated in direct contact with triplicate monolayer populations of chemotherapeutic-resistant mammary adenocarcinoma (SKBr-3) for period of 182-hours. Anti-neoplastic cytotoxicity was detected and measured using a MTT cell vitality assay and values reported as a percentage of matched negative reference controls (100%).

Anti-Neoplastic Cytotoxicityof Dual Combinations of Selectively "Targeted" Gemcitabine and Epirubicin Immunochemotherapeutics

Figure 5 Relative anti-neoplastic cytotoxicity for the dual-combination of covalent gemcitabine and epirubicin immunochemotherapeutics against chemotherapeutic-resistant human mammary adenocarcinoma.

Legends: Left-Panel (■) gemcitabine-(C4-amide)-[anti-EGFR], and (◆) gemcitabine-(C4-amide)-[anti-EGFR] with epirubicin-(C3-amide)- [anti-HER2/neu]. Right-Panel (■) gemcitabine-(C4-amide)-[anti-EGFR] with epirubicin-(C3-amide)-[anti-HER2/neu]; and (◆) gemcitabine-(C4- amide)-[anti-EGFR] with gemcitabine-(C4-amide)-[anti-HER2/neu]. Dual-combinations of covalent immunochemotherapeutics were formulated at gradient 50/50 chemotherapeutic-equivalent concentrations. Both individual and dual covalent immunochemotherapeutic combinations were incubated in direct contact with triplicate monolayer populations of chemotherapeutic-resistant mammary adenocarcinoma (SKBr-3) over a period of 182-hours. Anti-neoplastic cytotoxicity was detected and measured using a MTT cell vitality assay relative to matched negative reference controls.

Relative Anti-Neoplastic Cytotoxicityof Covalent Gemcitabineand Epirubicin Immunochemotherapeutics

Figure 6 Relative anti-neoplastic cytotoxicity for gemcitabine and epirubicin against chemotherapeutic-resistant human mammary adenocarcinoma as a function of challenge duration.

Legends: (..●..) gemcitabine-(C4-amide)-[anti-EGFR] following a 182-hour incubation period;[98] (--◆--) gemcitabine-(C4-amide)-[anti-HER2/neu] following a 96-hour incubation period;[98] (--☐--)* gemcitabine-(C5- carbonate)-[thiolated anti-HER2/neu] following a 182-hour incubation period;[97] (Δ) gemcitabine-(C4-amide)-[anti-HER2/neu] following a 96- hour incubation period;[99] (-.-◆-.-) epirubicin-(C3-amide)-[anti-HER2/ neu] following a 96-hour incubation period;[102] (●) epirubicin-(C3- amide)-SS-[anti-HER2/neu] following a 96-hour incubation period;[160] (Δ)* epirubicin-(C13-imino)-[thiolated-anti-HER2/neu] following a 96- hour incubation period;[85] and (❍)* epirubicin-(C3-amide)-[thiolatedanti- HER2/neu] following a 96-hour incubation period.[66] Covalent gemcitabine or epirubicin immunochemotherapeutics were formulated at gradient chemotherapeutic-equivalent concentrations and incubated with triplicate monolayer populations of chemotherapeutic-resistant mammary adenocarcinoma (SKBr-3). Anti-neoplastic cytotoxicity was detected and measured using a MTT cell vitality assay relative to matched negative reference controls. Note: (*) = incorporation of aeromatic ring structure into synthetic bond structure of covalent immunochemotherapeutic; and (-SS- ) designates incorporation of a potentially cleavable disulfide bond into the structure of covalent immunochemotherapeutic

Influence of Contact Incubation Period on Anti-Neoplastic Cytotoxic Potency of Gemcitabine and Epirubicin In Populations of Chemotherapeutic Resistant Mammary Adenocarcinoma (SKBr-3)

Figure 7Relative anti-neoplastic cytotoxicity for gemcitabine and epirubicin against chemotherapeutic-resistant human mammary adenocarcinoma as a function of challenge duration.

Legends: (◆) gemcitabine following a 96-hour incubation period; (■) gemcitabine following a 182-hour incubation period; and (▲) epirubicin following a 96-hour incubation period. Gemcitabine or epirubicin formulated at gradient chemotherapeutic-equivalent concentrations were incubated in direct contact with triplicate monolayer populations of chemotherapeuticresistant mammary adenocarcinoma (SKBr-3). Anti-neoplastic cytotoxicity was detected and measured using a MTT cell vitality assay relative to matched negative reference controls.

Collective Anti-Neoplastic Cytotoxicity of Mebendazole In Combination with Selectively "Targeted" Gemcitabin and Epirubicin Delivery

Figure 8 Relative anti-neoplastic cytotoxicity of gemcitabine-(C4-amide)- [anti-EGFR] in dual-combination with epirubicin-(C3-amide)-[anti-HER2/ neu] with and without mebendazole.

Legends: (◆) gemcitabine-(C4-amide)-[anti-EGFR] with epirubicin-(C3- amide)-[anti-HER2/neu] following a 182-hour incubation period; (▲) gemcitabine-( C4-amide)-[anti-EGFR] and epirubicin-(C3-amide)-[anti-HER2/ neu] in combination with mebendazole following a 182-hour incubation period; and (■) gemcitabine-(C4-amide)-[anti-EGFR] and epirubicin-(C3- amide)-[anti-HER2/neu] in combination with mebendazole following a 96- hour incubation period. Mean numerical results for (■) and (▲) are nearly identical so their marker legends are visually superimposed in the figure illustration. The covalent immunochemotherapeutic dual-combination formulated at gradient 50/50 chemotherapeutic-equivalent concentrations (+/- mebendazole 0.15μM fixed concentration) were incubated in direct contact with triplicate monolayer populations of chemotherapeutic-resistant mammary adenocarcinoma (SKBr-3) over a period of either 96-hours or 182-hours. Anti-neoplastic cytotoxicity was detected and measured using a MTT cell vitality assay relative to matched negative reference controls.

Relative Anti-Neoplastic Cytotoxic Potency of Benzimidazole Tubulin/Microtubule Inhibitors

Figure 9 Relative anti-neoplastic cytotoxicity of benzimidazoles against chemotherapeutic-resistant human mammary adenocarcinoma.

Legends: (◆) albendazole; (▲) flubendazole; and (■) mebendazole. Benzimidazole tubulin/microtubule inhibitors formulated at gradient molarequivalent concentrations were incubated in direct contact with triplicate monolayer populations of chemotherapeutic-resistant mammary adenocarcinoma (SKBr-3) over a period of 96-hours. Anti-neoplastic cytotoxicity was detected and measured using a MTT cell vitality assay relative to matched negative reference controls.

Influence of Contact Incubation Period on Mebendazole Anti- Neoplastic Cytotoxic Potency

Figure 10 Relative anti-neoplastic cytotoxicity of mebendazole against chemotherapeutic-resistant mammary adenocarcinoma as a function of challenge duration (incubation period).

Legends: (■) mebendazole following a 96-hour incubation period; and (◆) mebendazole following a 182-hour incubation period. Mebendazole formulated at gradient molar-equivalent concentrations was incubated in direct contact with triplicate monolayer populations of chemotherapeutic- resistant mammary adenocarcinoma (SKBr-3) over a period of either 96-hours or 182-hours. Anti-neoplastic cytotoxicity was detected and measured using a MTT cell vitality assay relative to matched negative reference controls.