Dexamethasone-(C₂₁-phosphoramide)-[anti-EGFR]: molecular design, synthetic organic chemistry reactions, and antineoplastic cytotoxic potency against pulmonary adenocarcinoma (A549)

Figures & data

Figure 1 Organic chemistry reaction scheme for the synthesis of dexamethasone-(C₂₁-phosphoramide)-[anti-EGFR].

Notes: Phase I, Phase II, and Phase III reaction schemes. Phase 1: reaction of the dexamethasone C₂₁-monophosphate group with 1-ethyl-3-[3-dimethylaminopropyl] carbodiimide to transiently form a reactive dexamethasone-C₂₁-monophosphate carbodiimide ester intermediate complex; Phase II: a transient Phase I reactive intermediate is rapidly converted to a Phase II dexamethasone-C₂₁-phosphorylimidazolide amine-reactive intermediate in the presence of imidazole. Phase III: the Phase II dexamethasone-C₂₁-phosphorylimidazolide amine-reactive intermediate is reacted with the ε-amine of lysine residues within the amino acid sequence of anti-EGFR monoclonal immunoglobulin, resulting in the synthesis of a covalent dexamethasone-(C₂₁-phosphoramide)-[anti-EGFR] immunopharmaceutical.
Abbreviations: EGFR, epidermal growth factor receptor type 1; IgG, immunoglobulin G.

Figure 2 Evaluation of dexamethasone-(C₂₁-phosphoramide)-[anti-EGFR] by analytical HP-TLC for the detection of residual dexamethasone not covalently bound to anti-EGFR immunoglobulin.

Notes: Lane 1: dexamethasone; Lane 2: dexamethasone (molar excess) and Phase II dexamethasone-phosphorylimidazolide amine-reactive intermediate; and Lane 3: Phase III covalent dexamethasone-(C₂₁-phosphoramide)-[anti-EGFR] immunopharmaceutical following serial microfiltration (MWCO =10 kDa). Standardized dexamethasone equivalent concentrations of dexamethasone-(C₂₁-phosphoramide)-[anti-EGFR], the dexamethasone-phosphorylimidazolide amine-reactive intermediate, and dexamethasone were applied to HP-TLC plates (silica gel, 250 µm thickness, UV 254 nm indicator) and developed utilizing a propanol/ethanol/H₂O/glacial acetic acid (17:5:5:1, v/v) mobile phase. Identification of any residual dexamethasone or unreacted dexamethasone-phosphorylimidazolide in the Phase III covalent dexamethasone-(C₂₁-phosphoramide)-[anti-EGFR] immunopharmaceutical was subsequently determined by direct UV illumination. High concentrations of monoclonal IgG as a molecular component of covalent of the dexamethasone-(C₂₁-phosphoramide)-[anti-EGFR] immunopharmaceutical effectively quench UV detection of any chemotherapeutic moiety at the application origin.
Abbreviations: EGFR, epidermal growth factor receptor type 1; HP-TLC, high-performance thin layer chromatography.

Figure 3 Characterization of the molecular weight profile for the covalent immuno-pharmaceutical dexamethasone-(C₂₁-phosphoramide)-[anti-EGFR] relative to reference control anti-EGFR monoclonal immunoglobulin fractions and conventional molecular weight standards.

Notes: Lane 1: murine antihuman EGFR monoclonal immunoglobulin. Lane 2: dexamethasone-(C₂₁-phosphoramide)-[anti-EGFR]. The covalent dexamethasone-C₂₁-phosphate immunopharmaceutical and monoclonal IgG fractions were size-separated by nonreducing SDS-PAGE followed by lateral transfer onto sheets of nitrocellulose membrane to facilitate detection with HRPO protein G conjugate. Subsequent analysis entailed incubation of membranes with a HRPO chemiluminescent substrate and the acquisition of autoradiography images.
Abbreviations: EGFR, epidermal growth factor receptor type 1; IgG, immuno-globulin G.

Figure 4 Detection of total immunoglobulin in the form of dexamethasone-(C₂₁-phosphoramide)-[anti-EGFR] selectively bound to the exterior surface membrane of pulmonary adenocarcinoma.

Notes: Covalent dexamethasone-(C₂₁-phosphoramide)-[anti-EGFR] immunopharmaceutical formulated at gradient IgG-equivalent concentrations were incubated in direct contact with triplicate monolayer populations of chemotherapeutic-resistant human pulmonary adenocarcinoma (A549) over a 4-hour time period. Total IgG bound to the exterior surface membrane was then detected and measured by cell-ELISA.
Abbreviations: EGFR, epidermal growth factor receptor type 1; IgG, immunoglobulin G.

Figure 5 Relative antineoplastic cytotoxic potency of dexamethasone-(C₂₁-phosphoramide)-[anti-EGFR] against chemotherapeutic-resistant pulmonary adenocarcinoma.

Notes: (▲) Dexamethasone-(C₂₁-phosphoramide)-[anti-EGFR]; (■) dexam ethasone; and (◆) IgG anti-EGFR immunoglobulin. Formulated in triplicate at gradient standardized (dexamethasone equivalent) concentrations, both dexamethasone-(C₂₁-phosphoramide)-[anti-EGFR] and dexamethasone were individually incubated in direct contact with monolayer populations of chemotherapeutic-resistant pulmonary adenocarcinoma (A549) for a period of 192 hours. Antineoplastic cytotoxic potency was measured using a MTT cell vitality assay relative to matched negative reference controls.
Abbreviations: EGFR, epidermal growth factor receptor type 1; IgG, immunoglobulin G.