Fludarabine- (C2-methylhydroxyphosphoramide)- [anti-IGF-1R]: Synthesis and Selectively Targeted Anti-Neoplastic Cytotoxicity against Pulmonary Adenocarcinoma (A549)

Abstract

Introduction Many if not most conventional small molecular weight chemotherapeutics are highly potent against many forms of neoplastic disease. Unfortunately, majority of an administered dose unintentionally diffuses passively into normal tissues and healthy organ systems following intravenous administration. One strategy for both increasing potency and reducing dose-limited sequela is the selective “targeted” delivery of conventional chemotherapeutic agents. Materials and Methods The fludarabine-(C2- methylhydroxyphosphoramide)-[anti-IGF-1R] was synthesized by initially reacting fludarabine with a carbodiimide to form a fludarabine carbodiimide phosphate ester intermediate that was subsequently reacted with imidazole to create an amine-reactive fludarabine- (C2-phosphorylimidazolide) intermediate. Monoclonal anti-IGF-1R immunoglobulin was combined with the amine-reactive fludarabine- (C2-phosphorylimidazolide) intermediate resulting in the synthesis of covalent fludarabine-(C2-methylhydroxyphosphoramide)- [anti-IGF-1R] immunochemotherapeutic. Residual fludarabine and un-reacted reagents were removed by serial microfiltration (MWCO 10,000) and monitored by analytical-scale HP-TLC. Retained IGF-1R binding-avidity of fludarabine-(C2- methylhydroxyphosphoramide)-[anti-IGF-1R] was established by cell-ELISA using pulmonary adenocarcinoma cell (A549) which over-expresses IGF-1R and EGFR. Anti-neoplastic cytotoxic potency of fludarabine-(C2-methylhydroxyphosphoramide)-[anti- IGF-1R] was determined against pulmonary adenocarcinoma (A549) using an MTT-based vitality stain methodology. Results The fludarabine molar-incorporation-index for fludarabine- (C2-methylhydroxyphosphoramide)-[anti-IGF-R1] was 3.67:1 while non-covalently bound fludarabine was not detected by analytical scale HP-TLC following serial micro-filtration. Size-separation fludarabine-(C2-methylhydroxyphosphoramide)-[anti- IGF-1R] by SDS-PAGE with chemo luminescent autoradiography detected only a single 150-kDa band. Cell-ELISA of fludarabine- (C2-methylhydroxyphosphoramide)-[anti-IGF-1R] measuring total immunoglobulin bound to exterior surface membranes of pulmonary adenocarcinoma (A549) increased with elevations in immunoglobulin-equivalent concentrations of the covalent fludarabine immunochemotherapeutic. Between the fludarabine-equivalent concentrations of 10−10 M and 10−5 M both fludarabine-(C2- methylhydroxyphosphoramide)-[anti-IGF-1R] and fludarabine had ex-vivo anti-neoplastic cytotoxic potency levels that increased rapidly between the fludarabine-equivalent concentrations of 10−6 M and 10−5 M where cancer cell death percentages increased from 24.4% to a maximum of 94.7% respectively. Conclusion The molecular design and organic chemistry reaction schemes were developed for synthesizing fludarabine-(C2- methylhydroxyphosphoramide)-[anti-IGF-1R] which possessed both properties of selective “targeted” delivery and anti-neoplastic cytotoxic potency equivalent to fludarabine chemotherapeutic.

Gemcitabine‐(5′‐phosphoramidate)‐[anti‐IGF‐1R]: molecular design, synthetic organic chemistry reactions, and antineoplastic cytotoxic potency in populations of pulmonary adenocarcinoma (A549)

“…Relative effectiveness of the multistage organic chemistry reaction scheme implemented for the synthesis of gemcitabine-(5′-phosphoramidate)-[anti-IGF-1R] is in part demonstrated by attainment of a 2.67:1 gemcitabine:IgG molar incorporation index. Such qualities are further substantiated by comparative experimental results from analogous synthesis methods in related investigations where 1-ethyl-3 -[3-dimethylaminopropyl]carbodiimide in combination with imidazole has been used to covalently bond fludarabine, [79] dexamethasone-C 21 -phosphate, and other phosphorylated pharmaceutical analogs to monoclonal IgG fractions. The gemcitabine:IgG molar incorporation index of 2.67:1 for gemcitabine-(5′-phosphoramidate)-[anti-IGF-1R] was comparatively greater or equivalent to values obtained in previous investigations utilizing other covalent bond forming agents for the synthesis of covalent immunochemotherapeutics such as (i) gemcitabine-(C 5methylcarbamate)-[anti-HER2/neu] (Gem:IgG = 1.1-to1); [75] (ii) gemcitabine-(C 4amide)-[anti-HER2/neu] (Gem:IgG = 2.78-1); [76] (iii) epirubicin-(C 3amide)-[anti-HER2/neu] (Epi:IgG = 0.275-1); [38] (iv) epirubicin-(C 3amide)-[anti-EGFR] (Epi:IgG = 0.407-1); [38] and (vi) epirubicin-(C 13imino)-[anti-HER2/neu] (Epi:IgG = 0.400-1).…”

“…Exceptions are chemotherapeutic-resistant metastatic melanoma M21 (covalent daunorubicin immunochemotherapeutics synthesized using antichondroitin sulfate proteoglycan 9.2.27 surface marker) [68,71,156] ; chemotherapeutic-resistant mammary carcinoma MCF-7AdrR (covalent anthracycline-ligand chemotherapeutics utilizing epidermal growth factor (EGF) or an EDF fragment) [157] ; and chemotherapeutic-resistant mammary adenocarcinoma (SKBr-3) populations (epirubicin-anti-HER2/neu and epirubicin-anti-EGFR, gemcitabine-anti-HER 2/neu), [38,64,75,76,92] and chemotherapeutic-resistant pulmonary adenocarcinoma A549 (fludarabine-(C 2 -methylhydroxyphosphoramide)-[anti-IGF-1R]). [79] A high degree of probability exists that in vivo, gemcit abine-(5 ′ -phosphoramidate)-[anti-IGF1R], gemcitabine-(C 2methylcarbamate)-[anti-HER2/neu], [155] and gemcitabine-(C 2 -methylhydroxylamide)-[anti-EGFR] [76] would provide planes of antineoplastic cytotoxic potency that would be at least equivalent to, if not surpass that of gemcitabine chemotherapeutic due to several contributing parameters. More specifically, under in vivo conditions, it is anticipated that the antineoplastic cytotoxicity of gemcitabine-(5′-phosphoramidate)-[anti-IGF-1R] would be further complemented by the attributes and advantages of (i) enhanced pharmacokinetic profiles; (ii) greater cytosol concentrations within 'targeted' cancer cell populations over time, all in collective concert with; and (iii) stimulation of the endogenous host immune responses of antibodydependent cell cytotoxicity (ADCC), complement-mediated cytolysis (CMC), and opsonization/phagocytosis subsequent to formation of membrane IgG-antigen complexes.…”

“…The organic chemistry reaction regimens as described from a methodology perspective are similar to those illustrated in previous research investigations that for the first time delineated two different and separate organic chemistry reaction regimens for the synthesis of covalent gemcitabine immunochemotherapeutics. [75,76] The organic chemistry reaction scheme utilized to synthesize covalent gemcitabine-(5′phosphoramidate)-[anti-IGF-1R] immunochemotherapeutic was founded on organic chemistry reaction regimens initially employed in synthesis regimens for the synthetic production of fludarabine-(5′-phosphoramidate)-[anti-IGF-1R] [79] and dexamethasone-(C 21-…”

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

“…The molecular design and a corresponding organic chemistry reaction scheme have been delineated to enable a multiphase regimen for synthesizing a covalent dexamethasone-(C 21 -phosphoramide)-[anti-EGFR] immunocorticosteroid. Analogous organic chemistry reaction schemes have been used to synthesize the covalent immunochemotherapeutics, fludarabine-(C 2 -methylhydroxyphosphoramide)-[anti-IGF-1R], 41 and gemcitabine-(C 2 -phosphoramide)-[anti-IGF-1R]. Removal of residual unreacted dexamethasone or reagents by serial microfiltration of a highly concentrated reaction mixture formulation of dexamethasone-(C 21 -phosphoramide)-[anti-EGFR] was determined by standardized high-performance thin layer chromatography (HP-TLC) analysis.…”