Synthesis and Biological Evaluation of EC72:  A New Folate-Targeted Chemotherapeutic

Abstract

A novel folate conjugate of mitomycin C, herein referred to as EC72, was designed and evaluated for biological activity against FR-positive cells and tumors. EC72 was produced by coupling folic acid-gamma-cysteine to 7-N-modified MMC via a disulfide bond. This water soluble conjugate was found to retain high affinity for FR-positive cells, and it produced dose responsive activity in vitro against a panel of folate receptor (FR)-positive cell lines. EC72's activity was considered to be targeted and specific for the FR since (i) excess folic acid blocked biological activity, and (ii) FR-negative cell lines were unresponsive to this drug. Initial in vivo tests confirmed EC72's activity in both syngeneic and xenograft models, and this activity occurred in the apparent absence of gross or pathological toxicity. These results are significant, since daily dosing of EC72 for more than 30 consecutive days yielded no evidence of myelosuppression or toxicity to major organs, including the FR-positive kidneys. The latter observation supports published data, indicating that the apically oriented kidney proximal tubule FRs function to salvage folates prior to their excretion and to return these molecules back into systemic circulation. Overall, EC72's performance in vitro and in vivo warrants further preclinical study before this novel targeted chemotherapeutic is considered for clinical investigation.

Evaluation of disulfide reduction during receptor-mediated endocytosis by using FRET imaging

“…Because higher potencies are achieved generally when the drug is released from its attached targeting ligand (i.e., folic acid) in an unaltered form (30)(31)(32)(33), the data presented here suggest that disulfide bridges between folate and its therapeutic payload might constitute suitable linkers for site-specific drug release. This argument is supported by the much more potent antitumor activity of a disulfide-linked folate-mitomycin C conjugate relative to an analogus amide-linked conjugate (34,35). Similarly, a disulfidebridged folate-Pseudomonas exotoxin conjugate exhibited cytotoxicity that was four orders of magnitude more than the same conjugate connected by a thioether linkage (36).…”

Synthesis and evaluation of a novel ligand for folate-mediated targeting liposomes

“…Folic acid (folate) is a high affinity ligand for the FRs that retains high FR affinity upon derivatization via one of its carboxyl group. Due to its small size and ready availability, folate has become one of the most investigated targeting ligands for tumor-specific drug delivery (Curiel et al, 1999;Gabizon et al, 2004;Hilgenbrink et al, 2005;Ke et al, 2003;Kim et al, 2007;Leamon et al, 2005;Zhang et al, 2006). Folate has been incorporated into liposomes via conjugation to lipophilic anchors.…”

DOTA Conjugate with an Albumin-Binding Entity Enables the First Folic Acid–Targeted 177Lu-Radionuclide Tumor Therapy in Mice

“…Among these, the most successful approach was the conjugation of folic acid to highly toxic low-molecular-weight chemotherapeutics (5,12). With several of these folate-based drug conjugates, respectable anticancer effects were achieved in preclinical (13)(14)(15)(16) and clinical studies (17). If b 2 -particle-emitting radioisotopes are used for targeted therapy, cancer cells are irradiated not only by decays taking place at or within the targeted cells but also by decays in neighboring or distant cells by the so-called cross-fire effect (18).…”