(675b) Self-Assembling Protides Nanofibers and Their Application to Anticancer Drug Delivery

Nucleoside drugs are an essential class of therapeutics for the treatment of viral infections and certain cancers. The biological activity of these molecules originates from their antimetabolite character and ability to behave as disruptive analogs of the naturally occurring endogenous nucleoside metabolites used for nucleic acid synthesis and other cellular processes. Although widely represented in antiviral therapeutic regimes, fewer nucleoside drugs are potent against cancers and even fewer are effective in the treatment of solid tumors.

Gemcitabine (2’,2’-difluoro 2’-deoxycitidine, dFdC) is a nucleoside antimetabolite widely used in the treatment of advanced or metastatic cancers of the breast, lung, ovaries, and pancreas alone or in combination with other cytotoxic agents. dFdC is an analog of the naturally occurring nucleoside cytidine and exerts its cytotoxic effect after metabolic activation through masked chain termination in DNA synthesis and through inhibition of ribonucleotide reductase. However, multiple steps are required for the internalization and metabolic activation of the parent nucleoside introducing mechanisms for chemotherapeutic resistance. Additionally, dFdC has poor pharmacokinetic properties due to its rapid inactivation by circulating and hepatic deaminases resulting in more than half of the administered dose being excreted as the inactive metabolite after 24 hours. dFdC is also associated with pulmonary and vascular off-target toxicities due to systemic administration.

To address the current therapeutic limitations associated with the dosing, administration, and off-target toxicities of dFdC, we have developed a unique approach for the localized delivery of self-assembling dFdC prodrugs to the diseased site. Previously, we examined the utility of the nucleoside phosphoramidate moiety as a regulatory motif in small molecule self-assembly. Aromatic peptides functionalized with nucleoside phosphoramidates were found to self-assemble into highly regular supramolecular nanofibers which were responsive to HINT1 enzyme, the activity of which triggered a structural transition at the nanoscale through nanofiber association and bundling. Utilizing the same self-assembling molecular scaffold, we have substituted the natural nucleoside moiety within the nucleoside phopshoramidate motif with dFdC to generate pro-drug therapeutic bearing supramolecular nanofibers. The dFdC pro-nucleotide nanofibers were found to form hydrogels in a divalent cation dependent manner to generate implantable devices for in situ delivery. Additionally, the pro-nucleotide nanofibers could be combined with additional physically or chemically crosslinked hydrogels for injectable administration. The in vitro cytotoxic properties were investigated in cancer cell lines originating from malignancies commonly treated with dFdC clinically. Our studies indicate that the self-assembling dFdC pronucleotides possess low micromolar toxicity and are potentially able to circumvent clinical mechanisms of dFdC resistance due to avoidance of the need for active transport and bypass of the rate limiting initial phosphorylation step. This work presents the first demonstration of a self-assembling phosphoramidate pronucleotide derivative for potential injectable delivery.