Synthesis and characterisation of a nucleotide based pro-drug formulated with a peptide into a nano-chemotherapy for colorectal cancer

Abstract

Recent studies in colorectal cancer patients (CRC) have shown that increased resistance to thymidylate synthase (TS) inhibitors such as 5-fluorouracil (5-FU), reduce the efficacy of standard of care (SoC) treatment regimens. The nucleotide pool cleanser dUTPase is highly expressed in CRC and is an attractive target for potentiating anticancer activity of chemotherapy. The purpose of the current work was to investigate the activity of P¹, P⁴-di(2',5'-dideoxy-5'-selenouridinyl)-tetraphosphate (P₄-SedU₂), a selenium-modified symmetrically capped dinucleoside with prodrug capabilities that is specifically activated by dUTPase. Using mechanochemistry, P₄-SedU₂ and the corresponding selenothymidine analogue P₄-SeT₂ were prepared with a yield of 19% and 30% respectively. The phosphate functionality facilitated complexation with the amphipathic cell-penetrating peptide RALA to produce nanoparticles (NPs). These NPs were designed to deliver P₄-SedU₂ intracellularly and thereby maximise in vivo activity. The NPs demonstrated effective anti-cancer activity and selectivity in the HCT116 CRC cell line, a cell line that overexpresses dUTPase; compared to HT29 CRC cells and NCTC-929 fibroblast cells which have reduced levels of dUTPase expression. In vivo studies in BALB/c SCID mice revealed no significant toxicity with respect to weight or organ histology. Pharmacokinetic analysis of blood serum showed that RALA facilitates effective delivery and rapid internalisation into surrounding tissues with NPs eliciting lower plasma C_max than the equivalent injection of free P₄-SedU₂, translating the in vitro findings. Tumour growth delay studies have demonstrated significant inhibition of growth dynamics with the tumour doubling time extended by >2weeks. These studies demonstrate the functionality and action of a new pro-drug nucleotide for CRC.

Keywords: Anticancer; Dinucleoside tetraphosphate; Mechanochemsitry; Nanoparticles; Non-viral; RALA; Selenium; dUTPase.