RESUMO
Most antiviral and anticancer nucleosides are prodrugs that require stepwise phosphorylation to their triphosphate nucleotide form for biological activity. Monophosphorylation may be rate-limiting, and the nucleotides may be unstable and poorly internalized by target cells. Effective targeting and delivery systems for nucleoside drugs, including oligonucleotides used in molecular therapeutics, could augment their efficacy. The development of a carrier designed to effect selective transmembrane internalization of nucleotides via the asialoglycoprotein receptor (ASGPr) is now reported. In this work, the polycationic, polygalactosyl drug delivery carrier heptakis[6-amino-6-deoxy-2-O-(3-(1-thio-ß-D-galactopyranosyl)-propyl)]-ß-cyclodextrin hepta-acetate salt (GCyDAc), potentially a bifunctional carrier of (poly)nucleotides, was modeled by molecular docking in silico as an ASGPr-ligand, then synthesized for testing. The antivirals arabinosyl adenine (araA, vidarabine, an early generation antiviral nucleoside), arabinosyl adenine 5'-monophosphate (araAMP), and 12-mer-araAMP (p-araAMP) were selected for individual formulation with GCyDAc to develop this concept. Experimentally, beta cyclodextrin was decorated with seven protonated amino substituents on the primary face, and seven thiogalactose residues on its secondary face. AraA, araAMP, and p-araAMP were individually complexed with GCyDAc and complex formation for each drug was confirmed by differential scanning calorimetry (DSC). Finally, the free drugs and their GCyDAc complexes were evaluated for antiviral activity using ASGPr-expressing HepAD38 cells in cell culture. In this model, araA, araAMP, and p-araAMP showed relative antiviral potencies of 1.0, 1.1, and 1.2, respectively. In comparison, GCyDAc-complexes of araA, araAMP, and p-araAMP were 2.5, 1.3, and 1.2 times more effective than non-complexed araA in suppressing viral DNA production. The antiviral potencies of these complexes were minimally supportive of the hypothesis that ASGPr-targeted, CyD-based charge-association complexation of nucleosides and nucleotides could effectively enhance antiviral efficacy. GCyDAc was non-toxic to mammalian cells in cell culture, as determined using the MTS proliferation assay.
RESUMO
PURPOSE: Cyclodextrins (CDs) have been identified as a viable alternative to viral vectors for use in therapeutic applications. Here, the stability of the complex formed between the a multiply charged, cationic, fully substituted heptakis-(6-amino-2-galactosyl)cyclodextrin (BCDX12) with a multiply charged 12-mer hexachlorofluorescein tagged arabinopolynucleotide (Hex-PAH) have been evaluated. METHODS: The stability of complexes of Hex-PAH and BCD-X12 was studied with respect to mole ratio (1:1, 1:2, and 1:5 Hex-PAH:BCD-X12), pH, buffer concentration, temperature, and agitation using capillary electrophoresis with laser induced fluorescence detection (CE/LIF). Two neutral CDs and an additional cationic CD were also tested under the same analytical conditions to determine their ability to form complexes. RESULTS: Hex-PAH:BCDX12 complexes at mole ratios of 1:2 were stable in 10 mM (160 mM total borate concentration) sodium tetraborate buffer at pH 7.5 and at temperatures of 4 degrees C and 25 degrees C over 48 hours. However, the Hex-PAH:BCD-X12 complex was less stable at 37 degrees C and at higher buffer concentrations and pH values. Strong vortex mixing prior to analysis was found to disrupt the complex. Of the four CDs tested for their ability to complex with Hex-PAH, only BCDX12 formed stable complexes with Hex-PAH under the test conditions. CONCLUSIONS: Capillary electrophoresis was found to be well suited to test the stability of cyclodextrin-nucleotide complexes. CE/LIF indicated that only a single Hex- PAH:BCD-X12 complex was formed at all formulation ratios, and that the complexes were electrophoretically identical to each other, and increasing the molar ratio beyond 1:2 did not contribute measurably to complex stability. Storage temperature and agitation conditions were found to influence complex stability. Since no stable complexes were formed with neutral cyclodextrins, the results support the hypothesis of a 'charge associated' complex rather than an inclusion complex, although inclusion complexes cannot be excluded on the basis of these studies.
