Kinetics of phosphorothioate oligonucleotide metabolism in biological fluids

The in vitro stability and metabolism of GEM®91, a 25mer phosphorothioate antisense oligonucleotide complementary to the gag mRNA region of HIV-1, was investigated using capillary electrophoresis (CE). The in vitro degradation of the parent compound at 37°C was followed over the course of 120 h in human plasma. A CE method using laser-induced fluorescence detection was able to detect 5′-end intact metabolites including the parent compound extracted from biological fluids. Because the primary metabolic pathway is believed to be via 3′-exonuclease activity, the results of this study were compared with the stability of the compound in a solution containing 3′-exonuclease. The numerical solution of sequential first-order reactions was used to obtain kinetic parameters. Exonuclease digestion of the parent compound, as measured using an automated CE-UV instrument, yielded striking similarities between the two in vitro systems as well as between in vitro and in vivo systems.