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Nucleic Acids Research, Vol 25, Issue 18 3615-3620, Copyright © 1997 by Oxford University Press

ARTICLES

Kinetics of phosphorothioate oligonucleotide metabolism in biological fluids

M Gilar, A Belenky, DL Smisek, A Bourque and AS Cohen
Hybridon, Inc., 620 Memorial Drive, Cambridge, MA 02139, USA.

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 degrees 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.
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