A novel oligodeoxynucleotide inhibitor of thrombin. I. In vitro metabolic stability in plasma and serum.
OBJECTIVE
To determine the degradation rates and pathways of GS-522, a potent oligodeoxynucleotide (GGTTGGTGTGGTTGG) inhibitor of thrombin, in serum and plasma.


METHODS
A stability-indicating, anion-exchange HPLC method was developed and used to determine concentrations of GS-522 and metabolites.


RESULTS
In monkey plasma at 2 microM or below, the degradation of GS-522 can be fit to a first-order exponential with a kpobs approximately 0.01 min-1. At 3 microM and above the degradation process deviates from a monoexponential decay profile. An initial fast degradation process is followed by a slower phase with an observed rate constant equal to that observed at 2 microM and below. In monkey serum, the KM and Vmax are 8.4 microM and 0.87 microM min-1, respectively.


CONCLUSIONS
The kinetics are consistent with an equilibrium binding of GS-522 to prothrombin in plasma (Kd = 50 nM) which saturates at GS-522 concentrations > 2 microM. Compared to a scrambled sequence (GGTGGTGGTTGTGGT), with no defined tertiary structure, GS-522 is 4-fold more stable in serum. The metabolic profile in plasma is consistent with a 3'-exonuclease catalyzed hydrolysis of GS-522.
Shaw
J P
JP
Gilead Sciences, Inc., Foster City, California 94404, USA.
Fishback
J A
JA
Cundy
K C
KC
Lee
W A
WA
D052157:Aptamers, Nucleotide; D001483:Base Sequence; D004305:Dose-Response Relationship, Drug; D004355:Drug Stability; D006801:Humans; D066298:In Vitro Techniques; D008969:Molecular Sequence Data; D009841:Oligonucleotides / Q000378:metabolism* / Q000494:pharmacology*; D004364:Pharmaceutical Preparations / Q000378:metabolism*; D013917:Thrombin / Q000187:drug effects*
D052157:Aptamers, Nucleotide; D009841:Oligonucleotides; D004364:Pharmaceutical Preparations; C081676:thrombin aptamer; D013917:Thrombin