A NOVEL OLIGODEOXYNUCLEOTIDE INHIBITOR OF THROMBIN .1. IN-VITRO METABOLIC STABILITY IN PLASMA AND SERUM

Purpose. To determine the degradation rates and pathways of GS-522, a potent oligodeoxynucleotide (GGTTGGTGTGGTTGG) inhibitor of thrombin, i n serum and plasma. Methods. A stability-indicating, anion-exchange HP LC method was developed and used to determine concentrations of GS-522 and metabolites. Results. In monkey plasma at 2 mu M or below, the de gradation of GS-522 can be fit to a first-order exponential with a k(o bs)(p) similar to 0.01 min(-1). At 3 mu M and above the degradation pr ocess deviates from a monoexponential decay profile. An initial fast d egradation process is followed by a slower phase with an observed rate constant equal to that observed at 2 mu M and below. In monkey serum, the K-M and V-max are 8.4 mu M and 0.87 mu M min(-1), respectively. C onclusions. The kinetics are consistent with an equilibrium binding of GS-522 to prothrombin in plasma (K-d = 50 nM) which saturates at GS-5 22 concentrations > 2 mu M. Compared to a scrambled sequence (GGTGGTGG TTGTGGT), with no defined tertiary structure, GS-522 is 4-fold more st able in serum. The metabolic profile in plasma is consistent with a 3' -exonuclease catalyzed hydrolysis of GS-522.