A novel oligodeoxynucleotide inhibitor of thrombin. I. 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, 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.