Reversibility in Mice of FXI Antisense-Induced Anticoagulation by Administration of FXI Protein Concentrate or a Sense Oligonucleotide.

Antisense drug technology is an effective approach for specifically targeting coagulation factors expressed in the liver. We have shown previously that this approach can be used to produce potent antithrombotic activity against a range of coagulation factors, most notably FXI. Due to their long tissue half-life, 2^nd-Generation antisense oligonucleotides (ASOs) can be administered infrequently (once/week – once/month) at therapeutically effective doses in humans by subcutaneous injection. However, because of their long tissue half-life, selecting targets that produce antithrombotic activity without increased risk of bleeding is an important consideration. In addition, antidote strategies to reverse the action of ASO anticoagulant activity is also an important consideration. We have shown that ASO targeting of FXI is an effective and safe approach for the treatment of thromboembolic disease in a variety of mouse models. Here, we investigate the reversibility of FXI ASO activity in mice using two strategies;

1. by studying the effects of a sense oligonucleotide that is complimentary to our FXI ASO, and

2. by determining the reversibility of FXI ASO activity using human FXI protein concentrate.

Administration of a single subcutaneous dose of FXI ASO (ISIS 404071) at 50 mg/kg resulted in a time-dependent reduction in FXI mRNA levels in liver which corresponded with a prolongation of aPTT. Onset of action for aPTT prolongation was within 2 days, peaked at 4 days, and lasted for a total of 11–12 days. Chronic treatment (twice weekly for three weeks) led to a longer duration of action that lasted approximately 14 days. The ability of a sense oligonucleotide (SO) to reverse these effects was investigated by a single subcutaneous injection of SO at 90 mg/kg 2 days after chronic (3 week at 40 mg/kg twice weekly) FXI ASO treatment. SO treatment resulted in a significant shortening of FXI ASO duration of action, as measured by prolongation of aPTT. Recovery of 50% aPTT prolongation was shortened from a duration of three days to less than 24 hours following SO treatment. aPTT was completely normalized within 4–5 days following SO treatment (vs 14 days without SO treatment). These effects on aPTT reversibility correlated well with the reversibility of FXI mRNA depletion following SO treatment. Reversibility of FXI ASO anticoagulation activity was also examined using human FXI protein concentrate. Anticoagulation activity induced by chronic treatment with FXI ASO was rapidly and completely reversed following a single intravenous administration of purified human FXI (20 μg). These results demonstrate that the anticoagulation effect of FXI ASO treatment can be reversed by two alternative strategies; first, by using a sequence-specific sense oligonucleotide to neutralize the ASO inhibitory effect on target FXI mRNA levels or, second, by treating with purified human FXI protein concentrate. These results further strengthen the rationale for a FXI ASO strategy as a novel approach to antithrombotic therapy.