ALBERT

Description: Existing anticoagulants effectively inhibit the activity of coagulation factors of the extrinsic and common pathway but have substantial limitations and can cause severe bleeding complications. Here we describe a novel therapeutic approach to thrombosis treatment. We have developed and characterized the efficacy and safety of selective second-generation antisense oligonucleotides (ASOs) targeting coagulation factor XI (FXI), a member of the intrinsic coagulation pathway. Systemic treatment of mice with FXI ASO led to a potent, specific, and dose-dependent reduction of FXI mRNA levels in the liver with corresponding reductions in plasma levels of FXI protein and activity. FXIASO treatment produced potent, dose-dependent antithrombotic activity in various venous and arterial thrombosis models, comparable with warfarin or enoxaparin. However, unlike warfarin or enoxaparin, FXI inhibition did not cause bleeding. Coadministration of FXI ASO with enoxaparin or the antiplatelet drug clopidogrel produced improved antithrombotic activity without increased bleeding. Finally, plasma-derived FXI concentrate was shown to effectively and rapidly reverse the anticoagulant effect of FXI antisense therapy. These results support the concept that inhibition of FXI through antisense therapy might serve as a new and effective strategy for the treatment and prevention of venous thromboembolism with improved specificity and safety.

Description: Abstract 1149 Human clinical efficacy has been demonstrated with antisense oligonucleotides (ASOs) in several disease indications, including dyslipidemia, cancer, diabetes and multiple sclerosis. Coagulation factors are attractive targets for antisense therapeutics for several reasons. The liver is principally responsible for production of coagulation factors and is an organ which is highly sensitive to ASO drugs. ASOs are highly specific, and thus we can selectively evaluate multiple coagulation factors to determine their roles in thrombosis, bleeding, and other processes. Employing antisense technology we selectively reduced levels of several individual clotting factors and compared the relative risk/benefit profiles. Antithrombotic activity was determined following ferric chloride induced thrombosis in the inferior vena cava and bleeding tendency was measured by blood volume loss following tail nick. ASOs targeting several clotting factors were studied, including factors FII, FVII, FIX, FXI and FXII. Previously we characterized the beneficial effects of targeting FXI, demonstrating antithrombotic effects without increased bleeding risk. Here, we describe the effects of targeting another member of the intrinsic pathway, FXII. FXII ASO treatment resulted in a dose-dependent and specific reduction in FXII mRNA levels in liver. These reductions in FXII levels correlated well with a prolongation of aPTT with no effects on PT prolongation. The anticoagulant effect of FXII ASO treatment also correlated well with antithrombotic activity in a FeCl3 induced IVC thrombosis mouse model across a wide dose range. In addition to the venous thrombosis model, FXII ASO treatment was effective across a wide dose range in a model of arterial thrombosis. Furthermore, FXII ASO treatment was well tolerated and no prolongation of tail bleeding time was observed at any dose tested, indicating a broad safety margin for FXII ASO targeting (in mice). In addition, FXII inhibition was protective in a mouse TF driven pulmonary embolism model. Inhibiting coagulation factors with ASOs is highly specific and offers an attractive approach to identifying optimal antithrombotic targets as well as human therapeutics for the treatment of coagulation related disorders with the potential for improved safety profiles. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 2255 Complications associated with increased bleeding risk are the main limitations with current anticoagulant therapy. Inhibition of factor XI (FXI), a component of the intrinsic coagulation pathway, has received considerable interest because of the potential to produce sufficient anticoagulant activity without bleeding. The objective of these studies were to characterize the efficacy and safety of ISIS-FXrx and to assess the bleeding risk associated with FXI depletion under experimental surgical settings in cynomolgus monkeys. ISIS-FXIRx was selected from a series of 2nd-generation (2'-O-methoxyethyl) antisense oligonucleotides (ASOs) targeted to FXI which were evaluated for both tolerability and efficacy in cynomolgus monkeys (25 mg/kg twice per week for 13 weeks, SC). ISIS-FXIRx emerged as the most efficacious among the FXI ASOs evaluated, producing marked reductions of FXI RNA in liver, and FXI protein and activity levelsin plasma with a good tolerability profile. A more extensive dose-response evaluation of ISIS-FXrx (4,8,12 and 40 mg/kg/wk, SC) was conducted over a 13-week treatment period in cynomolgus monkeys to further assess tolerability and activity. ISIS-FXIRx produced a dose-dependent reduction in plasma FXI activity (〉80% at 4 weeks of treatment at 40 mg/kg) with a concomitant increase in aPTT (33%). No effects on PT, platelets and no evidence of bleeding were observed after 13 weeks of treatment with ISIS-FXrx. ASO treatment was well tolerated at all doses tested and only produced changes typical for this chemical class of ASOs (e.g., basophilic granules in multiple tissues due do drug accumulation). ISIS-FXIRx (20 mg/kg, SC for 6 weeks) was also evaluated for bleeding risk in two experimental surgical models in cynomolgus monkeys; a tail amputation model and gum laceration model. Enoxaparin (2 mg/kg), known to increase the risk of bleeding, produced a statistically significant (p

Description: Abstract 2250 It has recently been demonstrated that the intrinsic pathway is an important contributor to pathologic intravascular thrombosis, suggesting that targeting this pathway may yield effective antithrombotic agents. Furthermore, FXI deficiency is not associated with spontaneous bleeding in humans, and deficiency of a contact factor does not cause abnormal bleeding. We have previously shown that treatment with FXI Antisense Oligonucleotides (ASOs) produces potent, dose-dependent antithrombotic activity in various venous and arterial murine thrombosis models. In the current study we characterize the antithrombotic effects of FXI ASOs in a non-human primate model. Subcutaneous administration of FXI ASOs in cynomologus monkeys resulted in a dose-and time-dependant decrease in FXI mRNA levels in liver (up to 90%), decreased FXI protein and activity levels measured in plasma, and prolonged activated partial thromboplastin times (aPTT). Importantly, FXI depletion in monkeys was not associated with an increased risk of bleeding. Recent studies demonstrated that complete inhibition (〉99%) of FXI by a monoclonal antibody reduced thrombin generation and prevented vascular occlusion in a collagen-coated graft inserted into a baboon arteriovenous shunt (Tucker et al., Blood 2009). In the present study, we set out to determine the relationship between FXI levels and antithrombotic activity, and in particular, the minimal level of FXI reduction required to produce an antithrombotic effect in a baboon thrombosis model. Using the FXI monoclonal antibody, we demonstrate that ∼50% inhibition of FXI levels produces antithrombotic activity in baboons. Next we applied baboon-specific FXI ASOs and demonstrated reductions in FXI protein levels and activity, with corresponding increases in aPTT levels. Baboons were then treated with FXI ASOs in a manner that would produce ∼50% reduction of FXI protein levels and measured anti-thrombotic activity. Similar to the antibody approach, ASO-mediated reduction of FXI plasma levels ≥ 50% resulted in a potent antithrombotic effect. Furthermore, reductions in FXI levels do not increase bleeding times in baboons. These results further support the development of FXI ASOs for antithrombotic therapy with the potential for a superior safety profile compared to currently available anticoagulants. Disclosures: Crosby: Isis Pharmaceuticals: Employment. Zhao:Isis Pharmaceuticals: Employment. Gao:Isis Pharmaceuticals: Employment. Revenko:Isis Pharmaceuticals: Employment. MacLeod:Isis Pharmaceuticals: Employment. Gruber:Aronora, LLC: Consultancy, Equity Ownership. Monia:Isis Pharmaceuticals: Employment.

Description: Recent studies indicate that the plasma contact system plays an important role in thrombosis, despite being dispensable for hemostasis. For example, mice deficient in coagulation factor XII (fXII) are protected from arterial thrombosis and cerebral ischemia-reperfusion injury. We demonstrate that selective reduction of prekallikrein (PKK), another member of the contact system, using antisense oligonucleotide (ASO) technology results in an antithrombotic phenotype in mice. The effects of PKK deficiency were compared with those of fXII deficiency produced by specific ASO-mediated reduction of fXII. Mice with reduced PKK had ∼ 3-fold higher plasma levels of fXII, and reduced levels of fXIIa-serpin complexes, consistent with fXII being a substrate for activated PKK in vivo. PKK or fXII deficiency reduced thrombus formation in both arterial and venous thrombosis models, without an apparent effect on hemostasis. The amount of reduction of PKK and fXII required to produce an antithrombotic effect differed between venous and arterial models, suggesting that these factors may regulate thrombus formation by distinct mechanisms. Our results support the concept that fXII and PKK play important and perhaps nonredundant roles in pathogenic thrombus propagation, and highlight a novel, specific and safe pharmaceutical approach to target these contact system proteases.