ALBERT

Description: The Tissue Factor-Factor VIIa complex is the initiating protease in the extrinsic pathway of the coagulation cascade. Knockout of Factor VII in mice results in embryonic lethality. However, only humans with severe Factor VII deficiency exhibit any coagulopathy. In order to determine the potential of Factor VII as an anticoagulant target and the utility of antisense oligonucleotides (ASOs) as Factor VII inhibitors, ASOs were designed to promote RNaseH mediated degradation of Factor VII RNA and evaluated in mice. Factor VII is predominantly produced in the liver, a tissue that is tractable for antisense mediated target depletion. Following three weeks of treatment, the Factor VII ASO was well tolerated and supported significant reduction of Factor VII RNA and protein in mouse liver (ED50 of 3.5 mg/kg for RNA and 2.8 mg/kg for protein). Greater than 95% reduction in Factor VII RNA levels was observed at a dose of 25 mg/kg, administered twice weekly. Following three weeks exposure to 100 mg/kg/wk, prothrombin times (PT) from platelet poor mouse plasma was more than doubled, compared to untreated animals, while activated partial thromboplastin times (aPTT) was only increased 16%. Antithrombotic activity of Factor VII ASO was determined following ferric chloride injury to the inferior vena cava. ED50 for inhibition of thrombosis was 24 mg/kg/wk. The effects of Factor VII depletion on hemostatic control were also determined using a mouse tail nick model. No increase in blood loss was detected, compared to untreated animals, up to a weekly exposure of 80 mg/kg, the highest dose utilized in the study. Compared to warfarin, ASO mediated depletion of Factor VII promoted equivalent protection from thrombosis following ferric chloride injury while maintaining better hemostatic control. These findings support the development of an ASO drug targeting Factor VII as an anticoagulant.

Description: Abstract 2101 Poster Board II-78 The absence of abnormal bleeding associated with congenital deficiencies of the intrinsic coagulation pathway factor, FXI, suggests that this pathway is not important for normal blood coagulation in vivo. However, recent work in mice and higher species demonstrates that the intrinsic pathway is an important contributor to pathologic intravascular thrombosis, suggesting that targeting this pathway may yield effective antithrombotic agents with high safety. We have previously demonstrated that second generation antisense oligonucleotides (ASOs) targeting the intrinsic pathway member FXI were potent antithrombotic agents and that this activity was achieved in several mouse models of thrombosis without any increase in bleeding risk (Zhang et al. ASH 2008). Here we characterize the pharmacological activity, chemical SAR and safety profile of FXI antisense oligonucleotides administered subcutaneously in Cynomologus monkeys. Our first monkey study was designed to address the PK/PD kinetics of FXI ASOs from a single chemical series (20 mers, 5-10-5 MOE Gapmers). Two dose escalation regimes were evaluated (1) 5mg/kg (3wks), 10mg/kg (3 wks) followed by 25 mg/kg (6wks) and (2) 5mg/kg (3wks) followed by 10mg/kg (9 wks). The subsequent study was designed to evaluate chemical SAR around the active ASOs identified in the study 1. FXI antisense oligonucleotides FXI-AS1, FXI-AS2 demonstrated dose and time dependent pharmacologic activity, including, decreased FXI mRNA in liver (up to 90%), decreased FXI protein levels and FXI activity measured in plasma, with a maximal inhibition of 〉80% observed at 25mg/kg on both schedules, prolonged activated partial thromboplastin times (aPTT), maximal aPTT ratio of 2.O, but no change in prothrombin time (PT) as expected. Similar to previous studies in mouse the anticoagulant activity observed from FXI depletion in monkeys was not associated with increased bleeding risk as assessed in a skin bleeding time test. Additionally, FXI ASOs had no deleterious effects on organ weights, platelets, or on measures of liver and kidney function. The second study confirmed the effects of FXI-AS1 and FXI-AS2 and demonstrated that shorter ASOs with decreased gap size were equally potent. These results further support the development of FXI ASOs as human therapeutics for the treatment of coagulation related disorders with the potential for improved safety profiles. Disclosures: MacLeod: Isis Pharmaceuticals: Employment. Crosby:Isis Pharmaceuticals, Inc.: Employment. Zhao:Isis Pharmaceuticals, Inc.: Employment. Gao:Isis Pharmaceuticals, Inc.: Employment. May:Isis Pharmaceuticals, Inc.: Employment. Zhang:Isis Pharmaceuticals, Inc.: Consultancy. Lowenberg:Isis Pharmaceuticals, Inc.: Consultancy. Levi:Isis Pharmaceuticals, Inc.: Consultancy. Monia:Isis Pharmaceuticals, Inc.: Employment.

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: The ability to safely and effectively neutralize anticoagulant activity is of particular importance in the case of long acting drugs. While antisense oligonucleotides (ASOs) benefit from their slow clearance rate from liver (t1/2 approx 10 days in mice) which permits infrequent dosing, the potential necessity to reverse ASO drug activity presents a challenge. ISIS 401025 is a second generation ASO targeting prothrombin RNA that has previously been shown to reduce thrombin generation and prolong PT and aPTT as a result of depleting prothrombin mRNA levels in mice. To explore the possibility of developing an “antidote” strategy for ASO-mediated anticoagulation, we designed a sense oligonucleotide, ISIS 405277, complementary to the ISIS 401025 sequence and examined its ability to reverse the effects of prior treatment with ISIS 401025 in mice. A PT-INR of 3.0 was established after 3 weeks of treatment with ISIS 401025 at a weekly dose of 60 mg/kg, corresponding to a reduction of prothrombin mRNA transcript levels of 96%. A single injection of the sense oligonucleotide, ISIS 405277, resulted in a dose-dependent reversal of INR to 1.6, 1.1, and 0.9 three days after injection of 30, 60, and 90 mg/kg, respectively. Corresponding prothrombin transcript levels in liver tissue were returned to 18.4%, 27.8%, and 38.9% of normal levels, respectively. Results from a study designed to determine the kinetics of reversal of anticoagulation indicated that a 50% reduction of PT-INR 4.3, established following 3 weeks of treatment with ISIS 401025, required 11 days in the absence of subsequent treatment, while the reduction was achieved in 2.2 days following a single administration of 90 mg/kg of sense oligonucleotide. When mice were pretreated with an ASO targeting prothrombin that was not complementary to the sense oligonucleotide (ISIS 40527), sense oligonucleotide treatment was unable to reverse either target depletion or anticoagulation, indicating a sequence-specific antidote effect. These results demonstrate for the first time that subsequent administration of a sense strand oligonucleotide can neutralize ASO mediated target RNA degradation in animals, and demonstrates its potential utility in reversing ASO-based activity for anticoagulation.

Description: Numerous potential intervention points exist within coagulation pathways for development of novel anticoagulant agents. To help determine the most suitable target for the discovery of novel antithrombotic agents, we have implemented an antisense strategy to specifically deplete levels of various coagulation factors in mice. Here we report the effects of antisense oligonucleotides (ASOs) targeting factors II, VII, and IX on target RNA and protein levels in mice, as well as PT/aPTT. ISIS 401025 targeting factor II, ISIS 403102 targeting factor VII, and ISIS 402618 targeting factor IX dose-dependently suppressed their respective target transcript levels in a specific manner in primary mouse hepatocytes. Following subcutaneous administration to mice, each ASO reduced target RNA levels in liver and target protein levels in plasma in a specific and dose-dependent fashion (ED95 of approximate 25 mg/kg for each ASO). In conjunction with these effects, clotting times were also prolonged in a similar dose responsive fashion. Both PT and aPTT were increased following factor II inhibition and a PT-INR of 2.5 was achieved at 25 mg/kg, corresponding to a reduction in prothrombin transcript of 95%. As expected following factor VII ASO treatment, PT was significantly increased, while aPTT was unchanged. Conversely, following factor IX ASO treatment, aPTT was marginally but significantly prolonged, while PT was unchanged. These results demonstrate the capability of ASOs to dose-dependently and specifically modulate coagulation factor activity depletion and support the ASO approach as a novel strategy for the discovery of novel anticoagulation agents. Studies are in progress to assess relative safety and to expand the anticoagulation profile in mice for each of the factors under study.

Description: Abstract 4181 Human clinical efficacy has been demonstrated with antisense oligonucleotides (ASOs) in several disease indications, including dyslipidemia, cancer, diabetes and multiple sclerosis. Second generation ASOs show potent pharmacology in many tissues, with liver being one of the most sensitive to ASO action. As the liver is responsible for production of many of the factors involved in blood coagulation we systematically designed and evaluated ASOs selective for several members of the coagulation cascade. ASOs targeting coagulation cascade members of both the intrinsic and extrinsic pathway produced highly specific target knockdown in liver. An overall aim of this study was to evaluate the contribution of the various factors to the coagulation process as well as to assess their relative risk/benefit relationships. The “risk” component was determined by bleeding tendency as measured by blood volume loss following tail nick, and the “benefit” component was determined by antithrombotic effect following ferric chloride induced thrombosis in the inferior vena cava. We have previously reported the activity of FVII and FXI ASOs in a ferric chloride mouse model without increased bleeding risk. Here we identify additional promising new antithrombotic targets. FII and FIX ASOs were able to reduce thrombosis, but inhibition of these targets had deleterious effects on the bleeding tendency. The ASO effects on PT and aPTT will be presented. The approach used in this study will be used to further characterize targets for antithrombotic potential and determine their ability to be combined with antiplatelet therapies. In addition to the characterization of antithrombotic targets, these ASOs will be evaluated and developed as potent, selective and potentially safer human antithrombotic therapeutics. Disclosures: Crosby: Isis Pharmaceuticals, Inc.: Employment. Gao:Isis Pharmaceuticals, Inc.: Employment. Zhao:Isis Pharmaceuticals, Inc.: Employment. Bhattacharjee:Isis Pharmaceuticals, Inc.: Employment. May:Isis Pharmaceuticals, Inc.: Employment. Zhang:Isis Pharmaceuticals, Inc.: Consultancy. MacLeod:Isis Pharmaceuticals: Employment. Monia:Isis Pharmaceuticals, Inc.: Employment.

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: 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, 2nd-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; by studying the effects of a sense oligonucleotide that is complimentary to our FXI ASO, and 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.

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.