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Next Generation Antithrombotic Therapy: Focus on Antisense Therapy against Coagulation Factor XI Camilla Mattiuzzi, MD3
1 Laboratory of Clinical Chemistry and Hematology, Academic Hospital
of Parma, Parma, Italy 2 Clinical Pharmacology, Medical Faculty Mannheim, Ruprecht-Karls University Heidelberg, Mannheim, Germany 3 Service of Clinical Governance, General Hospital of Trento, Trento, Italy 4 Department of Haematology, Institute of Clinical Pathology and Medical Research (ICPMR), Pathology West, Westmead Hospital, Westmead, New South Wales, Australia
Address for correspondence Giuseppe Lippi, MD, U.O. Diagnostica Ematochimica, Azienda Ospedaliero-Universitaria di Parma, Via Gramsci, 14, 43126 - Parma, Italy (e-mail: [email protected]; [email protected]).
Semin Thromb Hemost 2015;41:255–262.
Abstract
Keywords
► ► ► ►
anticoagulant therapy antisense therapy factor XI venous thromboembolism ► thrombosis
Although the current therapeutic armamentarium of venous thrombosis encompasses the use of vitamin K antagonists, heparins, and direct oral anticoagulants, these drugs have several important drawbacks. Antisense oligonucleotides are relatively short single-stranded nucleic acid sequences, which hybridize with a target messenger RNA (mRNA) and suppress protein synthesis. Coagulation factor XI is a key player in blood coagulation, and thus represents a potential target for antisense therapy. The available evidence reviewed in this article suggests that factor XI antisense oligonucleotides may be more effective than conventional anticoagulants in preventing the onset and propagation of thrombosis, do not require factor measurement since the reduction of mRNA synthesis appears dose-dependently, robustly, and stably decreased for 3 to 5 weeks after the end of administration, with an incidence of major bleeding that is at least not greater than that associated with warfarin or low-molecular-weight heparin therapy. Despite conceptual simplicity, rational design, and relatively inexpensive cost, the preliminary findings in animal models and in patients undergoing knee surgery need to be validated in other prospective trials and cost-effective analyses before this attractive treatment option can be advocated as a new paradigm in prevention and treatment of venous thrombosis.
An Introduction to the Concept of Antisense Therapy Antisense oligonucleotides are commonly defined as unmodified or chemically modified single-stranded nucleic acid sequences with relatively short length (i.e., between 8 and 50 nucleotides), which can be specifically designed to hybridize with a target messenger RNA (mRNA) through specific
published online February 18, 2015
Issue Theme Anticoagulant Therapy: Present and Future; Guest Editor: Job Harenberg, MD.
hydrogen bonding or hydrophobic interactions.1 The term “antisense” derives from the fact that the oligonucleotide sequence is complementary to that of another specific mRNA sequence, the latter being named the “sense” sequence. The binding between an antisense oligonucleotide and its target mRNA occurs with a remarkably high degree of fidelity, and is then followed by a modulation of mRNA function by a series
Copyright © 2015 by Thieme Medical Publishers, Inc., 333 Seventh Avenue, New York, NY 10001, USA. Tel: +1(212) 584-4662.
DOI http://dx.doi.org/ 10.1055/s-0035-1546466. ISSN 0094-6176.
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Giuseppe Lippi, MD1 Job Harenberg, MD2 Emmanuel J. Favaloro, PhD, FFSc (RCPA)4
Antisense Therapy against Coagulation Factor XI
Lippi et al. annulling its final concentration in tissues, blood, and other biological fluids. This evidence has increasingly supported the construction of a large number of antisense oligonucleotides over the past decade, with the aim of preventing or treating a variety of human disorders including cardiovascular disease,3 cancer,4 autoimmune diseases,5 Duchenne muscular dystrophy,6 asthma,7 and severe viral infections such as those caused by human immunodeficiency virus8 or hepatitis C virus.9 Since venous thromboembolism (VTE) is characterized by a complex and multifactorial pathogenesis that involves a variety of prothrombotic factors,10 it is thus predictable that antisense therapy may find several indications also for the treatment of this condition.
Fig. 1 Current mechanisms of antisense therapy. Ago2, argonaute 2; DS RNase, double strand RNase; RNase H, ribonuclease H.
of mechanisms that involve either interference with RNA function without degradation (i.e., inhibition of translation initiation, inhibition or promotion of exon inclusion, inhibition of polyadenylation, disruption of RNA structure) or direct RNA degradation by endogenous enzymes such as ribonucleases (i.e., ribonuclease [RNase] H), argonaute 2, ribozymes, double-stranded RNases or enzymatic cleavage designed into the oligonucleotide (►Fig. 1).2 The consequence of the binding between the antisense oligonucleotide and its target mRNA is an interference in specified protein synthesis and a final reduction in the concentration of the protein level. Intuitively appealing, the use of antisense oligonucleotides would therefore be effective to abolish to various extents the synthesis of a target protein, thus ultimately reducing or even
Antisense Therapy against Coagulation Factor XI Coagulation factor XI is a key player of the intrinsic pathway of blood coagulation, since it acts as the physiological activator of coagulation factor IX,11 but also directly and indirectly activates thrombin activatable fibrinolysis inhibitor, a crucial protein for clot stabilization (►Fig. 2).12 The evidence that patients with congenital factor XI deficiency have a reduced risk of VTE13 presented an ideal background to initiate several studies aimed to reduce the concentration of this clotting factor in those at risk,14 inclusive of some trials using antisense oligonucleotides. The first trial using an antisense oligonucleotide selectively targeting coagulation factor XI was published by Zhang et al, in 2010.15 Specifically, 20-long antisense nucleotides were developed and then chemically modified with phosphorothioate in the backbone and 2’-O-methoxyethyl on the wings with a central deoxy gap (“5-10-5” design). The
Fig. 2 The role of factor XI in blood coagulation. F, coagulation factor; TAFI, thrombin-activatable fibrinolysis inhibitor; TF, tissue factor.
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oligonucleotides were administered to 8-week-old BALB/c and C57BL/6 mice. Subcutaneous injections of the antisense oligonucleotides were effective to determine a dose-dependent decrease of factor XI mRNA in liver, with maximal reduction up to 98% observed at the highest dose (50 mg/ kg). Factor XI concentration and activity were contextually decreased by more than 90% between days 2 and 7, with progressive return to basal levels between days 14 and 28. Even more importantly, the administration of the antisense oligonucleotides produced an effective, dose-dependent antithrombotic activity in various models of thrombosis, including FeCl3-induced inferior vena cava thrombosis, FeCl3-induced mesenteric vein thrombosis and stenosis-induced inferior vena cava thrombosis, which was comparable to that obtained with both warfarin and enoxaparin treatment. However, at variance with warfarin or enoxaparin, the administration of antisense oligonucleotides did not generate excessive bleeding in a tail-bleeding assay. Que Liu et al first investigated the safety, tolerability, pharmacokinetics, and pharmacodynamics of single and multiple doses of an antisense inhibitor of factor XI in healthy volunteers.16 In the single ascending-dose study, eight subjects were administered with 50, 100, 200, and 300 mg/kg of antisense inhibitor as a single subcutaneous or as multiple subcutaneous injections. The activity and concentration of factor XI were significantly reduced in the 200 and 300 mg cohorts 1-week after dosing. In the double blind multiple ascending-dose study, nine volunteers were randomized to 50 mg antisense inhibitor (three doses in week 1 followed by once weekly dosing for 5 weeks), nine volunteers to 100 mg antisense inhibitor (three doses in week 1 followed by once weekly dosing for 5 weeks), nine volunteers to 200 mg antisense inhibitor (three doses in week 1 followed by once weekly dosing for 5 weeks), whereas nine volunteers received placebo. Even in this study a robust, sustained, and dosedependent reduction in activity and concentration of factor XI was observed, with maximum reduction observed 1 to 2 weeks postdosing. In particular, the activity and concentration of factor XI were reduced by 92 and 100% after 6 weeks dosing. The hepatic half-life of factor XI antisense inhibitor was found to be approximately 20 days. No significant complications were observed (i.e., bleeding, change of vital signs, electrocardiogram, or alteration of hepatic and renal function), except for an increased rate of mild injection site reactions (33% compared with 10% in placebo group). Younis et al tested a single-stranded antisense oligonucleotide against the rhesus and human mRNA transcript of factor XI in 2 to 5 years old cynomolgus monkeys of Asian origin.17 The antisense oligonucleotide was administered to the animals via a 2-week loading dose (4, 8, 12, or 40 mg/kg) followed by once weekly dosing for up to 13 weeks. The liver expression of coagulation FXI mRNA decreased in a dose- and duration-dependent manner, with a substantial reduction of 50 and 90% by 6 weeks of treatment in the 12 and 40 mg/kg dose groups, respectively. The plasma activity of factor XI was concomitantly reduced by 25% at 8 days in the 40 mg/kg dose group, with maximal 75% reduction at day 21 and maintenance throughout for the following 13 weeks. Interestingly,
Lippi et al.
monkeys treated with the antisense oligonucleotide did not experience substantial increases in bleeding time or blood volume loss after partial tail amputation or gum or skin laceration, at variance with animals treated with enoxaparin, which had instead a two to threefold increase in both bleeding time and blood volume loss. No direct toxic reactions were in evidence after antisense oligonucleotide administration. Crosby et al investigated the effect of a 20-long antisense oligonucleotide in baboons, treated with a dosage of 25 mg/ kg, three times weekly for a total period of 7 weeks.18 The plasma activity and concentration of factor XI were similarly reduced by 50% in all animals by day 25, achieving a maximum inhibition of approximately 70% at the end of each infusion period. The anticoagulant effect was then evaluated using a model of thrombus formation on collagen-coated graft segments, and it was found that in antisense oligonucleotide treated baboons the platelet accumulation in propagated thrombus was significantly reduced by approximately 40% compared with placebo. The safety of factor XI inhibition with antisense oligonucleotide treatment was then evaluated as duration of bleeding after a standardized skin incision. Interestingly, bleeding time values overlapped in control animals and in those receiving the antisense oligonucleotide. In a subsequent animal study, Yau et al developed and administrated a 20-long antisense oligonucleotide in rabbits at 15 mg/kg twice-weekly dose.19 After 4 weeks of treatment, the antisense oligonucleotide was effective to reduce factor XI mRNA expression, factor XI plasma concentration, and activity by 84, 96, and 99%, respectively. The anticoagulant effect was then explored in a rabbit model of thrombosis, involving catheter implantation in the jugular vein until occlusion for a maximum of 35 days. Compared with control, the administration of the antisense oligonucleotide was effective to significantly prolong the mean time to catheter occlusion by more than twofold. van Montfoort et al also tested the effect of antisense oligonucleotide therapy in 4-week-old male Apoe / mice on C57Bl/6 background.20 In brief; the animals were administered with intraperitoneal injection (50 mg/kg) of factor XI antisense oligonucleotides every 3 to 4 days for a total of 10 days (i.e., three injections). Compared with control mice receiving nonsense oligonucleotides, the activity of factor XI was reduced by 64% in those administered with antisense oligonucleotides. The anticoagulant effect was then assessed by carotid plaque rupture induced by ultrasound application and measurement of acute thrombus formation. The treatment with antisense oligonucleotides was effective to decrease thrombus propagation and fibrin deposition. Even more interestingly, the platelet aggregates also appeared more unstable than those generated in animals receiving nonsense oligonucleotides. The mouse tail bleeding assay also generated identical results in animals receiving antisense and nonsense oligonucleotides. The results of these studies have lead the way to the first in-human, open-label, and parallel-group study, in which 300 patients undergoing elective primary unilateral total knee arthroplasty were assigned to receive one of two doses of Seminars in Thrombosis & Hemostasis
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Antisense Therapy against Coagulation Factor XI
Antisense Therapy against Coagulation Factor XI
Lippi et al.
factor XI antisense oligonucleotide (100, 200, or 300 mg) or enoxaparin 40 mg once daily.21 The antisense oligonucleotide used in this study binds to factor XI mRNA in the liver and produces a ribonuclease H1 (RNase H1)–mediated degradation mRNA (►Fig. 1), thus limiting the synthesis and reducing the concentration of factor XI. The treatment with antisense oligonucleotide against factor XI was started 36 days before surgery with three subcutaneous doses during the 1st week and four once-weekly doses. On the day of surgery, patients received a dose 6 hours postoperatively, with the final dose given on day 39. As in the previous animal trials, the mean factor XI activity was remarkably lower in patients treated with 200-mg antisense oligonucleotide (0.38 0.01 U/mL) and 300-mg antisense oligonucleotide (0.20 0.01 U/mL) compared with those receiving enoxaparin (0.93 0.02 U/ mL). The activity of factor XI remained consistently reduced up to 5 weeks after the end of treatment. Compared with the enoxaparin controls, the frequency of incident VTE up to 3 months after knee arthroplasty was significantly reduced by the administration of 300-mg antisense oligonucleotide (26%; p < 0.001), but not in those receiving 200-mg antisense oligonucleotide (4%; p ¼ 0.59). The rate of major or clinically relevant nonmajor bleeding was also marginally lower in patients receiving both 200-mg antisense oligonucleotide (6%; p ¼ 0.09) and 300-mg antisense oligonucleotide (6%; p ¼ 0.16) compared with those receiving enoxaparin. The frequency of injection site-related adverse event was; however, lower in patients receiving enoxaparin (3%) than in those receiving both 200-mg antisense oligonucleotide (22%; p < 0.001) and 300-mg antisense oligonucleotide (32%; p < 0.001).
procedures.29 Like warfarin, DOACs are also orally administered, have a lower risk of major bleeding compared with conventional anticoagulants, and only require laboratory monitoring in specific clinical circumstances such as in the presence of thrombotic or hemorrhagic episodes, in patients undergoing urgent invasive procedures, in those with extreme of body weight, as well as for suspicion of overdosage and intoxication.25 The relative shortcomings of these drugs are represented by the need of frequent (daily) administration, the metabolic interplay with other pharmacological agents (especially antibiotics),30 the challenging and relatively expensive measurement process,31–33 and current lack of antidotes. As such, the development of novel pharmacological agents that may be effective to prevent (or treat) thrombosis, which are relatively inexpensive, do not require continuous administration and display better safety profiles should be regarded as a highly attractive perspective.34 Antisense therapy against coagulation factor XI potentially fulfills all these important considerations. The available evidence suggests that antisense oligonucleotides may be more effective than conventional anticoagulants in preventing the onset and propagation of thrombosis, do not require factor XI measurement since the reduction of mRNA synthesis appears to be dose-dependently, robustly, and stably decreased for 3 to 5 weeks after the end of administration, with an incidence of major bleeding that is at least not greater than that caused by treatment with either warfarin or LMWH.
Additive Value of Antisense Therapy to Vitamin K Antagonists, Heparin, and Direct Oral Anticoagulants
Interestingly, antisense therapies against other coagulation factors are in development (►Fig. 2), also with promising results. Yau et al produce a 20-long, factor XII antisense oligonucleotide, which was then administrated to rabbits.19 After 4 weeks of treatment, the antisense oligonucleotide was effective to reduce factor XII mRNA expression, factor XII plasma levels, and activity by 97, 97, and 99%, respectively. The anticoagulant effect was also investigated in a rabbit model of thrombosis, involving catheter implantation in the jugular vein until occlusion for a maximum of 35 days. Compared with control, the administration of the antisense oligonucleotide was effective to significantly prolong the mean time to catheter occlusion by more than twofold. Similar results were obtained by Bird et al using prekallikrein antisense oligonucleotide treatment in mice,35 demonstrating that plasma kallikrein suppression is also effective to prevent thrombus formation in mice with minimal bleeding.
Venous thrombosis is a major health care issue, especially in hospitalized patients and in those undergoing major and orthopedic surgery.22,23 The current armamentarium of pharmacological agents used to prevent and treat VTE entails old-generation drugs, such as vitamin K antagonists (especially warfarin) or low-molecular-weight heparin (LMWH), along with specific inhibitors of coagulation factors II and X, conventionally known as direct oral anticoagulants (DOACs).24,25 Although the clinical usefulness of these pharmacological agents has been now clearly established, their use in the real world is still plagued by several drawbacks. Warfarin and other vitamin K antagonists can be orally administered but display a narrow therapeutic window, and thus require ongoing dose adjustment by means of regular laboratory monitoring to prevent under or overcoagulation.26,27 LMWH is equally effective as warfarin, does not require constant laboratory monitoring, but can only be administered by repeated (i.e., once or twice daily) subcutaneous injections, and is hence an inconvenient means of longterm anticoagulation.28 Both vitamin K antagonists and LMWH are also associated with a significant risk of bleeding, especially in patients undergoing surgery and other invasive Seminars in Thrombosis & Hemostasis
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Other Targets for Antisense Therapy in Venous Thromboembolism
Potential Limitations of Antisense Therapy The results published so far (summarized in ►Table 1) do not yet make a compelling case for replacing conventional anticoagulants with factor XI antisense therapy (►Table 2).36 Preliminary findings in animal models and in patients undergoing a highly prothrombotic scenario such as knee surgery need to be validated in other prospective trials, and in different
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Authors
Cohort
Dosage
Maximum plasma factor XI reduction
Clinical endpoints
References
Zhang et al, 2010
Mice
50 g/kg
• Activity: > 90% • Concentration: > 90%
• Antithrombotic activity against FeCl3-induced inferior vena cava thrombosis, FeCl3-induced mesenteric vein thrombosis and stenosis-induced inferior vena cava thrombosis • Lower bleeding than enoxaparin or warfarin
15
Que Liu et al, 2011
Humans
50–300 mg/kg
• Activity: 92% • Concentration: > 100%
None
16
Younis et al, 2012
Cynomolgus monkeys
4–40 mg/kg
• Activity: 75%
None
17
Crosby et al, 2013
Baboons
25 mg/kg
• Activity: 70% • Concentration: 70%
• Platelet accumulation in propagated thrombus significantly reduced by 40% • No major bleeding compared with placebo
18
Yau et al, 2014
Rabbits
15 mg/kg
• Activity: 99% • Concentration: 96%
• Prolongation of mean time of jugular vein catheter occlusion
19
van Montfoort et al, 2014
Mice
50 mg/kg
• Activity: 64%
• Decreased thrombus propagation and fibrin deposition after carotid plaque rupture • No major bleeding compared with placebo
20
Büller et al, 2014
Humans
100–300 mg
• Activity (200 mg): 59% • Activity (300 mg): 78%
• Rate of incident venous thromboembolism up to 3 mo after elective primary unilateral total knee arthroplasty significantly reduced by administration of 300-mg antisense oligonucleotide compared with enoxaparin • Marginally but not significantly lower bleeding than enoxaparin
21
clinical scenarios. In particular, it should be clearly established whether such a degree of factor XI suppression may be really associated with a safety profile throughout the maintenance of antisense therapy, as well as the exact level of inhibition required to maintain a safe balance between preventing thrombosis and managing bleeding risk. The clinical effectiveness should be also compared against the future standard of therapy that will be represented by DOACs. Despite conceptual simplicity, rational design, and relatively inexpensive cost, cost-effective analyses should be planned to establish if antisense therapy is economically advantageous over treatment with conventional anticoagulants or DOACs. The management of bleeding episodes in patients receiving factor XI antisense therapy is another potential drawback.37 This is due to several factors, such as the long elimination half-life of antisense
oligonucleotides, the suppression of factor XI activity by more than 50% without evidence of recovery for up to 1 month after administration of the last dose, combined with the current lack of effective antagonists and antidotes. Therefore, in analogy with other anticoagulant drugs, such as Idraparinux, (Sanofi-Aventis, Toulouse, France)38,39 the long duration of the anticoagulant effect may be effective to prevent thromboembolism but can also be associated with an increased risk of major hemorrhages, which would require a continuative factor replacement therapy until the bleeding has resolved. A final concern emerges from the potential side effects of this innovative therapy. Beside the considerable high frequency of injection site-related adverse event observed in humans, the potential risk of potential profibrinolytic effects, desensitization, and teratogenesis40 of antisense drug Seminars in Thrombosis & Hemostasis
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Table 1 Synthesis of studies on antisense therapy against coagulation factor XI
Antisense Therapy against Coagulation Factor XI
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Table 2 Potential limitations of antisense therapy • Validation needed in other prospective trials and in different clinical scenarios • Identification of the best compromise of protein suppression for preventing thrombosis and maintaining a safety profile • Clinical effectiveness tested against direct oral anticoagulants • Cost-effectiveness still uncertain • High potential risk of bleeding due to: – Long elimination half-life of oligonucleotides – Long-lasting suppression of protein concentration – Lack of antagonists and antidotes • Potential adverse reactions – Injection site-related adverse event – Teratogenesis – Profibrinolytic effects – Desensitization
13 Salomon O, Steinberg DM, Zucker M, Varon D, Zivelin A, Seligsohn
14
15
16
17
18
19
technology, specifically during long-term therapy, requires further exploration and clarification. Finally, the potential development of antisense molecules with higher affinity to the target factor XI due to the relative complex molecular structure of the antisense molecule should be considered as an attractive perspective for the future.
20
21
22
References 1 Dias N, Stein CA. Antisense oligonucleotides: basic concepts and 2
3 4 5
6
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8
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11 12
mechanisms. Mol Cancer Ther 2002;1(5):347–355 Bennett CF, Swayze EE. RNA targeting therapeutics: molecular mechanisms of antisense oligonucleotides as a therapeutic platform. Annu Rev Pharmacol Toxicol 2010;50:259–293 Lippi G, Favaloro EJ. Antisense therapy in the treatment of hypercholesterolemia. Eur J Intern Med 2011;22(6):541–546 Castanotto D, Stein CA. Antisense oligonucleotides in cancer. Curr Opin Oncol 2014;26(6):584–589 Mourich DV, Marshall NB. Antisense approaches to immune modulation for transplant and autoimmune diseases. Curr Opin Pharmacol 2005;5(5):508–512 Falzarano MS, Passarelli C, Ferlini A. Nanoparticle delivery of antisense oligonucleotides and their application in the exon skipping strategy for Duchenne muscular dystrophy. Nucleic Acid Ther 2014;24(1):87–100 Gauvreau GM, Boulet LP, Cockcroft DW, et al. Antisense therapy against CCR3 and the common beta chain attenuates allergeninduced eosinophilic responses. Am J Respir Crit Care Med 2008; 177(9):952–958 Lu X, Yu Q, Binder GK, et al. Antisense-mediated inhibition of human immunodeficiency virus (HIV) replication by use of an HIV type 1-based vector results in severely attenuated mutants incapable of developing resistance. J Virol 2004;78(13):7079–7088 de Jong YP, Jacobson IM. Antisense therapy for hepatitis C virus infection. J Hepatol 2014;60(1):227–228 Lippi G, Franchini M. Pathogenesis of venous thromboembolism: when the cup runneth over. Semin Thromb Hemost 2008;34(8): 747–761 Duga S, Salomon O. Congenital factor XI deficiency: an update. Semin Thromb Hemost 2013;39(6):621–631 Vercauteren E, Gils A, Declerck PJ. Thrombin activatable fibrinolysis inhibitor: a putative target to enhance fibrinolysis. Semin Thromb Hemost 2013;39(4):365–372
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U. Patients with severe factor XI deficiency have a reduced incidence of deep-vein thrombosis. Thromb Haemost 2011; 105(2):269–273 Löwenberg EC, Meijers JC, Monia BP, Levi M. Coagulation factor XI as a novel target for antithrombotic treatment. J Thromb Haemost 2010;8(11):2349–2357 Zhang H, Löwenberg EC, Crosby JR, et al. Inhibition of the intrinsic coagulation pathway factor XI by antisense oligonucleotides: a novel antithrombotic strategy with lowered bleeding risk. Blood 2010;116(22):4684–4692 Que Liu ED, Claudette B, Shuting X, et al. ISIS-FXIRx, a novel and specific antisense inhibitor of factor XI, caused significant reduction in FXI antigen and activity and increased aPTT without causing bleeding in healthy volunteers. Blood 2011;118:209 Younis HS, Crosby J, Huh JI, et al. Antisense inhibition of coagulation factor XI prolongs APTT without increased bleeding risk in cynomolgus monkeys. Blood 2012;119(10):2401–2408 Crosby JR, Marzec U, Revenko AS, et al. Antithrombotic effect of antisense factor XI oligonucleotide treatment in primates. Arterioscler Thromb Vasc Biol 2013;33(7):1670–1678 Yau JW, Liao P, Fredenburgh JC, et al. Selective depletion of factor XI or factor XII with antisense oligonucleotides attenuates catheter thrombosis in rabbits. Blood 2014;123(13):2102–2107 van Montfoort ML, Kuijpers MJ, Knaup VL, et al. Factor XI regulates pathological thrombus formation on acutely ruptured atherosclerotic plaques. Arterioscler Thromb Vasc Biol 2014;34(8):1668–1673 Büller HR, Bethune C, Bhanot S, et al; the FXI-ASO TKA Investigators. Factor XI antisense oligonucleotide for prevention of venous thrombosis. N Engl J Med 2015;372(3):232–240 Raskob GE, Angchaisuksiri P, Blanco AN, et al; ISTH Steering Committee for World Thrombosis Day. Thrombosis: a major contributor to global disease burden. Semin Thromb Hemost 2014;40(7):724–735 Dahl OE, Harenberg J, Wexels F, Preissner KT. Arterial and venous thrombosis following trauma and major orthopedic surgery: molecular mechanisms and strategies for intervention. Semin Thromb Hemost 2015;41(2):141–145 Zolfaghari S, Harenberg J, Froelich L, Wehling M, Weiss C. Development of a tool to identify patients’ preference for vitamin K antagonist or direct oral anticoagulant therapy. Semin Thromb Hemost 2014; 40(1):121–128 Lippi G, Favaloro EJ. Recent guidelines and recommendations for laboratory assessment of the direct oral anticoagulants (DOACs): is there consensus? Clin Chem Lab Med 2015;53(2):185–197 Favaloro EJ, Lippi G. The new oral anticoagulants and the future of haemostasis laboratory testing. Biochem Med (Zagreb) 2012; 22(3):329–341 Riva N, Ageno W. Pros and cons of vitamin K antagonists and nonvitamin K antagonist oral anticoagulants. Semin Thromb Hemost 2015;41(2):178–187 Agnelli G, Prandoni P, Di Minno G, et al. Thromboprophylaxis with low molecular weight heparins. An assessment of the methodological quality of studies. Semin Thromb Hemost 2015;41(2): 113–132 Favaloro EJ, Lippi G, Koutts J. Laboratory testing of anticoagulants: the present and the future. Pathology 2011;43(7):682–692 Lippi G, Favaloro EJ, Mattiuzzi C. Combined administration of antibiotics and direct oral anticoagulants: a renewed indication for laboratory monitoring? Semin Thromb Hemost 2014;40(7): 756–765 Tripodi A, Di Iorio G, Lippi G, Testa S, Manotti C. Position paper on laboratory testing for patients taking new oral anticoagulants. Consensus document of FCSA, SIMeL, SIBioC and CISMEL1). Clin Chem Lab Med 2012;50(12):2137–2140 Harenberg J, Kraemer S, Du S, et al. Determination of direct oral anticoagulants from human serum samples. Semin Thromb Hemost 2014;40(1):129–134
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Patients’ serum and urine as easily accessible samples for the measurement of non-vitamin K antagonist oral anticoagulants. Semin Thromb Hemost 2015;41(2):228–236 34 Bane CE Jr, Gailani D. Factor XI as a target for antithrombotic therapy. Drug Discov Today 2014;19(9):1454–1458 35 Bird JE, Smith PL, Wang X, et al. Effects of plasma kallikrein deficiency on haemostasis and thrombosis in mice: murine ortholog of the Fletcher trait. Thromb Haemost 2012;107(6):1141–1150 36 Flaumenhaft R. Making (anti)sense of factor XI in thrombosis. N Engl J Med 2015;372(3):277–278
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Extended prophylaxis of venous thromboembolism with idraparinux. N Engl J Med 2007;357(11):1105–1112 39 Harenberg J, Vukojevic Y, Mikus G, Joerg I, Weiss C. Long elimination half-life of idraparinux may explain major bleeding and recurrent events of patients from the van Gogh trials. J Thromb Haemost 2008;6(5):890–892 40 Crooke ST. Antisense Drug Technology: Principles, Strategies, and Applications. 2nd ed. New York, NY: CRC Press; 2007
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Next Generation Antithrombotic Therapy: Focus on Antisense Therapy against Coagulation Factor XI Camilla Mattiuzzi, MD3
1 Laboratory of Clinical Chemistry and Hematology, Academic Hospital
of Parma, Parma, Italy 2 Clinical Pharmacology, Medical Faculty Mannheim, Ruprecht-Karls University Heidelberg, Mannheim, Germany 3 Service of Clinical Governance, General Hospital of Trento, Trento, Italy 4 Department of Haematology, Institute of Clinical Pathology and Medical Research (ICPMR), Pathology West, Westmead Hospital, Westmead, New South Wales, Australia
Address for correspondence Giuseppe Lippi, MD, U.O. Diagnostica Ematochimica, Azienda Ospedaliero-Universitaria di Parma, Via Gramsci, 14, 43126 - Parma, Italy (e-mail: [email protected]; [email protected]).
Semin Thromb Hemost 2015;41:255–262.
Abstract
Keywords
► ► ► ►
anticoagulant therapy antisense therapy factor XI venous thromboembolism ► thrombosis
Although the current therapeutic armamentarium of venous thrombosis encompasses the use of vitamin K antagonists, heparins, and direct oral anticoagulants, these drugs have several important drawbacks. Antisense oligonucleotides are relatively short single-stranded nucleic acid sequences, which hybridize with a target messenger RNA (mRNA) and suppress protein synthesis. Coagulation factor XI is a key player in blood coagulation, and thus represents a potential target for antisense therapy. The available evidence reviewed in this article suggests that factor XI antisense oligonucleotides may be more effective than conventional anticoagulants in preventing the onset and propagation of thrombosis, do not require factor measurement since the reduction of mRNA synthesis appears dose-dependently, robustly, and stably decreased for 3 to 5 weeks after the end of administration, with an incidence of major bleeding that is at least not greater than that associated with warfarin or low-molecular-weight heparin therapy. Despite conceptual simplicity, rational design, and relatively inexpensive cost, the preliminary findings in animal models and in patients undergoing knee surgery need to be validated in other prospective trials and cost-effective analyses before this attractive treatment option can be advocated as a new paradigm in prevention and treatment of venous thrombosis.
An Introduction to the Concept of Antisense Therapy Antisense oligonucleotides are commonly defined as unmodified or chemically modified single-stranded nucleic acid sequences with relatively short length (i.e., between 8 and 50 nucleotides), which can be specifically designed to hybridize with a target messenger RNA (mRNA) through specific
published online February 18, 2015
Issue Theme Anticoagulant Therapy: Present and Future; Guest Editor: Job Harenberg, MD.
hydrogen bonding or hydrophobic interactions.1 The term “antisense” derives from the fact that the oligonucleotide sequence is complementary to that of another specific mRNA sequence, the latter being named the “sense” sequence. The binding between an antisense oligonucleotide and its target mRNA occurs with a remarkably high degree of fidelity, and is then followed by a modulation of mRNA function by a series
Copyright © 2015 by Thieme Medical Publishers, Inc., 333 Seventh Avenue, New York, NY 10001, USA. Tel: +1(212) 584-4662.
DOI http://dx.doi.org/ 10.1055/s-0035-1546466. ISSN 0094-6176.
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Giuseppe Lippi, MD1 Job Harenberg, MD2 Emmanuel J. Favaloro, PhD, FFSc (RCPA)4
Antisense Therapy against Coagulation Factor XI
Lippi et al. annulling its final concentration in tissues, blood, and other biological fluids. This evidence has increasingly supported the construction of a large number of antisense oligonucleotides over the past decade, with the aim of preventing or treating a variety of human disorders including cardiovascular disease,3 cancer,4 autoimmune diseases,5 Duchenne muscular dystrophy,6 asthma,7 and severe viral infections such as those caused by human immunodeficiency virus8 or hepatitis C virus.9 Since venous thromboembolism (VTE) is characterized by a complex and multifactorial pathogenesis that involves a variety of prothrombotic factors,10 it is thus predictable that antisense therapy may find several indications also for the treatment of this condition.
Fig. 1 Current mechanisms of antisense therapy. Ago2, argonaute 2; DS RNase, double strand RNase; RNase H, ribonuclease H.
of mechanisms that involve either interference with RNA function without degradation (i.e., inhibition of translation initiation, inhibition or promotion of exon inclusion, inhibition of polyadenylation, disruption of RNA structure) or direct RNA degradation by endogenous enzymes such as ribonucleases (i.e., ribonuclease [RNase] H), argonaute 2, ribozymes, double-stranded RNases or enzymatic cleavage designed into the oligonucleotide (►Fig. 1).2 The consequence of the binding between the antisense oligonucleotide and its target mRNA is an interference in specified protein synthesis and a final reduction in the concentration of the protein level. Intuitively appealing, the use of antisense oligonucleotides would therefore be effective to abolish to various extents the synthesis of a target protein, thus ultimately reducing or even
Antisense Therapy against Coagulation Factor XI Coagulation factor XI is a key player of the intrinsic pathway of blood coagulation, since it acts as the physiological activator of coagulation factor IX,11 but also directly and indirectly activates thrombin activatable fibrinolysis inhibitor, a crucial protein for clot stabilization (►Fig. 2).12 The evidence that patients with congenital factor XI deficiency have a reduced risk of VTE13 presented an ideal background to initiate several studies aimed to reduce the concentration of this clotting factor in those at risk,14 inclusive of some trials using antisense oligonucleotides. The first trial using an antisense oligonucleotide selectively targeting coagulation factor XI was published by Zhang et al, in 2010.15 Specifically, 20-long antisense nucleotides were developed and then chemically modified with phosphorothioate in the backbone and 2’-O-methoxyethyl on the wings with a central deoxy gap (“5-10-5” design). The
Fig. 2 The role of factor XI in blood coagulation. F, coagulation factor; TAFI, thrombin-activatable fibrinolysis inhibitor; TF, tissue factor.
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oligonucleotides were administered to 8-week-old BALB/c and C57BL/6 mice. Subcutaneous injections of the antisense oligonucleotides were effective to determine a dose-dependent decrease of factor XI mRNA in liver, with maximal reduction up to 98% observed at the highest dose (50 mg/ kg). Factor XI concentration and activity were contextually decreased by more than 90% between days 2 and 7, with progressive return to basal levels between days 14 and 28. Even more importantly, the administration of the antisense oligonucleotides produced an effective, dose-dependent antithrombotic activity in various models of thrombosis, including FeCl3-induced inferior vena cava thrombosis, FeCl3-induced mesenteric vein thrombosis and stenosis-induced inferior vena cava thrombosis, which was comparable to that obtained with both warfarin and enoxaparin treatment. However, at variance with warfarin or enoxaparin, the administration of antisense oligonucleotides did not generate excessive bleeding in a tail-bleeding assay. Que Liu et al first investigated the safety, tolerability, pharmacokinetics, and pharmacodynamics of single and multiple doses of an antisense inhibitor of factor XI in healthy volunteers.16 In the single ascending-dose study, eight subjects were administered with 50, 100, 200, and 300 mg/kg of antisense inhibitor as a single subcutaneous or as multiple subcutaneous injections. The activity and concentration of factor XI were significantly reduced in the 200 and 300 mg cohorts 1-week after dosing. In the double blind multiple ascending-dose study, nine volunteers were randomized to 50 mg antisense inhibitor (three doses in week 1 followed by once weekly dosing for 5 weeks), nine volunteers to 100 mg antisense inhibitor (three doses in week 1 followed by once weekly dosing for 5 weeks), nine volunteers to 200 mg antisense inhibitor (three doses in week 1 followed by once weekly dosing for 5 weeks), whereas nine volunteers received placebo. Even in this study a robust, sustained, and dosedependent reduction in activity and concentration of factor XI was observed, with maximum reduction observed 1 to 2 weeks postdosing. In particular, the activity and concentration of factor XI were reduced by 92 and 100% after 6 weeks dosing. The hepatic half-life of factor XI antisense inhibitor was found to be approximately 20 days. No significant complications were observed (i.e., bleeding, change of vital signs, electrocardiogram, or alteration of hepatic and renal function), except for an increased rate of mild injection site reactions (33% compared with 10% in placebo group). Younis et al tested a single-stranded antisense oligonucleotide against the rhesus and human mRNA transcript of factor XI in 2 to 5 years old cynomolgus monkeys of Asian origin.17 The antisense oligonucleotide was administered to the animals via a 2-week loading dose (4, 8, 12, or 40 mg/kg) followed by once weekly dosing for up to 13 weeks. The liver expression of coagulation FXI mRNA decreased in a dose- and duration-dependent manner, with a substantial reduction of 50 and 90% by 6 weeks of treatment in the 12 and 40 mg/kg dose groups, respectively. The plasma activity of factor XI was concomitantly reduced by 25% at 8 days in the 40 mg/kg dose group, with maximal 75% reduction at day 21 and maintenance throughout for the following 13 weeks. Interestingly,
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monkeys treated with the antisense oligonucleotide did not experience substantial increases in bleeding time or blood volume loss after partial tail amputation or gum or skin laceration, at variance with animals treated with enoxaparin, which had instead a two to threefold increase in both bleeding time and blood volume loss. No direct toxic reactions were in evidence after antisense oligonucleotide administration. Crosby et al investigated the effect of a 20-long antisense oligonucleotide in baboons, treated with a dosage of 25 mg/ kg, three times weekly for a total period of 7 weeks.18 The plasma activity and concentration of factor XI were similarly reduced by 50% in all animals by day 25, achieving a maximum inhibition of approximately 70% at the end of each infusion period. The anticoagulant effect was then evaluated using a model of thrombus formation on collagen-coated graft segments, and it was found that in antisense oligonucleotide treated baboons the platelet accumulation in propagated thrombus was significantly reduced by approximately 40% compared with placebo. The safety of factor XI inhibition with antisense oligonucleotide treatment was then evaluated as duration of bleeding after a standardized skin incision. Interestingly, bleeding time values overlapped in control animals and in those receiving the antisense oligonucleotide. In a subsequent animal study, Yau et al developed and administrated a 20-long antisense oligonucleotide in rabbits at 15 mg/kg twice-weekly dose.19 After 4 weeks of treatment, the antisense oligonucleotide was effective to reduce factor XI mRNA expression, factor XI plasma concentration, and activity by 84, 96, and 99%, respectively. The anticoagulant effect was then explored in a rabbit model of thrombosis, involving catheter implantation in the jugular vein until occlusion for a maximum of 35 days. Compared with control, the administration of the antisense oligonucleotide was effective to significantly prolong the mean time to catheter occlusion by more than twofold. van Montfoort et al also tested the effect of antisense oligonucleotide therapy in 4-week-old male Apoe / mice on C57Bl/6 background.20 In brief; the animals were administered with intraperitoneal injection (50 mg/kg) of factor XI antisense oligonucleotides every 3 to 4 days for a total of 10 days (i.e., three injections). Compared with control mice receiving nonsense oligonucleotides, the activity of factor XI was reduced by 64% in those administered with antisense oligonucleotides. The anticoagulant effect was then assessed by carotid plaque rupture induced by ultrasound application and measurement of acute thrombus formation. The treatment with antisense oligonucleotides was effective to decrease thrombus propagation and fibrin deposition. Even more interestingly, the platelet aggregates also appeared more unstable than those generated in animals receiving nonsense oligonucleotides. The mouse tail bleeding assay also generated identical results in animals receiving antisense and nonsense oligonucleotides. The results of these studies have lead the way to the first in-human, open-label, and parallel-group study, in which 300 patients undergoing elective primary unilateral total knee arthroplasty were assigned to receive one of two doses of Seminars in Thrombosis & Hemostasis
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Antisense Therapy against Coagulation Factor XI
Antisense Therapy against Coagulation Factor XI
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factor XI antisense oligonucleotide (100, 200, or 300 mg) or enoxaparin 40 mg once daily.21 The antisense oligonucleotide used in this study binds to factor XI mRNA in the liver and produces a ribonuclease H1 (RNase H1)–mediated degradation mRNA (►Fig. 1), thus limiting the synthesis and reducing the concentration of factor XI. The treatment with antisense oligonucleotide against factor XI was started 36 days before surgery with three subcutaneous doses during the 1st week and four once-weekly doses. On the day of surgery, patients received a dose 6 hours postoperatively, with the final dose given on day 39. As in the previous animal trials, the mean factor XI activity was remarkably lower in patients treated with 200-mg antisense oligonucleotide (0.38 0.01 U/mL) and 300-mg antisense oligonucleotide (0.20 0.01 U/mL) compared with those receiving enoxaparin (0.93 0.02 U/ mL). The activity of factor XI remained consistently reduced up to 5 weeks after the end of treatment. Compared with the enoxaparin controls, the frequency of incident VTE up to 3 months after knee arthroplasty was significantly reduced by the administration of 300-mg antisense oligonucleotide (26%; p < 0.001), but not in those receiving 200-mg antisense oligonucleotide (4%; p ¼ 0.59). The rate of major or clinically relevant nonmajor bleeding was also marginally lower in patients receiving both 200-mg antisense oligonucleotide (6%; p ¼ 0.09) and 300-mg antisense oligonucleotide (6%; p ¼ 0.16) compared with those receiving enoxaparin. The frequency of injection site-related adverse event was; however, lower in patients receiving enoxaparin (3%) than in those receiving both 200-mg antisense oligonucleotide (22%; p < 0.001) and 300-mg antisense oligonucleotide (32%; p < 0.001).
procedures.29 Like warfarin, DOACs are also orally administered, have a lower risk of major bleeding compared with conventional anticoagulants, and only require laboratory monitoring in specific clinical circumstances such as in the presence of thrombotic or hemorrhagic episodes, in patients undergoing urgent invasive procedures, in those with extreme of body weight, as well as for suspicion of overdosage and intoxication.25 The relative shortcomings of these drugs are represented by the need of frequent (daily) administration, the metabolic interplay with other pharmacological agents (especially antibiotics),30 the challenging and relatively expensive measurement process,31–33 and current lack of antidotes. As such, the development of novel pharmacological agents that may be effective to prevent (or treat) thrombosis, which are relatively inexpensive, do not require continuous administration and display better safety profiles should be regarded as a highly attractive perspective.34 Antisense therapy against coagulation factor XI potentially fulfills all these important considerations. The available evidence suggests that antisense oligonucleotides may be more effective than conventional anticoagulants in preventing the onset and propagation of thrombosis, do not require factor XI measurement since the reduction of mRNA synthesis appears to be dose-dependently, robustly, and stably decreased for 3 to 5 weeks after the end of administration, with an incidence of major bleeding that is at least not greater than that caused by treatment with either warfarin or LMWH.
Additive Value of Antisense Therapy to Vitamin K Antagonists, Heparin, and Direct Oral Anticoagulants
Interestingly, antisense therapies against other coagulation factors are in development (►Fig. 2), also with promising results. Yau et al produce a 20-long, factor XII antisense oligonucleotide, which was then administrated to rabbits.19 After 4 weeks of treatment, the antisense oligonucleotide was effective to reduce factor XII mRNA expression, factor XII plasma levels, and activity by 97, 97, and 99%, respectively. The anticoagulant effect was also investigated in a rabbit model of thrombosis, involving catheter implantation in the jugular vein until occlusion for a maximum of 35 days. Compared with control, the administration of the antisense oligonucleotide was effective to significantly prolong the mean time to catheter occlusion by more than twofold. Similar results were obtained by Bird et al using prekallikrein antisense oligonucleotide treatment in mice,35 demonstrating that plasma kallikrein suppression is also effective to prevent thrombus formation in mice with minimal bleeding.
Venous thrombosis is a major health care issue, especially in hospitalized patients and in those undergoing major and orthopedic surgery.22,23 The current armamentarium of pharmacological agents used to prevent and treat VTE entails old-generation drugs, such as vitamin K antagonists (especially warfarin) or low-molecular-weight heparin (LMWH), along with specific inhibitors of coagulation factors II and X, conventionally known as direct oral anticoagulants (DOACs).24,25 Although the clinical usefulness of these pharmacological agents has been now clearly established, their use in the real world is still plagued by several drawbacks. Warfarin and other vitamin K antagonists can be orally administered but display a narrow therapeutic window, and thus require ongoing dose adjustment by means of regular laboratory monitoring to prevent under or overcoagulation.26,27 LMWH is equally effective as warfarin, does not require constant laboratory monitoring, but can only be administered by repeated (i.e., once or twice daily) subcutaneous injections, and is hence an inconvenient means of longterm anticoagulation.28 Both vitamin K antagonists and LMWH are also associated with a significant risk of bleeding, especially in patients undergoing surgery and other invasive Seminars in Thrombosis & Hemostasis
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Other Targets for Antisense Therapy in Venous Thromboembolism
Potential Limitations of Antisense Therapy The results published so far (summarized in ►Table 1) do not yet make a compelling case for replacing conventional anticoagulants with factor XI antisense therapy (►Table 2).36 Preliminary findings in animal models and in patients undergoing a highly prothrombotic scenario such as knee surgery need to be validated in other prospective trials, and in different
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Authors
Cohort
Dosage
Maximum plasma factor XI reduction
Clinical endpoints
References
Zhang et al, 2010
Mice
50 g/kg
• Activity: > 90% • Concentration: > 90%
• Antithrombotic activity against FeCl3-induced inferior vena cava thrombosis, FeCl3-induced mesenteric vein thrombosis and stenosis-induced inferior vena cava thrombosis • Lower bleeding than enoxaparin or warfarin
15
Que Liu et al, 2011
Humans
50–300 mg/kg
• Activity: 92% • Concentration: > 100%
None
16
Younis et al, 2012
Cynomolgus monkeys
4–40 mg/kg
• Activity: 75%
None
17
Crosby et al, 2013
Baboons
25 mg/kg
• Activity: 70% • Concentration: 70%
• Platelet accumulation in propagated thrombus significantly reduced by 40% • No major bleeding compared with placebo
18
Yau et al, 2014
Rabbits
15 mg/kg
• Activity: 99% • Concentration: 96%
• Prolongation of mean time of jugular vein catheter occlusion
19
van Montfoort et al, 2014
Mice
50 mg/kg
• Activity: 64%
• Decreased thrombus propagation and fibrin deposition after carotid plaque rupture • No major bleeding compared with placebo
20
Büller et al, 2014
Humans
100–300 mg
• Activity (200 mg): 59% • Activity (300 mg): 78%
• Rate of incident venous thromboembolism up to 3 mo after elective primary unilateral total knee arthroplasty significantly reduced by administration of 300-mg antisense oligonucleotide compared with enoxaparin • Marginally but not significantly lower bleeding than enoxaparin
21
clinical scenarios. In particular, it should be clearly established whether such a degree of factor XI suppression may be really associated with a safety profile throughout the maintenance of antisense therapy, as well as the exact level of inhibition required to maintain a safe balance between preventing thrombosis and managing bleeding risk. The clinical effectiveness should be also compared against the future standard of therapy that will be represented by DOACs. Despite conceptual simplicity, rational design, and relatively inexpensive cost, cost-effective analyses should be planned to establish if antisense therapy is economically advantageous over treatment with conventional anticoagulants or DOACs. The management of bleeding episodes in patients receiving factor XI antisense therapy is another potential drawback.37 This is due to several factors, such as the long elimination half-life of antisense
oligonucleotides, the suppression of factor XI activity by more than 50% without evidence of recovery for up to 1 month after administration of the last dose, combined with the current lack of effective antagonists and antidotes. Therefore, in analogy with other anticoagulant drugs, such as Idraparinux, (Sanofi-Aventis, Toulouse, France)38,39 the long duration of the anticoagulant effect may be effective to prevent thromboembolism but can also be associated with an increased risk of major hemorrhages, which would require a continuative factor replacement therapy until the bleeding has resolved. A final concern emerges from the potential side effects of this innovative therapy. Beside the considerable high frequency of injection site-related adverse event observed in humans, the potential risk of potential profibrinolytic effects, desensitization, and teratogenesis40 of antisense drug Seminars in Thrombosis & Hemostasis
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Table 1 Synthesis of studies on antisense therapy against coagulation factor XI
Antisense Therapy against Coagulation Factor XI
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Table 2 Potential limitations of antisense therapy • Validation needed in other prospective trials and in different clinical scenarios • Identification of the best compromise of protein suppression for preventing thrombosis and maintaining a safety profile • Clinical effectiveness tested against direct oral anticoagulants • Cost-effectiveness still uncertain • High potential risk of bleeding due to: – Long elimination half-life of oligonucleotides – Long-lasting suppression of protein concentration – Lack of antagonists and antidotes • Potential adverse reactions – Injection site-related adverse event – Teratogenesis – Profibrinolytic effects – Desensitization
13 Salomon O, Steinberg DM, Zucker M, Varon D, Zivelin A, Seligsohn
14
15
16
17
18
19
technology, specifically during long-term therapy, requires further exploration and clarification. Finally, the potential development of antisense molecules with higher affinity to the target factor XI due to the relative complex molecular structure of the antisense molecule should be considered as an attractive perspective for the future.
20
21
22
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6
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mechanisms. Mol Cancer Ther 2002;1(5):347–355 Bennett CF, Swayze EE. RNA targeting therapeutics: molecular mechanisms of antisense oligonucleotides as a therapeutic platform. Annu Rev Pharmacol Toxicol 2010;50:259–293 Lippi G, Favaloro EJ. Antisense therapy in the treatment of hypercholesterolemia. Eur J Intern Med 2011;22(6):541–546 Castanotto D, Stein CA. Antisense oligonucleotides in cancer. Curr Opin Oncol 2014;26(6):584–589 Mourich DV, Marshall NB. Antisense approaches to immune modulation for transplant and autoimmune diseases. Curr Opin Pharmacol 2005;5(5):508–512 Falzarano MS, Passarelli C, Ferlini A. Nanoparticle delivery of antisense oligonucleotides and their application in the exon skipping strategy for Duchenne muscular dystrophy. Nucleic Acid Ther 2014;24(1):87–100 Gauvreau GM, Boulet LP, Cockcroft DW, et al. Antisense therapy against CCR3 and the common beta chain attenuates allergeninduced eosinophilic responses. Am J Respir Crit Care Med 2008; 177(9):952–958 Lu X, Yu Q, Binder GK, et al. Antisense-mediated inhibition of human immunodeficiency virus (HIV) replication by use of an HIV type 1-based vector results in severely attenuated mutants incapable of developing resistance. J Virol 2004;78(13):7079–7088 de Jong YP, Jacobson IM. Antisense therapy for hepatitis C virus infection. J Hepatol 2014;60(1):227–228 Lippi G, Franchini M. Pathogenesis of venous thromboembolism: when the cup runneth over. Semin Thromb Hemost 2008;34(8): 747–761 Duga S, Salomon O. Congenital factor XI deficiency: an update. Semin Thromb Hemost 2013;39(6):621–631 Vercauteren E, Gils A, Declerck PJ. Thrombin activatable fibrinolysis inhibitor: a putative target to enhance fibrinolysis. Semin Thromb Hemost 2013;39(4):365–372
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Extended prophylaxis of venous thromboembolism with idraparinux. N Engl J Med 2007;357(11):1105–1112 39 Harenberg J, Vukojevic Y, Mikus G, Joerg I, Weiss C. Long elimination half-life of idraparinux may explain major bleeding and recurrent events of patients from the van Gogh trials. J Thromb Haemost 2008;6(5):890–892 40 Crooke ST. Antisense Drug Technology: Principles, Strategies, and Applications. 2nd ed. New York, NY: CRC Press; 2007
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33 Harenberg J, Du S, Krämer S, Weiss C, Krämer R, Wehling M.
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