Trends in Cardiovascular Medicine
Volume 15, Issue 1 , Pages 41-45, January 2005

The potential of aptamers as anticoagulants

  • Shahid M. Nimjee

      Affiliations

    • University Program of Genetics, Duke University Medical Center, Durham, North Carolina, USA
    • Department of Surgery, Division of Experimental Surgery, Duke University Medical Center, Durham, North Carolina, USA
    • ,
  • Christopher P. Rusconi

      Affiliations

    • Department of Surgery, Division of Experimental Surgery, Duke University Medical Center, Durham, North Carolina, USA
    • Regado Biosciences Inc., Research Triangle Park, North Carolina, USA
    • ,
  • Robert A. Harrington

      Affiliations

    • Duke Clinical Research Institute, Duke University Medical Center, Durham, North Carolina, USA
    • ,
  • Bruce A. Sullenger

      Affiliations

    • University Program of Genetics, Duke University Medical Center, Durham, North Carolina, USA
    • Department of Surgery, Division of Experimental Surgery, Duke University Medical Center, Durham, North Carolina, USA
    • Corresponding Author InformationAddress correspondence to: Bruce A. Sullenger, Box 2601 DUMC, Durham, NC 27710, USA. Tel.: (+1) 919-684-6344

Article Outline

Useful additional options for anticoagulant therapy have been introduced over the last 15 years, including low-molecular-weight heparins and direct thrombin inhibitors. Despite these impressive advances, a need for safer effective anticoagulants remains. Aptamers represent a therapeutic modality that has the potential to address this unmet need. Aptamers are small nucleic acid molecules that function as direct protein inhibitors, much like monoclonal antibodies. Aptamers are delivered by parenteral administration, can be formulated to possess a very short or sustained half-life, and are purported to be nonimmunogenic. Perhaps most relevant to the development of safer anticoagulant therapies, recent studies have shown that antidotes can be rationally designed to control the pharmacologic effects of aptamers in vivo, paving the way for a new class of antidote-controlled therapeutics. This review discusses the limitations of current anticoagulant therapies, the properties of aptamers and how these properties can be exploited to address the unmet needs within this therapeutic class, and the progress to date in developing new aptamer-based anticoagulant therapies.

 

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Limitations of current anticoagulant therapy 

Given the central role of thrombosis in the pathobiology of acute ischemic heart disease, injectable (intravenous [i.v.] or subcutaneous [s.c.]) anticoagulants have become the foundation of medical treatment for patients presenting with acute coronary syndromes (unstable angina and myocardial infarction) and for those undergoing coronary revascularization procedures, either percutaneously or surgically (Harrington et al., 2004, Popma et al., 2004). Currently available anticoagulants include unfractionated heparin (UFH), the low-molecular-weight heparins (LMWHs), an indirect Factor Xa inhibitor (fondaparinux), and the direct thrombin inhibitors (DTIs) (e.g., recombinant hirudin, bivalirudin, and argatroban). The present paradigm both for anticoagulant use and for continued antithrombotic drug development is to establish a balance between efficacy (reducing the risk of ischemic events) and safety (minimizing the risk of bleeding) (Harrington 2003). Each of the available agents carries an increased risk of bleeding relative to placebo. Interestingly, the DTI bivalirudin has been shown to have a net therapeutic benefit that includes superior efficacy as well as a reduced risk of bleeding compared with UFH (Lincoff et al. 2003).

Generally, cardiovascular clinicians have been willing to trade off an increased risk of bleeding when a drug can reduce the ischemic complications of either the acute coronary syndromes or the coronary revascularization procedures. However, recent data have suggested that bleeding events, particularly those that require blood transfusion, may carry inherent consequences other than just the limited but well-known infectious disease risks (Rao et al. 2004). An anticoagulant that can limit that risk while preserving anti-ischemic benefit is appealing.

UFH has additional limitations that newer anticoagulants address. It has complex pharmacokinetics that make the predictability of its use in individual patients challenging. This means that clinicians need to monitor and adjust the use of UFH through the use of frequent activated partial thromboplastin times (aPTTs). The relationship between aPTTs and clinical outcomes (both ischemic and bleeding) is U shaped and speaks to the complexities of using and monitoring UFH in clinical practice (Granger et al. 1996). UFH binds to a number of plasma proteins that are abundant at the site of disrupted atherosclerotic plaque and may neutralize its effects. For physiochemic reasons, UFH has poor activity against clot-bound thrombin. UFH is associated with two unusual but potentially catastrophic complications—heparin-induced thrombocytopenia (HIT) and HIT with thrombosis (HITT) (Warkentin and Greinacher 2004). Finally, the immediate antidote to UFH therapy is the use of protamine. Through electrical charge neutralization, protamine can reverse the anticoagulant effects of UFH. However, the dose predictability is challenging, and there are serious side effects that can be seen with its use (Hirsh et al. 2001).

The LMWHs have improved on the predictability of UFH dosing and do not require lab-based monitoring as part of their routine use. HIT and HITT are observed less frequently with the LMWHs relative to UFH, but they have not eliminated this risk. Two of the three commercially available DTIs, lepirudin and argatroban, are specifically approved for use in patients who have developed or have a history of HIT. Bivalirudin is approved for use as an anticoagulant during percutaneous coronary intervention (PCI) and therefore provides an attractive alternative to UFH in patients who have HIT.

All currently available anticoagulants increase the risk of bleeding relative to placebo, although bivalirudin is associated with less bleeding in a PCI population than is UFH. There are no direct and clear antidotes to the anticoagulant effects of the LMWHs, nor of the DTIs. These patients are managed by administering blood products (including clotting factor). The ability of protamine to reverse the effects of the LMWHs is even less predictable than that seen with UFH; in part, this is due to the subcutaneous dosing of the LMWHs. For practical purposes, there is limited ability to effectively use protamine as an antidote to the LMWHs (Jones et al. 2002).

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Aptamers as therapeutic agents 

Aptamers, whose root word derives from the word aptus (“fit”) are single- stranded oligonucleotides that fold into a specific three-dimensional structure that enables them to directly bind to and inhibit a protein target. Aptamers are generated by an iterative, in vitro selection process termed SELEX (Systematic Evolution of Ligands by EXponential enrichment; Figure 1) (Ellington and Szostak, 1990, Tuerk and Gold, 1990). SELEX technology has enabled the isolation of aptamers to a myriad of target proteins (Gold et al. 1995). In December 2004, Eyetech, in conjunction with Pfizer, received approval from the Food and Drug Administration for their antivascular endothelial growth factor (VEGF) aptamer pegaptanib sodium (Macugen) to treat age-related macular degeneration. The development of this drug has paved the way for the future development of therapeutic aptamers for other indications (Eyetech Study Group, 2002, Eyetech Study Group, 2003, Gragoudas et al., 2004, Ruckman et al., 1998).

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  • Figure 1. 

    Systemic evolution of ligands by exponential enrichment (Systematic Evolution of Ligands by EXponential enrichment; SELEX). A DNA or RNA library is incubated with a protein target and those ligands that bind to the target are separated from those that do not. The bound pool is then reverse transcribed (in the case of the RNA pool) and amplified in preparation for a subsequent round of selection. This process is repeated until there is no increased affinity of the enriched pool for the protein target. RT-PCR, reverse transcriptase-polymerase chain reaction.

Aptamers possess several properties that make them potentially quite suitable for use as anticoagulants. First, aptamers typically exhibit high affinity and specific binding to their target protein, with dissociation constants in the high picomolar to low nanomolar range, and specificity constants of 103 or greater when one compares aptamer binding with the target versus related nontargeted proteins (Jellinek et al., 1995, Rusconi et al., 2000, Rusconi et al., 2002). Thus, as has been demonstrated by us in the discovery of anti-Factor VIIa and anti-Factor IXa aptamers (Rusconi et al., 2000, Rusconi et al., 2002, Rusconi et al., 2004), as well as others in the discovery of antithrombin aptamers (Bock et al., 1992, DeAnda et al., 1994, Griffin et al., 1993), aptamer technology is well suited for the discovery of direct-acting, potent anticoagulants. Moreover, the high affinity of most aptamers should allow for therapeutic dosing at submicromolar levels, which should reduce potential nonspecific effects. Second, aptamers are purported to be nonimmunogenic. Their small size and similarity to endogenous molecules theoretically makes them poor antigens, and the lack of antigenicity of aptamers has been supported by recent clinical studies (Eyetech Study Group, 2002, Eyetech Study Group, 2003). If, as suggested by these early clinical trials, low or no immunogenicity is indeed a class property of aptamers, then aptamer-based anticoagulants are unlikely to induce autoimmune conditions such as those that resemble HIT or HITT, or to be subject to potential antibody reactions as are peptide and polypeptide-based anticoagulants. At a minimum, such drugs could potentially be useful in the anticoagulation of patients with HIT/HITT. Third, the pharmacokinetic properties of aptamers are tunable and, to date, aptamers have exhibited predictable pharmacokinetics with well-behaved pharmacokinetic/pharmacodynamic (PK/PD) relationships, suggesting aptamer-based anticoagulants may require less monitoring than does heparin. In fact, aptamers can be formulated to possess very short half-lives (minutes) or can be easily conjugated to high-molecular-weight polyethylene glycol to provide compounds with much longer half-lives (∼9–12 h following bolus i.v. or s.c. injection) (Tucker et al. 1999). Thus, within this class of compounds, one can envision anticoagulants with PK/PD properties similar to bivalirudin or to enoxaparin, depending on the intended applications of the drug. Finally, as discussed subsequently, antidotes can be rationally designed to control the pharmacologic activity of aptamers. This property is unique to aptamers, and provides a means to extend the heparin–protamine paradigm to a whole new class of direct-acting, specific anticoagulants (Rusconi et al. 2002). In addition, this drug–antidote design technology is generalizable; thus, it broadly offers the possibility of novel antidote-controlled antithrombotics.

As parenteral agents, the most likely initial indications for aptamer-based anticoagulants are for use in hospitalized patients, particularly those undergoing interventional procedures or surgeries. For this constellation of indications, the ideal anticoagulant would be deliverable by i.v. or s.c. injection, immediately therapeutic, easily dosed so as not to require frequent monitoring, and immediately and predictably reversible. As discussed above, the chemical properties of aptamers afford great flexibility in drug design, particularly with respect to the need for novel, rapidly reversible anticoagulants. Two aptamer-based anticoagulant agents in development are designed specifically to meet the need for rapid-onset, rapid-offset agents. Interestingly, the two agents use very different mechanisms to achieve controllable anticoagulation. The first agent, a direct thrombin inhibitor termed ARC-183 (Archemix Corp., Cambridge MA), is a very short half-life agent, similar to bivalirudin, the pharmacology of which can be controlled by rate of infusion and can be rapidly reversed by cessation of infusion. The second agent—an antidote-controlled, direct, Factor IXa (FIXa) inhibitor termed REG1 (Regado Biosciences, Inc., Research Triangle Park, NC)—is a medium-duration-of-effect anticoagulant that can be rapidly and durably reversed by bolus injection of a specific antidote to the drug. The remainder of this review discusses the development to date of these two agents and their potential to address the need for safer, effective anticoagulants.

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Anticoagulant aptamers in development 

Thrombin is a key intermediate in the coagulation cascade, because it activates upstream procoagulant factors to amplify the coagulation reaction and converts fibrinogen to fibrin, the building block of the fibrin matrix of blood clots (Figure 2) (Coughlin 2000). In the early 1990s, scientists at Gilead Sciences discovered a family of single-stranded DNA aptamers that exhibited binding affinities between 2 and 200 nM to thrombin (Figure 3A) (Bock et al. 1992). The most potent of these aptamers, initially termed GS-522 and now called ARC-183, is a 15-residue DNA oligonucleotide of approximately 5000 Da.

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  • Figure 2. 

    Cell-based model of coagulation. In this model of coagulation, thrombin (Factor IIa; FIIa) and Factor IXa (FIXa) are circled to illustrate where the antithrombin DNA aptamer and anti-FIXa RNA aptamer respectively bind to inhibit coagulation. TF, Tissue factor; VA, Factor Va; vWF, von Willebrand factor.

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  • Figure 3. 

    (A) Thrombin aptamer ARC-183. The sequence of the DNA aptamer to thrombin. (B) Aptamer 9.3tC inhibits Factor IXa (FIXa) by folding into a specific structure. Upon administration of antidote 5-2C, the aptamer binds to the antidote by Watson-Crick base pairing, and in so doing, loses its structure and activity.

In vitro analysis of the antithrombin activities of ARC-183 demonstrated that this aptamer inhibits thrombin activity by preventing the thrombin-catalyzed conversion of fibrinogen to fibrin. In addition, it was found that this aptamer could inhibit both free and clot-bound thrombin, and that it was able to prolong clotting times in pure fibrinogen and plasma assays (Bock et al. 1992).

The pharmacologic properties of this thrombin aptamer have been evaluated in a number of animal models. The aptamer prolonged the prothrombin time (PT) in both cynomolgus monkeys and in sheep undergoing hemofiltration with an extracorporeal circuit (Griffin et al. 1993).

In a canine cardiopulmonary bypass (CPB) model, animals received either 0.3 or 0.5 mg/kg per min infusion of thrombin aptamer and exhibited increased PT, aPTT, and activated clotting time (ACT)that subsequently returned to baseline after the infusion of the aptamer ceased. Pharmacokinetic analysis revealed that the elimination half-life of the drug was approximately 1.9 min pre- and post-CPB and 7.7 min during CPB. There was no significant difference in fibrinogen or platelet consumption between the heparin and thrombin aptamer groups, nor was there significant hemorrhage, hematoma formation, or oozing from the surgical site (DeAnda et al. 1994). These data illustrated the potential of aptamers to function as anticoagulants in a clinical scenario. In August 2004, Archemix and Nuvelo announced on their Web sites(http://www.archemix.comthe initiation of Phase I, healthy, volunteer trials of the thrombin aptamer, with the goal of future development of the compound for use in patients undergoing coronary artery bypass graft surgery on CBP.

Factor IX (FIX) is a serine protease that is essential for robust Factor Xa (FXa) and thrombin generation, as evidenced by the fact that the absence or deficiency in FIX results in a bleeding diathesis, hemophilia B. As shown in Figure 2, FIXa, in complex with its requisite cofactor Factor VIIIa, is the catalyst of Factor X (FX) activation on the surface of activated platelets, the primary surface on which thrombin generation occurs (Figure 2) (Schmidt and Bajaj 2003). Rusconi et al. (2002) isolated a specific RNA ligand to FIXa that completely blocked FX cleavage by the enzyme complex and increased aPTT in a dose-dependent manner to a maximum equal to that of plasma that was FIX deficient (<1% FIX activity) without any effect on PT.

One of the greatest causes of morbidity associated with anticoagulation therapy is bleeding (Ginsberg et al., 2001, Moll and Roberts, 2002). Many reversal strategies for anticoagulation involve pharmacokinetic manipulation. This, however, is problematic, because pathophyisologic differences between patients produces differential capacity to clear drugs and subsequent unpredictability in anticoagulation and reversal. Because these drugs are primarily cleared by blood-based metabolism, the possibility exists that such drugs may be metabolized in the region of blood stasis to below therapeutic levels, leading to the unintended reversal of the drug and thereby thrombosis. Thus, antidote control of anticoagulation is paramount in establishing truly safe therapies for all patient populations. In order to achieve this end, an antidote RNA oligonucleotide was synthesized to reverse the activity of anti-FIXa aptamer 9.3t, which ultimately produced the first rationally designed drug–antidote pair in anticoagulant therapy. An aptamer inhibits its target by folding into a specific conformation and binding to the protein; therefore, any change in this shape would render the aptamer functionally inactive. Utilizing the principle of Watson-Crick base pairing, a second RNA molecule was constructed that was complementary to 9.3t (Figure 3B). The antidote oligonucleotide 5-2C inhibited the activity of the aptamer rapidly and sustainably compared with controls (Rusconi et al. 2002). In plasma from patients with HIT, where heparin therapy is contraindicated, 9.3t effectively prolonged the aPTT and was efficiently reversed by its matched antidote (Rusconi et al. 2002).

In vivo models have also validated the clinical potential of FIXa aptamer and its matched antidote as a reversible anticoagulant. The aptamer Ch-9.3t (aptamer 9.3t conjugated to a cholesterol moiety at its 5′ end to increase its circulating half-life) prolonged ACT and aPTT in a porcine systemic anticoagulation model, exhibiting a blood half-life of ∼60 to 90 min. Antidote 5-2C reversed >95% of aptamer function within 10 min in these animals (Rusconi et al. 2004).

A murine arterial injury model showed that the FIXa aptamer could maintain the patency of a vessel that underwent a thrombotic insult (Rusconi et al. 2004). Furthermore, in a mouse-tail transection bleeding model, animals treated with high levels of Ch-9.3t exhibited significantly more blood loss compared with controls. However, no increased hemorrhage was observed in aptamer-treated animals that received antidote 5-2C immediately after tail transection compared with control animals (Rusconi et al. 2004). Regado Biosciences has developed an optimized version of this anti-FIXa aptamer–antidote pair, termed REG1, and anticipates initiation of Phase I healthy volunteer testing of this aptamer–antidote pair in the first half of 2005.

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Conclusions 

Despite the addition of new effective anticoagulants to the clinical arsenal, there remains a need for safer, effective anticoagulants. As a class of compounds, aptamer-based anticoagulants have the potential to address this unmet need, because they offer the potential to develop potent, direct-acting, specific, and—most importantly—controllable agents. The two aptamer-based anticoagulants in development, ARC-183 and REG1, highlight the flexibility engendered by the chemical structure of aptamers, because both potentially offer unique solutions to the need for control over anticoagulant therapy, but achieve this control with fundamentally different approaches—a short half-life for ARC-183 versus antidote control for REG1. To determine whether either compound will truly offer clinicians the safe, effective, and controllable anticoagulant agents they need requires further clinical testing.

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PII: S1050-1738(05)00003-4

doi:10.1016/j.tcm.2005.01.002

Trends in Cardiovascular Medicine
Volume 15, Issue 1 , Pages 41-45, January 2005

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