Inhibitors of propagation of coagulation: factors V and X

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1 British Journal of Clinical Pharmacology DOI: /j x Inhibitors of propagation of coagulation: factors V and X Vincenzo Toschi 1 & Maddalena Lettino 2 1 Department of Hematology and Blood Transfusion, Thrombosis Center, San Carlo Borromeo Hospital, Milan and 2 Coronary Care Unit, Department of Cardiology, Fondazione IRCCS Policlinico San Matteo, Pavia, Italy Correspondence Dr Vincenzo Toschi MD, Department of Hematology and Blood Transfusion, Thrombosis Center, A.O. Ospedale San Carlo Borromeo, Via Pio II, Milano, Italy. Tel.: Fax: vincenzo.toschi1@alice.it Keywords acute coronary syndromes, atrial fibrillation, factor Va inhibitors, factor Xa inhibitors, stroke, venous thromboembolism Received 30 December 2010 Accepted 5 April 2011 Accepted Article 4 May 2011 Cardiovascular diseases are still the most important cause of morbidity and mortality in western countries and antithrombotic treatment is nowadays widely used. Drugs able to reduce coagulation activation are the treatment of choice for a number of arterial and/or venous thromboembolic conditions. Some of the drugs currently used for this purpose, such as heparins (UFH or LMWH) and VKA, have limitations consisting of a narrow therapeutic window and an unpredictable response with the need of laboratory monitoring in order to assess their efficacy and safety. These drawbacks have stimulated an active research aimed to develop new drugs able to act on single factors involved in the coagulation network, with predictable response. Intense experimental and clinical work on new drugs has focused on synthetic agents, which could preferably be administered orally and at fixed doses. The most advanced clinical development with new anticoagulants has been achieved for those inhibiting FXa and some of them, like fondaparinux, are already currently used in clinical practice. Other agents, such as rivaroxaban, apixaban, otamixaban and edoxaban are under development and have already been studied or are currently under investigation in large scale phase III clinical trials for prevention and treatment of venous thromboembolism, atrial fibrillation and acute coronary syndromes. Some of them have proved to be more effective than conventional therapy. Data on some agents inhibiting FVa are still preliminary and some of these drugs have so far been considered only in patients with disseminated intravascular coagulation secondary to sepsis. Introduction Atherothrombosis and venous thromboembolism are still the leading cause of morbidity and mortality in western countries. Anticoagulant drugs are the mainstay in the treatment of a wide range of these conditions and, for more than 40 years, unfractionated heparin (UFH) and vitamin K antagonists (VKA) have been the sole anticoagulants available to clinicians. Despite the fact that UFH and warfarin, the most commonly used VKA, have been extensively used for both therapy and prevention of a wide variety of thrombotic conditions, these drugs have some important limitations. Specifically, warfarin is characterized by a delayed onset and offset of action, a narrow therapeutic window, an unpredictable response in patients due to genetic differences in its metabolism or in its pharmacologic target and interactions with food and drugs [1]. UFH is purified from animal (porcine) sources with potential future shortage in its supply, it has to be administered mainly by the intravenous route, and, more importantly, it 2011 The Authors British Journal of Clinical Pharmacology 2011 The British Pharmacological Society may be associated with a condition known as heparin induced thrombocytopenia (HIT), which may cause lifethreatening thrombotic complications [2, 3]. Both warfarin and UFH also need laboratory monitoring by INR and APTT, respectively, in order to assess their efficacy and safety in terms of thrombotic or haemorrhagic complications. Consequently, they also need frequent dose adjustments [4, 5]. The limitations of UFH and warfarin have led to the development of new anticoagulants for the prevention and treatment of arterial and venous thromboembolism. Low molecular weight heparins (LMWHs), derived from the depolymerization of UFH, are characterized by a shorter chain length, a longer half-life and a more predictable dose-response compared with UFH [6].This allows weightadjusted fixed doses with no need for laboratory monitoring. However, some limitations are still associated with LMWHs.These drugs are excreted by the kidney and, therefore, their use may be unsafe in patients with renal insufficiency with special cautions to be observed in subjects with moderate to severe renal failure. Moreover, LMWHs Br J Clin Pharmacol / 72:4 / / 563

2 V. Toschi & M. Lettino still need to be administered parenterally (mainly subcutaneously), and this may be a drawback especially in an outpatient setting. These drugs are only incompletely neutralized by protamine sulfate compared with UFH and, finally, LMWHs are also associated with HIT, although to a much lesser degree than UFH [7].These features prompted, in the past decade, an intense experimental and clinical investigation on new agents selectively targeting single factors involved in the activation of the coagulation network which could be administered at fixed doses and, in some conditions, even orally. More specifically, the most advanced clinical development on new anticoagulants has been achieved for those inhibiting FXa and FIIa and some of them have already been approved for clinical use. Data on some of the drugs inhibiting FVa are to be considered more preliminary.this review will focus on new antithrombotic drugs designed to antagonize inhibitors of propagation of coagulation activated factors V and X including those that have already been approved for clinical use in Europe such as fondaparinux, rivaroxaban and Drot AA. The coagulation network Activation of the coagulation network is a multi-step process characterized by enzymatic sequential activation of a series of circulating inactive proteins, and their interaction with platelets [8]. Upon breaking of the vasculature, platelets adhere at the site of injury and subendothelial cells express a cell-surface molecule called tissue factor (TF), which, upon binding to factor VII/VIIa (FVII/VIIa), forms an activated complex that comprises TF and FVIIa. According to the more recent view of coagulation process activation, this phase is called initiation and is characterized by generation of small amounts of activated factors. Specifically,TF-FVIIa complex activates both factor IX (FIX) to FIXa and factor X (FX) to FXa.The activated proteins play distinct roles in the ensuing reactions. FIXa associates with platelets, where it plays a role in the later stages of haemostasis, whereas FXa forms a complex with factor Va (FVa) to convert a small amount of prothrombin (FII) to thrombin (FIIa). The source of FVa for this reaction is likely protein released from platelets which adhere to collagen of the subendothelial matrix by glycoprotein VI. In fact, FVa can be released by platelets in a partially active form and does not require further activation to promote thrombin generation. In the subsequent step, called amplification, the thrombin formed in the initiation phase plays a key role in the activation of the coagulation, where it works as an amplifier by acting on platelets and proteins to facilitate platelet-driven thrombin generation. Platelet activation leads to exposure on their membrane of an increased amount of phosphatidylserine thus facilitating assembly of coagulation proteins on their surface and increasing their activity. In addition, thrombin can activate coagulation proteins with production of factor VIIIa (FVIIIa), FVa, FIXa and FXa which bind to the platelet surface. In a further step called propagation activated platelets and activated factors induce a burst of thrombin generation, which means that a large amount of prothrombin is converted to thrombin on the surface of activated platelets. Factor IXa, formed during the initiation phase, binds to activated platelets. In this phase also FVIIIa binds to the platelet membrane and the factor IXa-VIIIa complex activates FX on the platelet surface. This platelet surface-generated FXa forms a complex with platelet surface FVa and this complex, in the presence of prothrombin, promotes thrombin generation. Factors Xa and Va, together with platelet membrane phospholipids, form the so called prothrombinase complex. The burst of thrombin produced during the propagation phase leads to cleavage of fibrinogen with fibrin production and to the assembly of fibrin strands, which stabilize the initial platelet plug. In addition to its role in cleaving fibrinogen, thrombin activates factor XI (FXI) on the platelet surface. FXIa can in turn activate FIX to FIXa thus enhancing FXa generation. In the proposed model, FXI is not primary to the pathway which leads to thrombin generation, but contributes to the positive feedback loop to increase thrombin production. In a further step, thrombin activates a larger amount of all the coagulation proteins, thus amplifying the different reactions and catalyzes the conversion of more circulating fibrinogen to fibrin, which is the scaffolding of the clot, composed of platelets, red blood cells and leucocytes. It is important to underline that high levels of thrombin generated during the propagation phase bind to fibrin where it is functionally active being protected from inhibition by antithrombin (AT). Disruption of the plug brings fibrinogen into contact with the bound thrombin, where fibrin formation can be initiated immediately without the need of thrombin generation. The entire process is depicted in Figure 1. New anticoagulant drugs targeting coagulation factor X FXa is a good target for new anticoagulants. It plays a crucial role in the activation of the coagulation since it is able to activate prothrombin upon binding FVa (Figure 1) and it has been demonstrated that, when activated, 1 molecule of FXa can generate >1000 thrombin molecules [9]. Drugs inhibiting FXa have been extensively studied and a large body of both experimental and clinical data have been obtained. Drugs inhibiting FXa may act directly or indirectly via AT [10 13]. The need for AT binding in order to exert anti-fxa activity implies the formation of a drug-at complex. The complex has a high molecular weight and therefore it is able to inhibit circulating FXa but unable to enter into the clot and to block the functionally active, thrombus-bound, FXa. By contrast, drugs able to inhibit directly FXa are small molecules, capable of entering into the thrombus and therefore inhibiting both circu- 564 / 72:4 / Br J Clin Pharmacol

3 Anti-factor X and anti-factor V anticoagulants Steps in coagulation Coagulation cascade Drug II Initiation IIa X TF/VIIa IX IIa Drotrecogin ART-123 Propagation IIa VIIIa Xa Va II IXa IIa Fondaparinux Idraparinux Idrabiotaparinux Rivaroxaban Otamixaban Thrombin activity IIa Apixaban Edoxaban Betrixaban Fibrinogen Fibrin YM150 TAK442 Figure 1 Mechanism of activation of coagulation and new antithrombotic drugs Table 1 Main pharmcokinetic and pharmacodynamic characteristics of anti-factor Xa anticoagulants that have been tested or are currently under investigation in phase III studies Drug Anti-FXa activity Route of administration Bioavailability (%) Time to peak Half-life Renal excretion (%) Fondaparinux Indirect Subcutaneous h 17 h ~100 Idraparinux Indirect Subcutaneous h 130 h ~100 Otamixaban Direct Intravenous min 25 min <25 Rivaroxaban Direct Oral ~ h 7 11 h 66 Apixaban Direct Oral ~ h 8 14 h ~25 Edoxaban Direct Oral h 9 11 h ~33 lating and thrombus-bound FXa.This review will focus only on drugs which have reached phase II or III clinical trials and those already approved for clinical use.the main pharmacokinetic and pharmacodynamic characteristics of anti- FXa anticoagulants are reported in Table 1. Indirect factor Xa inhibitors: fondaparinux, idraparinux and idrabiotaparinux Like UFH and LMWH, this class of antithrombotic drugs needs AT in order to exert its pharmacological action which consists in inhibition of FXa activity and, consequently, of thrombin generation. Fondaparinux Fondaparinux is the first of a new class of synthetic antithrombotic agents inhibiting FXa. It is a fully synthetically produced pentasaccharide, which is identical to the sequence present in the native heparin molecule that binds to AT with high affinity and, by this mechanism, specifically inhibits FXa. The drug has been tested in a number of phase III clinical studies and it is licensed in EU for the prevention of venous thromboembolism (VTE) in patients who are scheduled for orthopaedic or general surgery and in those with medical conditions [7, 12 14]. Fondaparinux is also licensed for treatment of patients with VTE and in those with non-st and ST-segment elevation acute coronary syndromes (ACS). More specifically, fondaparinux proved superior to the LMWH enoxaparin in high risk orthopaedic surgery, such as hip fracture, elective hip surgery, total hip replacement and total knee replacement as demonstrated in a meta-analysis of the four large scale clinical studies PENTHIFRA, EPHESUS, PENTATHLON and PENTAMAKS [15]. Extended duration thromboprophylaxis with fondaparinux was also evaluated in the phase III PENTHIFRA-Plus study in patients who received surgery for Br J Clin Pharmacol / 72:4 / 565

4 V. Toschi & M. Lettino hip fracture [16]. Prolonging the duration of prophylaxis with fondaparinux, 2.5 mg once a day subcutaneously from 1 to 4 weeks after fracture, impressively decreased the rate of venographically documented DVT from 35% to 1.4% (P = ) and that of symptomatic VTE from 2.7% to 0.3% (P = 0.021) [16]. Studies on treatment of patients with VTE demonstrated that fondaparinux was equally effective and safe compared with enoxaparin or UFH in the treatment of deep vein thrombosis (DVT) or pulmonary embolism (PE) [17, 18]. Finally, a recent randomized trial on more than 3000 patients with superficial vein thrombosis of the legs, the CALISTO study [19], compared fondaparinux at a dose of 2.5 mg subcutaneously once a day for 45 days with placebo. The study demonstrated that active treatment significantly reduced the incidence of the primary efficacy outcome of symptomatic DVT, PE, extension to safenofemoral junction, recurrence of superficial vein thrombosis and death (0.9% in the active treatment group vs. 5.9% in the placebo group), with a relative risk reduction with fondaparinux of 85% (P < 0.001). No significant difference was observed in bleeding complications between the two groups. As far as treatment of patients with coronary disease is concerned, two randomized, large scale clinical trials assessed the potential clinical benefits of fondaparinux compared either with LMWH or to doseadjusted UFH/placebo in patients with non-st and ST-segment elevation ACS, respectively. The OASIS 5 and OASIS 6 trials [20, 21] globally enrolled more than patients, and a combined analysis of the data of the two studies, in relation to interventional management strategy, including patients, was recently published [22]. The analysis was performed on patients from the OASIS 5 and 6 trials who were randomized in a double-blind fashion to fondaparinux 2.5 mg daily or a heparin-based strategy (dose-adjusted UFH or enoxaparin). Results were stratified according to whether an early invasive, a delayed invasive or an initial conservative management strategy was performed. Results showed that fondaparinux was superior to heparin in reducing the composite of death, myocardial infarction or stroke (8.0% vs. 7.2%, respectively, P = 0.03) and death alone (4.3% vs. 3.8%, P = 0.05). Moreover, fondaparinux reduced major bleeding by 41% (3.4% vs. 2.1%, P < ) and had a more favourable net clinical outcome than heparin (11.1% vs. 9.3%,P < ).In patients treated with an invasive strategy, fondaparinux suppressed ischaemic events to an extent similar to heparin and reduced major bleeding by more than one-half, resulting in a superior net clinical outcome (10.8% vs. 9.4%, P = 0.008). A similar benefit also was observed in those treated with a conservative strategy (P < 0.001). Taken together these data indicate that, compared with a heparin-based strategy, fondaparinux reduced mortality, ischaemic events and major bleeding across the full spectrum of ACS and was associated with a more favourable net clinical outcome in patients undergoing either an invasive or a conservative management strategy. Both OASIS 5 and 6 showed that the main problem associated with fondaparinux in ACS may be catheter thrombosis during percutaneuous coronary intervention (PCI) which requires heparin flushes through the catheter. Interestingly, patients exposed to fondaparinux have been reported to develop anti-heparin/ platelet factor 4 antibodies. However, the drug does not bind to platelets or platelet factor 4 owing to absent or weak cross-reactivity with this epitope [23]. Consequently fondaparinux should not cause HIT and the drug has also proved effective in treating this disorder, as supported by several observational studies [24]. However, randomized trials should be carried out to support this indication. Idraparinux and idrabiotaparinux Idraparinux is a synthetic hypermethylated derivative of fondaparinux with high affinity for AT. The drug is eliminated by the kidney with a plasma half-life of h, thus allowing once weekly subcutaneous administration [25]. A main problem of the drug is the delayed elimination after prolonged administration, which may explain the high incidence of haemorrhagic complications observed in clinical trials [26]. For this reason idrabiotaparinux was developed in order to obtain a rapid inhibition of anticoagulant activity. Idrabiotaparinux differs from idraparinux in that it contains a biotin moiety that enables reversal of the anticoagulant effect with intravenous avidin. Avidin, a protein derived from egg white, binds the biotin moiety with high affinity to form a complex that is rapidly cleared by the kidney [27]. Idraparinux was tested in the phase III trial programme for treatment of VTE and for stroke prevention in atrial fibrillation (AF). The open-label, non-inferiority van Gogh trials compared idraparinux, administered at a dose of 2.5 mg subcutaneously, once weekly for 3 to 6 months with standard therapy with heparin (LMWH or UFH) followed by VKA in 2904 patients with acute symptomatic DVT and in 2215 patients with PE [28]. At 3 months, in DVT patients, the incidence of recurrent VTE was similar in the two treatment groups (2.9% vs. 3.0%, respectively) thus satisfying the prespecified non-inferiority criteria. Idraparinux was associated with a significantly lower rate of major plus clinically non-major bleeds than conventional therapy (4.0% vs. 7.0%, respectively, P = 0.004). However, in the PE patients idraparinux was less effective than conventional treatment with a rate of recurrent VTE of 3.4% compared with 1.6%, in the heparin group. Again, the incidence of clinically relevant bleeding was significantly lower in patients treated with idraparinux than in those treated with conventional therapy (5.8% vs. 8.2%, respectively, P = 0.02). The therapeutic efficacy of long-term treatment with idraparinux was tested in the van Gogh extension study which compared 6 months idraparinux (2.5 mg subcutaneously once weekly) with placebo in 1215 patients with DVT or PE who had been previously treated for 6 months with idraparinux or VKA [29]. The rate of recurrent VTE was significantly lower with idraparinux than with placebo (1.0% vs. 3.7%, respectively, P = 0.002). However the incidence of major 566 / 72:4 / Br J Clin Pharmacol

5 Anti-factor X and anti-factor V anticoagulants plus clinically relevant non-major bleeding was much higher in patients who received idraparinux compared to the placebo group (4.5% vs. 15%, respectively, P < 0.001). Bleeding events included three fatal intracranial haemorrhages in the idraparinux-treated group, thus suggesting that long-term treatment with idraparinux, although effective, could be associated with an increased risk of clinically significant haemorrhagic events. The results of the AMADEUS trial on patients with AF reinforced this concept. This study compared idraparinux (2.5 mg subcutaneously once weekly) with VKA, at a dose adjusted to maintain an INR of 2 to 3, for prevention of stroke and systemic embolism in patients with AF [30]. Despite the fact that the incidence of the primary composite end point with idraparinux was similar to that of VKA (0.9% vs. 1.3%), the trial was prematurely stopped after the randomization of 4576 patients for an excess of clinically relevant bleeding with idraparinux compared with VKA (19.7% vs. 11.7%, respectively,p < ), including a significant increase in intracranial haemorrhages (1.1 vs. 0.4%, respectively, P = 0.014). As already reported, although clinical studies clearly indicate that idraparinux is an effective anticoagulant, concerns about the risk of serious bleeding, due to its delayed elimination after prolonged administration [26], prompted the development of the biotinylated analogue idrabiotaparinux. One large randomized, double-blind, phase III clinical study with idrabiotaparinux was carried on in VTE. The EQUINOX trial is a bioequivalence study aimed to compare, during a 6 month treatment, the safety and efficacy of biotinylated idraparinux (3 mg) with idraparinux (2.5 mg) in 757 patients with symptomatic DVT [31]. Both drugs were given subcutaneously once weekly. Inhibition of FXa activity was also measured at different time points during the study. The efficacy outcome was recurrent VTE. The safety outcomes were clinically relevant bleeding and death. Recurrent VTE during the 6 months treatment period was 2.3% vs. 3.2% in the idrabiotaparinux and in the idraparinux groups, respectively. The rate of clinically relevant bleeding was 5.2% in the idrabiotaparinux vs. 7.3% in the idraparinux treated patients, whereas death occurred in 1.6% vs. 3.2% of patients who received idrabiotaparinux and idraparinux, respectively. Taken together data of EQUINOX study suggest that idrabiotaparinux and idraparinux are similarly effective and safe for DVT treatment and both drugs have similar time course of FXa inhibition. Moreover, the anticoagulant effect of idrabiotaparinux can be rapidly reversed after intravenous avidin infusion, thus confirming previously reported data demonstrating that the molecule, can be easily inactivated by its specific antidote [27, 32]. These data prompted two large phase III clinical trials on treatment of patients with PE and on prevention of stroke and systemic embolism in patients with AF. The CASSIOPEA trial is an international, multicentre, randomized, double-blind, double-dummy study comparing 3 to 6 months treatment with idrabiotaparinux (3.0 mg subcutaneously once weekly) vs. oral INR-adjusted warfarin in 3200 patients with symptomatic PE with or without symptomatic DVT. The enrolment phase has been completed and data are still to be published. The BOREALIS-AF trial is a multicentre, randomized, doubleblind, non-inferiority study comparing the efficacy and safety of once weekly subcutaneous idrabiotaparinux with oral adjusted-dose warfarin in the prevention of stroke and systemic thromboembolism in patients with AF. The study was prematurely stopped before completing the enrolment and the results are not yet available. Direct factor Xa inhibitors: otamixaban, rivaroxaban, apixaban and edoxaban Otamixaban The parenteral direct FXa inhibitor otamixaban has a rapid onset of action, a short half-life and a limited (<25%) renal excretion. It also has a predictable anticoagulant effect which obviates the need for routine coagulation monitoring [33]. Finally, unlike UFH or LMWH the drug does not cause HIT. For all these reasons otamixaban might be a good candidate to replace UFH or LMWH in patients with ACS who are scheduled or not for PCI [34]. The SEPIA-ACS 1- TIMI 42 trial is a phase II double-blind, triple-dummy,dose-finding study which compared five different intravenous regimens of otamixaban (0.100 to mg kg -1 h -1 ) with an active therapy of heparin plus eptifibatide for the prevention of major cardiovascular events in 3241 patients with non ST-segment elevation ACS and planned early invasive strategy [35]. Although the rate of primary efficacy outcome, a composite of death, myocardial infarction, urgent revascularization or bailout glycoprotein IIb/IIIa use at 7 days, did not significantly differ across the five different doses of otamixaban, or compared with the heparin plus eptifibatide arm, the rate of primary efficacy end point was lowest with the two intermediate otamixaban doses. The lowest dose of otamixaban was stopped early because of an excess of thrombotic complications. In any case the rate of thrombotic complications was numerically higher with all doses of otamixaban compared with heparin plus eptifibatide. Moreover, otamixaban was associated with a significant dosedependent increase in the primary safety outcome of noncoronary artery bypass graft surgery-related TIMI major or minor bleeding, though bleeding rates with intermediate otamixaban doses were similar to those with heparin plus eptifibatide. On the basis of these results a phase III clinical trial with otamixaban in ACS patients was proposed [36] and is actually ongoing. Rivaroxaban, apixaban and edoxaban These factor Xa inhibitors are small, orally active molecules, which are able to bind reversibly to the active site of FXa. Though numerous agents are currently under development, those more extensively studied in large scale clinical trials are rivaroxaban, apixaban and edoxaban. Table 2 summarizes the results of phase III trials with oral, direct anticoagulants so far completed and published. Br J Clin Pharmacol / 72:4 / 567

6 V. Toschi & M. Lettino Table 2 Phase III trials testing oral, direct anti-factor Xa anticoagulants already completed and published Study Clinical setting Drug (dose) Comparator Patients (n) Study design Primary efficacy outcome (% patients vs.controls, P) VTE prophylaxis RECORD 1 [45] Rivaroxaban 10 mg o.d., Enoxaparin 4541 Double-blind 1.1 vs. 3.7, P < Hip arthroplasty 5 weeks RECORD 2 [46] Rivaroxaban 10 mg o.d., Enoxaparin 2509 Doule-blind 2.0 vs. 9.3, P < Hip arthroplasty 5 weeks RECORD 3 [47] Rivaroxaban 10 mg o.d., Enoxaparin 2531 Doule-blind 9.6 vs. 18.9, P < Knee arthroplasty days RECORD 4 [48] Rivaroxaban 10 mg o.d., Enoxaparin 3148 Doule-blind 6.9 vs. 10.1, P = Knee arthroplasty days ADVANCE 1 [57] Apixaban Enoxaparin 3195 Double-blind 8.9 vs. 9.9, P = 0.06* Knee arthroplasty 2.5 mg b.i.d. Double-dummy ADVANCE 2 [58] Apixaban Enoxaparin 3057 Double-blind 15.1 vs. 24.4, P = Knee arthroplasty 2.5 mg b.i.d. ADVANCE 3 [59] Apixaban Enoxaparin 5407 Double-blind 1.4 vs. 3.9, P < Hip arthroplasty 2.5 mg b.i.d. Double-dummy VTE treatment EINSTEIN-DVT [54] Rivaroxaban 15 mg b.i.d. 1 week, 20 mg o.d. 3, 6 or 12 months Enoxaparin VKA 3449 Open label 2.1 vs. 3.0, P < AF stroke prophylaxis AVERROES [61] Apixaban ASA 5599 Double-blind 1.6 vs. 3.7, P < AF 2.5 mg b.i.d. *Non inferiority criteria not satisfied. o.d., once daily; b.i.d., twice daily. Rivaroxaban Rivaroxaban, is an oral direct selective FXa inhibitor, absorbed through the gastrointestinal tract, with a bioavailability of about 80%. The drug has a rapid onset of action, with a peak at 2 to 3 h, and a half-life of 7 to 11 h. Rivaroxaban is excreted by several modalities: one third is cleared unchanged by the kidney, one third is metabolized by the liver CYP3A4-dependent and independent pathway and then eliminated through the faeces, and one third is metabolized in the liver with the inactive metabolites then eliminated by the kidneys. Importantly, the drug is a substrate for the P-glycoprotein (P-gp) transporter. This efflux transporter is highly expressed in the intestine and kidney, and co-administration of potent P-gp inhibitors, such as quinidine or ketoconazole, can increase plasma concentrations of rivaroxaban by reducing its clearance. Consequently, these drugs may potentiate the anticoagulant action of rivaroxaban therefore increasing its prohaemorrhagic potential. Ketoconazole is also an inhibitor of CYP3A4 and its use is contraindicated in association with the drug [37 39]. By contrast, rivaroxaban can be co-administered with aspirin or clopidogrel and also with non-steroidal anti-inflammatory drugs [40, 41]. Prophylaxis of venous thromboemboembolism Rivaroxaban at total daily doses of 5 20 mg was successfully tested in phase II trials in the prophylaxis of VTE in patients scheduled for total knee and total hip replacement, showing to have similar efficacy and safety compared with enoxaparin [42 44]. The daily dose of 10 mg was judged to provide a satisfactory balance between efficacy and safety and was selected for the phase III RECORD trial programme, in which rivaroxaban was compared with enoxaparin 40 mg once a day, starting in the evening before surgery (RECORD 1, 2, and 3) or 30 mg twice daily (RECORD 4) in more than patients undergoing hip or knee arthroplasty (Table 2). All four of the RECORD trials were double-blind and rivaroxaban was given 6 to 8 h after surgical wound closure. In all the RECORD studies the primary efficacy end point was total VTE and more specifically symptomatic or asymptomatic DVT detected by mandatory, bilateral venography, non fatal PE and all cause mortality. The secondary efficacy end point was major VTE (a composite of proximal DVT, nonfatal PE and all VTE-related death). The primary safety end point was major bleeding, defined as fatal bleeding, bleeding into critical organ, bleeding requiring reoperation and clinically overt extra-surgical-site bleeding associated with a fall in haemoglobin level 2gdl -1 or requiring transfusion of 2 units of blood.the RECORD 1 trial compared rivaroxaban with enoxaparin in 4541 patients undergoing total hip replacement [45]. After about 5 weeks treatment, rivaroxaban significantly reduced the incidence of total VTE compared with enoxaparin for an equal duration of treatment (1.1% vs. 3.7%, respectively, P < 0 001). In the RECORD 2 trial, which 568 / 72:4 / Br J Clin Pharmacol

7 Anti-factor X and anti-factor V anticoagulants involved 2509 patients, 5 weeks treatment with rivaroxaban significantly reduced the incidence of total VTE compared with a 10 to 14 days regimen with enoxaparin, followed by 21 to 25 days of placebo (2.0% vs. 9.3%, respectively, P < 0.001) [46]. The RECORD 3 trial included 2531 patients undergoing knee arthroplasty. A 10 to 14 days treatment with rivaroxaban significantly reduced the rate of total VTE compared with an equal duration of treatment with enoxaparin (9.6% vs. 18.9%, respectively, P < 0.001) [47]. Finally, in the RECORD 4 trial, which involved 3148 patients undergoing knee arthroplasty, a 10 to 14 course treatment with rivaroxaban significantly reduced the incidence of total VTE compared with an equal duration of enoxaparin tested at the higher 30 mg twice daily dose (6.9% vs. 10.1%, respectively, P < 0.012) [48]. Notably, in both RECORD 2 and 3 trials, rivaroxaban significantly reduced the incidence of symptomatic VTE and death (which was a secondary end point), compared with enoxaparin, by 80% and 60%, respectively [46, 47]. Concerning bleeding events, rivaroxaban was not associated with an increase in haemorrhagic complications in each RECORD trial. However, two meta-analysis of the four trials indicate a small bur significant increase in major plus clinically relevant non-major bleeding with rivaroxaban compared with enoxaparin [36, 49, 50]. On the basis of these data rivaroxaban was approved in 2008 in Europe and Canada for the prevention of VTE in adult patients undergoing elective hip and knee replacement. A trial on the prophylaxis of VTE in hospitalized medically ill patients (MAGEL- LAN), comparing rivaroxaban 10 mg once daily, administered orally for about 35 days, with subcutaneous enoxaparin 40 mg once daily for 10 to 14 days, has now been completed after the enrolment of 8110 patients. The results of the study are currently under evaluation. Treatment of venous thromboemboembolism The efficacy and safety of rivaroxaban for the treatment of VTE was evaluated in two phase II clinical studies that enroled more than 1150 patients. The ODIXa study randomized 613 patients to twice daily doses of 10, 20 or 30 mg, or to a once daily dose of 40 mg rivaroxaban or to standard treatment with enoxaparin (1 mg kg -1 twice daily) followed by VKA for 12 weeks [51]. The primary efficacy end point, which was the reduction in thrombotic burden at 21 days without recurrence of VTE or VTE-related death, was observed and was not significantly different among the rivaroxaban dosage groups or in comparison with standard therapy. Specifically, 43.8% to 59.2% of patients receiving rivaroxaban achieved the primary efficacy end point vs. 45% of patients receiving standard treatment. Rates of symptomatic VTE recurrence and major bleeding were low and not significantly different among the different treatment groups [52]. In the second phase II study, the EINSTEIN-DVT trial, 543 patients with acute DVT were randomized to receive rivaroxaban 20, 30 or 40 mg once daily or standard therapy (LMWH and VKA). As observed in the previous study, the primary efficacy outcome at 3 months, which consisted of the composite of symptomatic, recurrent venous thromboembolic complications and asymptomatic decrease in thrombotic burden (as assessed by quantitative ultrasound and perfusion lung scanning), was not significantly different among the different treatment groups.the primary efficacy end point occurred in 5.4% to 6.6% of patients receiving rivaroxaban and in 9.9% of those allocated to standard therapy [53]. Considering the primary safety end point, the rate of any clinically relevant bleeding was 2.2% to 6.0% in the rivaroxaban treatment group and 8.8% in those receiving standard therapy.these data prompted the beginning of phase III studies. EINSTEIN-DVT and EINSTEIN-PE are large scale, multicentre, open-label, randomized trials performed in patients with confirmed DVT or PE, respectively, comparing rivaroxaban alone with subcutaneous enoxaparin followed by VKA.The rivaroxaban regimen used in these two trials was 15 mg twice daily for the first week, followed by 20 mg once daily, for a predefined treatment period of 3, 6 or 12 months.the EINSTEIN-EXT enrolled VTE patients who completed a 6 to 12 months treatment period with rivaroxaban or VKA. These patients were randomized to receive either rivaroxaban (20 mg once daily) or placebo for an additional 6 or 12 months. The primary efficacy and safety outcomes were, respectively, recurrent VTE and major or clinically relevant non-major bleeding.the results of the EINSTEIN-DVT study were recently published demonstrating that in 3449 patients with acute DVT rivaroxaban was not inferior to enoxaparin with respect both to primary efficacy (2.1% vs. 3.0%, P < 0.001) and to primary safety outcome (8.1% in each patient group) [54]. The other two studies on rivaroxaban in VTE were recently completed or are about to finish the recruitment, and data so far obtained are under evaluation. Stroke prevention in atrial fibrillation Based on the results of the phase II studies on treatment of VTE, the dose of 20 mg was chosen to perform the phase III, muticentre, double blind, clinical trial on stroke prevention in AF. The ROCKET-AF trial was then designed to compare rivaroxaban 20 mg once daily (15 mg once daily in patient with moderate renal insufficiency) with dose adjusted warfarin in patients with AF for whom VKA are indicated on the basis of the current guidelines. The study has been completed and the results, presented at the AHA Scientific Session in 2010, demonstrate the non-inferiority of rivaroxaban compared with warfarin in the prevention of stroke and systemic embolism, with a similar incidence of haemorrhagic complications. Treatment of acute coronary syndromes In the phase II ATLAS ACS-TIMI 46 trial rivaroxaban combined with aspirin was tested in secondary prevention of recurrences in patients with ACS [55]. The results of the study demonstrate that rivaroxaban combined to antiplatelet therapy Br J Clin Pharmacol / 72:4 / 569

8 V. Toschi & M. Lettino increases the risk of bleeding in a dose-dependent manner, but reduces the rate of death, myocardial infarction or stroke compared with antiplatelet therapy alone (3.9% vs. 5.5%,respectively,P = ).Taking into account the data on safety, efficacy and net clinical benefit, doses of 2.5 mg twice daily and 5 mg twice daily were selected for testing the drug in the large multicentre, randomized phase III trial, ATLAS ACS-TIMI 51. At present the study is ongoing. A total of patients are planned to be enroled and the trial primary completion date will be June Apixaban Apixaban is another orally active FXa inhibitor, rapidly absorbed by the gastrointestinal tract, with a maximal plasma concentration achieved at 3 h after oral administration and a half-life of 8 to 14 h [38]. The drug is excreted through different pathways and specifically by hepatic metabolism via CYP3A4 and renal excretion. Concomitant treatment with inhibitor of CYP3A4 is therefore contraindicated in patients treated with apixaban [46]. Notably, like rivaroxaban, apixaban is able to inhibit both FXa bound to the prothrombinase complex as well as the circulating free enzyme [52]. Prophylaxis of venous thromboembolism Apixaban was first tested in a phase II, dose-finding study, in patients undergoing total knee replacement, the APROPOS Study [56]. A total of 1217 patients were randomized to apixaban at doses of 5 to 20 mg once daily or to enoxaparin (30 mg twice daily) or open-label warfarin (titrated to an INR of ). Treatment lasted days. The primary efficacy outcome of composite of VTE (as assessed by mandatory venography) and all-cause mortality during treatment was significantly lower in all apixaban groups compared with enoxaparin (P < 0.02) and warfarin-treated patients (P < 0.001). The rate of major bleeding in the apixaban group was dose-related and ranged from 0 to 3.3%. No major bleeding was observed in the two comparator arms. On the basis of the results of this study the dose of 2.5 mg twice daily was selected for the phase III prevention trials.the ADVANCE programme has evaluated the efficacy and safety of apixaban at a dose of 2.5 mg twice daily (started in the morning of the day after surgery) with enoxaparin in patients with knee or hip replacement (Table 2). The ADVANCE 1 is a double-blind, double-dummy study, that has compared apixaban 2.5 mg twice daily with enoxaparin (30 mg twice daily) in 3195 patients undergoing total knee replacement. Both medications were started 12 to 24 h after surgery and continued for 10 to 14 days. The primary efficacy outcome rate (a composite of asymptomatic and symptomatic DVT, as assessed by bilateral venography, nonfatal PE, and death from any cause) was not different between the two treatment groups (8.9% vs. 9.9% in apixaban and enoxaparin groups, respectively), whereas the incidence of major bleeding was 0.7% with apixaban and 1.4% with enoxaparin (P = 0.053). However, despite similar efficacy between apixaban and enoxaparin, because of the low overall events rates, apixaban did not meet the prespecified non-inferiority criteria [57]. The ADVANCE 2 trial, which included 3057 patients undergoing total knee replacement, compared the same apixaban regimen of ADVANCE 1, with enoxaparin at a dose of 40 mg once daily [58]. Interestingly, in this trial apixaban significantly reduced the incidence of total VTE compared with enoxaparin (15.1% vs. 24.4%, respectively, P = 0.019). The rate of major or clinically relevant non-major bleeding was lower but not statistically significant in the apixaban group (3.5% vs. 4.8%). Finally, the ADVANCE 3 study compared 5 weeks treatment with apixaban 2.5 mg twice daily with enoxaparin 40 mg once daily in 5407 patients undergoing total hip replacement. [59]. The rate of primary efficacy outcome (a composite of asymptomatic or symptomatic DVT, nonfatal PE or death from any cause) was significantly lower in the apixaban group compared with enoxaparin (1.4% vs. 3.9%, respectively, P < 0.001) thus satisfying both non-inferiority and superiority criteria. The incidence of the composite outcome of major and clinically relevant non-major bleeding was not different in the two treatment groups (4.8% vs. 5.0%, respectively). Apixaban is also being evaluated for thromboprophylaxis in medical patients. In the ADOPT trial, a 30 days regimen of apixaban 2.5 mg twice daily is being compared with enoxaparin 40 mg once daily in patients with an acute medical illness. The expected number of patients to be enroled is The study is currently recruiting, and the estimated completion date is May A subpopulation of cancer patients will be included in this study. Treatment of venous thromboembolism The efficacy and safety of apixaban in the treatment of patients with VTE was assessed in the phase II BOTTICELLI study [60]. Five hundred consecutive patients with confirmed symptomatic DVT were randomized to receive either apixaban 5 or 10 mg twice daily, or 20 mg once daily, or conventional therapy with LMWH or fondaparinux, followed by a VKA for 84 to 91 days. The primary efficacy outcome (a composite of symptomatic recurrent VTE and asymptomatic deterioration of bilateral compression ultrasound or perfusion lung scan) was observed in 4.2% of patients receiving conventional therapy and in 6.0% and 5.6% of patients allocated to apixaban 5 mg and 10 mg twice daily, respectively. The rate of the primary efficacy endpoint was lowest (2.6%) in patients treated with apixaban 20 mg.the rate of major and clinically relevant non-major bleeding was also similar between apixaban and conventional therapy. These results prompted further evaluation of apixaban in phase III studies: the AMPLIFY and AMPLIFY-EX clinical trials. The AMPLIFY study, still ongoing, has as primary endpoint the evaluation of apixaban in preventing the recurrence or death in patients with VTE, whereas the AMPLIFY-EX, has the same end point in patients who are 570 / 72:4 / Br J Clin Pharmacol

9 Anti-factor X and anti-factor V anticoagulants candidates for long term treatment with VKA. Both studies will stop the enrolment in Stroke prevention in atrial fibrillation Two studies have been designed to investigate the efficacy and safety of apixaban in stroke prevention in patients with AF. The ARISTOTLE phase III study is still ongoing, comparing apixaban 5 mg twice daily with warfarin (dose adjusted to achieve an INR of 2.0 to 3.0) in patients with AF and at least one additional risk factor for stroke.the AVERROES trial, is a phase III, randomized, double-blind study, which compared the same apixaban dose (5 mg twice daily) with aspirin (81 to 324 mg day -1 ) in 5599 patients with AF who were unsuitable for, or intolerant of, VKA treatment (Table 2).The primary outcome was the occurrence of stroke or systemic embolism. The study was designed in order to determine whether apixaban was superior to aspirin. The study was prematurely concluded because of a clear benefit in favour of apixaban, and data were recently published [61]. Treatment with apixaban significantly reduced the risk of stroke or systemic embolic events by 54% compared with aspirin. The incidence of primary outcome was 1.6% per year among patients assigned to apixaban and 3.7% per year among those assigned to aspirin (P < 0.001).The benefit of treatment, however, occurred without a corresponding increase in the risk of bleeding. More specifically, the incidence of major bleeding was 1.4% and 1.2% per year in the apixaban and the aspirin group, respectively (P = 0.57) with 11 cases of intracranial bleeding with apixaban and 13 with aspirin. Interestingly, the risk of first hospitalization for cardiovascular causes was reduced with apixaban as compared with aspirin (12.6% vs. 15.5% per year, P < 0.001). These results clearly indicate that in patients with AF for whom treatment with VKA is unsuitable, apixaban might be an effective option to VKA in reducing risk of stroke or systemic embolism without significantly increasing the risk of major or intracranial bleeding. Treatment of acute coronary syndromes The APPRAISE study was a phase II, double-blind, placebo-controlled, dose-ranging clinical trial. Patients (1715) with recent ST-elevation or non-st-elevation ACS were randomized to 6 months of placebo or to one of four doses of apixaban: 2.5 mg twice daily, 10 mg once daily, 10 mg twice daily or 20 mg once daily. Nearly all patients received aspirin and 76% received clopidogrel.the primary outcome was major or clinically relevant non-major bleeding. A secondary outcome was a composite of cardiovascular death, myocardial infarction, severe recurrent ischaemia, and ischaemic stroke. At the recommendation of the Data Monitoring Committee, the two highest doses of apixaban were discontinued because of excess total bleeding in patients receiving apixaban plus dual antiplatelet therapy [62]. However, apixaban 2.5 mg twice daily resulted in lower rates of ischaemic events compared with placebo. The increase in bleeding was more pronounced and the reduction in ischaemic events was less evident in patients taking aspirin plus clopidogrel than in those taking aspirin alone. Further investigation of apixaban in patients at risk of recurrent ischaemic events is therefore warranted. Edoxaban Edoxaban (DU-176b), is an orally active drug, rapidly absorbed from the gastrointestinal tract,with a time to peak concentration of 1 2 h and a half-life of 9 11 h [63]. The drug is eliminated by a dual mechanism. About one third is excreted by the kidney and the reminder in the faeces. Edoxaban has been studied in a phase II trial in VTE prophylaxis and in stroke prevention in patients with AF. The phase II study in AF was undertaken to evaluate the safety and efficacy of the drug in Asian patients with AF and and at least one additional risk factor for stroke [63]. In a multicentre, active-controlled, double-blind edoxaban and open-label warfarin trial, a total of 235 patients from four Asian countries were randomly assigned to edoxaban 30 mg once daily, 60 mg once daily or warfarin doseadjusted to INR of 2.0 to 3.0 for 3 months. The primary endpoint was the incidence of bleeding events (major,clinically relevant non-major and minor). Secondary endpoints were thromboembolic events.the incidence of all bleeding events was 20.3% for 30 mg and 23.8% for 60 mg edoxaban, and 29.3% for warfarin,thus suggesting a trend for a reduction in the incidence of all bleeding events in AF patients treated with edoxaban compared with warfarin. Based on these results, the double-blind, double-dummy phase III, ENGAGE-AF-TIMI 48 trial, was initiated. The study, was designed to compare two doses of edoxaban (30 and 60 mg once daily) with conventional therapy with VKA in patients with AF. The study is still ongoing and no data are available at the moment. Other factor Xa inhibitors Other FXa inhibitors under development include drugs able to antagonize indirectly or directly activated FX. Some of these agents, briefly summarized in this review, are for parenteral use whereas other are administered orally. Semuloparin Semuloparin (AVE5026) is a new ultra low molecular weight heparin (ULMWH), with a molecular weight of 2400 Da. It has an indirect anti-fxa activity, with a ratio anti-fxa : anti-fiia higher than 30 and a half-life of h thus allowing once daily subcutaneous administration [64, 65]. A high anti-fxa to anti-fiia activity is associated with a high antithrombotic effect and a low prohaemorrhagic tendency. After a single subcutaneous dose AVE5026 is completely absorbed with a bioavailability of 98% compared with intravenous administration. The drug has a linear pharmacokinetic profile and after repeated dosing in elderly subjects a slight increase in accumulation of this agent was seen reflecting renal impairment [62]. In the phase II, parallel-group, double-blind, double-dummy, TREK study, 690 patients undergoing total knee replacement surgery, were randomized to receive a once daily Br J Clin Pharmacol / 72:4 / 571

10 V. Toschi & M. Lettino dose of AVE5026 (5, 10, 20, 40 or 60 mg) or enoxaparin 40 mg [66]. The primary efficacy end point was VTE until post-operative day 11, defined as DVT (objectively assessed, either symptomatic or asymptomatic), non-fatal PE and VTE-related death. The primary safety end point was the incidence of major and any bleeding.the results of the study demonstrated a significant dose response across the five AVE5026 doses for VTE prevention (P < ). The incidence of events was 40%, 44.1%, 15.6%, 13.6% and 5.3% for the AVE5026 subcutaneous doses of 5, 10, 20, 40 and 60 mg, respectively.the VTE rate in the enoxaparin group was 35.8%. A statistically significant dose response was also found for total bleeding events (P < ) and for major bleeding (P < 0.023) with rates ranging from 3.8% to 20.5% for the five AVE5026 dosing arms, compared with 5% in the enoxaparin group. A largescale phase III programme, the SAVE programme, which planned to enrol more than patients, has been carried on and already completed in knee and hip surgery, in abdominal surgery, in patients with cancer undergoing chemotherapy and in acutely ill patients. The results of these trials are not yet published though preliminary data indicate that semuloparin (20 mg once daily) proved superior to enoxaparin (40 mg once daily) in total hip replacement for total VTE and clinically relevant bleeding, and also to placebo for extended VTE prophylaxis in hip fracture surgery [67]. However, results on both efficacy and safety were inconclusive in total knee replacement and in hip fracture surgery compared with enoxaparin. A metaanalysis of three studies involving 4479 patients undergoing knee and hip surgery [67] suggested that semuloparin was superior in terms of efficacy (total VTE plus total death) compared with enoxaparin with a similar rate of clinically relevant bleeding.the superiority of semuloparin vs. enoxaparin was not confirmed after excluding asymptomatic distal DVT. M118 The indirect FXa inhibitor M118 is a new LMWH with a molecular weight of 6500 Da and a predominant anti-fxa activity (ratio anti-fxa : anti-fiia = 1.4). M118 has been specifically designed for use in ACS. The drug shows broad anticoagulant activity, including potent activity against both FXa (~240 IU ml -1 ) and thrombin (FIIa ~ 170 IU ml -1 ). M118 has a high bioavailability (78%) after subcutaneous administration, and predictable pharmacokinetics after subcutaneous and intravenous administration [68]. Other advantages of the drug are a half-life of approximately 1 h after intravenous bolus injection and 2 3 h after subcutaneous administration. Additionally, M118 seems not to activate platelets, its activity is monitorable by standard coagulation assays (anti-fxa, anti-fiia, APTT, and ACT) and is reversible with protamine sulfate (1 mg per 100 IU dose). For all these reasons the drug demonstrates potentially superior properties to conventional LMWHs [68, 69]. M118 has been tested in a phase II multicentre study [69] which included 503 patients undergoing PCI who were allocated in an open-label fashion to one of the following four arms: UFH 70 U kg -1 or M118 50, 75 or 100 IU kg -1. In all four arms the drug was administered by intravenous injection. The primary outcome (composite of death, myocardial infarction, repeat revascularization, stroke, thrombocytopenia, catheter thrombus, bailout use of glycoprotein IIb/IIIa inhibitor or any bleeding through day 30) occurred in 31.1% of patients randomized to UFH and in 22.7%, 28.3% and 30.1% of patients randomized to the M118 doses of 50, 75 and 100 IU kg -1, respectively. The adverse events profiles of M118 and UFH were comparable. The results of this study demonstrate that M118 is well tolerated and suitable to be used as an anticoagulant in patients undergoing elective PCI, though further studies are necessary to confirm its role in the treatment of patients with ACS. Betrixaban Betrixaban (PRT ), is an oral, direct, competitive, active site-directed, inhibitor of FXa that is able to block both free and prothrombinase bound FXa [70].The drug has a number of favourable pharmacokinetic and pharmacodynamic characteristics. It has a much higher specificity for FXa than for other coagulation proteins such as thrombin, which reduces its prohaemorrhagic potential. Betrixaban has an almost exclusive biliary excretion with a low renal clearance, corresponding to a urine excretion of about 5% of the administered dose. It can therefore be used in patients with renal impairment. Moreover the drug does not interact with the major cytochrome CYP450, and for this reason is has little or no interaction which other drugs. Finally, betrixaban has a half-life of about h therefore allowing patients to be maintained within the therapeutic range over the 24 h dosing period [71]. The drug has been tested in a phase II study, the EXPERT study, in 215 patients undergoing elective total knee replacement [72]. In the study patients were randomized to receive post-operatively betrixaban 15 or 40 mg orally twice daily or enoxaparin 30 mg subcutaneously every 12 h, respectively, for days. The betrixaban dosage was blind but enoxaparin was not. The incidence of the primary efficacy outcome, consisting of DVT (as assessed by mandatory venography), symptomatic proximal DVT or PE, was 20%, 15% and 10%, respectively, for betrixaban 15 mg, betrixaban 40 mg and enoxaparin. As far as safety outcomes are concerned, no bleeds were reported for betrixaban 15 mg, whereas clinically significant non-major bleeding was not different in the betriban 40 mg and enoxaparin groups (2.4% vs. 4.6%, respectively). One major bleed (2.3%) was observed in enoxaparin arm. These data indicate that betrixaban has a favourable efficacy and safety profile and therefore it may be an optimal alternative to LMWHs for VTE prophylaxis in patients undergoing major orthopaedic surgery. Betrixaban was also evaluated in patients with AF. A phase II study, EXPLORE-Xa, comparing the efficacy and safety of betrixaban vs. warfarin in AF patients has been completed and 572 / 72:4 / Br J Clin Pharmacol