Oral FXa Inhibitor Research Articles

Reversal of Heparin-Induced Anticoagulation By Andexanet Alfa, a Universal Antidote for Factor Xa Inhibitors

Background: Unfractionated heparin is used in the management of venous thromboembolism, stroke prevention in atrial fibrillation as well as in cardiac surgeries (e.g., valve replacement and other procedures requiring cardiopulmonary bypass). Bleeding is the major complication that may result from these therapies. Although protamine sulfate can be used to neutralize heparin, its administration can cause severe hypotensive and anaphylactic reactions. Andexanet alfa (AnXa) is a recombinant modified human fXa under development for the reversal of both direct and antithrombin III (ATIII)-dependent indirect fXa inhibitors. We previously reported nonclinical and Phase 2 clinical data in healthy subjects with enoxaparin, a low molecular weight heparin (LMWH). Here we report new in vitro data demonstrating that AnXa can also effectively reverse anticoagulation of unfractionated heparin.Methods: Inhibition of human fXa or thrombin (IIa) by ATIII in the presence of heparin was studied in an enzymatic assay in buffered solution. The reaction mixture contained fXa or IIa (20 nM), ATIII (200 nM), different amounts of unfractionated heparin and AnXa. At different time points, a small aliquot of the reaction mixture was taken and quenched into a 96-well assay plate containing protamine (50 µg/mL) and a chromogenic substrate (100 µM) for fXa (Spectrozyme-Xa) or IIa (S2238). Residual fXa or IIa activity was measured in a plate reader by monitoring the initial rate of substrate cleavage at 405 nm. Reversal of heparin anti-fXa and anti-IIa activities by AnXa in human plasma was evaluated using modified anti-fXa and anti-IIa chromogenic assays. Reversal of heparin-induced clotting prolongation of human plasma was measured by a 96-well format turbidity assay using an aPTT reagent and Ca2+. The reversal effect of AnXa on heparin anticoagulation and clot formation was further characterized by thromboelastography (TEG) in human plasma or whole blood. Reversal of heparin was compared to enoxaparin. Protamine sulfate was included as a control. Di-arginine piperazine (PER977), a positively charged small molecule, was also tested for comparison.Results: AnXa dose-dependently reversed the anti-fXa activity of heparin in the enzymatic assay and in human plasma. Interestingly, AnXa was also able to reverse the heparin anti-IIa activity in the same assays. In the enzymatic assay with IIa and ATIII, heparin accelerated the reaction of IIa inhibition by ~1000-fold. AnXa dose-dependently reversed the IIa inhibition to its basal level seen without heparin. The reversal effect of AnXa on IIa inhibition could be blocked by addition of a small molecule direct fXa inhibitor (e.g. rivaroxaban) in the same enzymatic assay. This suggests that interaction of AnXa with the primary binding site on the surface of heparin-activated ATIII (i.e., the reactive center loop (RCL)) might play a major role in the anti-IIa reversal as binding to the RCL was the minimal requirement for the inhibition of both fXa and IIa by ATIII. In human plasma, AnXa reversed the anti-IIa activity of heparin (0.8 IU/mL) with an estimated EC50 comparable to anti-fXa reversal (anti-fXa:126 nM; anti-IIa: 131nM). These results indicate that AnXa was able to compete with not only fXa, but also IIa, for binding to heparinized-ATIII and reverse both the anti-fXa and anti-IIa activities of heparin. In addition, AnXa dose-dependently and completely reversed prolongation of clotting by heparin (0.8 IU/mL) in human plasma as measured by the turbidity assay as well as parameters of heparin (1.0 IU/mL) anticoagulation as measured by TEG in either plasma or whole blood. As expected, protamine sulfate (5-10 µg/mL) neutralized heparin (1.0 IU/mL) anticoagulation in plasma in the TEG assay. At higher concentrations, protamine prevented clot formation, consistent with protamine's known anticoagulant effect when overdosed. No reversal activity was observed by PER977 in the same TEG assays using either plasma or whole blood.Conclusions: The current and previously reported data demonstrate that AnXa could be a universal fXa inhibitor antidote to reverse a broad range of newer direct oral fXa inhibitors, ATIII-dependent indirect inhibitors, such as fondaparinux and enoxaparin, as well as unfractionated heparin. The mechanism for reversal of ATIII-dependent inhibitors by AnXa is distinct from other positively charged molecules that bind directly to heparin. DisclosuresLu:Portola Pharmaceuticals, Inc.: Employment. Lin:Portola Pharmaceuticals, Inc.: Employment. Curnutte:Sea Lane Biotechnologies: Consultancy; 3-V Biosciences: Equity Ownership; Portola Pharmaceuticals, Inc.: Employment. Conley:Portola Pharmaceuticals, Inc.: Employment.

Engineered Factor Xa Variants Retain Procoagulant Activity Independent of Direct Factor Xa-Inhibitors

The venom of the Australian Elapid snake Pseudonaja textilis contains a prothrombin-activating complex that consists of factor Xa (FXa) and factor Va (FVa) homologs which are evolutionary adapted to derail the hemostatic system of its prey, leading to runaway coagulation. These adaptations include functional resistance to inactivation by the main inhibitors of coagulation, antithrombin and activated protein C. Further studies revealed that venom FXa, unlike other FXa species, is also resistant to inhibition by direct oral FXa-inhibitors such as rivaroxaban and apixaban (Ki >1000 nM for venom FXa vs. 1 nM for human FXa). Accordingly, venom FXa is able to support thrombin generation (TG) in FX-depleted plasma spiked with pharmacological concentrations (0.4-2 μM) of these FXa-inhibitors. While human FXa-initiated TG resulted in a 8-fold prolonged lag time and a 70% reduced thrombin peak, those parameters were within normal range in venom FXa-triggered TG.Venom FX homologs produced by Elapid snakes comprise a heterogeneous insertion between His91-Tyr99 (chymotrypsin numbering) in the serine protease domain. A recent crystal structure of one of these homologs shows that this insertion is in close proximity of the active site pocket. In contrast, P. textilis liver-derived plasma FX, which, when activated, is fully inhibited by the FXa-inhibitors (Ki10 nM), lacks this structural feature. We investigated whether the His91-Tyr99 insertion is at the basis of the reduced sensitivity of venom FXa towards FXa-inhibitors. To do so, we constructed and stably expressed human-snake FX chimeras (FX-A, -B, -C) that incorporate His91-Tyr99 insertions from three venom FXa homologs. The chimeric FX variants were purified by successive ion-exchange and hydrophobic interaction chromatography steps, and FXa was generated following RVV-X-activation and size-exclusion chromatography.Evaluation of the kinetic parameters of prothrombin conversion in the presence of saturating amounts of FVa and anionic phospholipids revealed that the chimeric FXa variants exhibit an up to ~4-fold enhanced affinity for prothrombin (Km 0.11-0.29 μM) as compared to recombinant human FXa (rhFXa; Km 0.41 μM). The rate of prothrombin activation was 3-10-fold reduced (kcat 118-370 min-1 vs. 1243 min-1 for rhFXa), which may be indicative of a modified active site conformation. Consistent with this, the rate of chimeric FXa inhibition by antithrombin was impaired (kapp 0.12-0.95 x 103 M-1 s-1 vs. 4.07 x 103 M-1 s-1 for rhFXa). Furthermore, the variant that was most poorly inhibited by antithrombin (variant A) also exhibited the lowest catalytic rate of prothrombin conversion and vice versa (variant C). Conversely, apixaban or edoxaban inhibition of the FXa variants assembled into prothrombinase led to the highest Ki for chimeric variant C (2.3 or 0.3 µM), followed by variants B (1.4 or 0.2 µM), and A (0.2 or 0.006 µM) compared to rhFXa (0.004 or 0.0005 µM). Evaluation of the inhibition of uncomplexed FXa variants employing peptidyl substrate conversion revealed a similar decrease in sensitivity to the FXa-inhibitors (≤500-fold). These data suggest that insertion of the snake venom His91-Tyr99 regions indeed results in impaired engagement of the FXa active site pocket.We next assessed whether chimeric variant C, which is most resistant to inhibition by the direct FXa-inhibitors, is able to restore thrombin generation in a plasma system in the presence of apixaban or edoxaban. While rhFXa-triggered (5 nM) thrombin formation in FX-depleted plasma was inhibited by 2 μM apixaban, initiation with FXa-C (5 nM) resulted in normal thrombin generation parameters (peak thrombin 98%). In addition, the zymogen form of variant C also supported tissue factor-initiated (2 pM) thrombin generation in FX-depleted plasma with inhibitor concentrations up to 6 μM (apixaban) or 2 μM (edoxaban). Under these conditions, little if any thrombin was formed with rhFX present (peak thrombin 5%). We obtained similar results when performing these experiments in normal pooled plasma.Taken together, these results show that chimeric FX is able to restore hemostasis in plasma inhibited by the direct FXa-inhibitors, both in the zymogen as well as protease state. As such, these variants have the potential to serve as rescue therapeutic agents to overcome the effect of FXa-inhibitors in case of potential life-threatening bleeding events or emergency surgical interventions. DisclosuresBos:Bayer Hemophilia Awards: Research Funding.

Reversal of anticoagulants: an overview of current developments.

Several new anticoagulants have entered the clinical arena or are under clinical development. These drugs include indirect (fondaparinux) and direct oral factor Xa inhibitors (rivaroxaban, apixaban, edoxaban, betrixaban), and the direct thrombin inhibitor dabigatran. Especially the oral direct FXa and FIIa inhibitors overcome many of the shortcomings of heparins and vitamin K antagonists (VKAs). They are administered orally at a fixed dose; regular monitoring is not necessary; interaction with other drugs or nutrition occur less than with VKAs and they are at least as effective as VKAs for most indications tested. They are associated with about 50 % less intracranial bleeding than VKAs. Nevertheless, they are still associated with bleeding complications. Bleeding can occur spontaneously or as a result of trauma or urgent surgery. In such situations rapid reversal of the anticoagulant effect is highly desirable. For unfractionated heparin protamine, and for VKAs prothrombin complex concentrates are available as specific antidotes. Under clinical development are: for the direct and indirect FXa inhibitors a modified recombinant FXa (andexanet alpha), which lacks enzymatic activity; and for dabigatran a Fab fragment of a monoclonal antibody (idarucizumab). In addition a small molecule (aripazine) has entered phase I clinical trials, which seems to inhibit nearly all anticoagulants but VKAs and argatroban. This review summarises the current options and strategies in development to antagonise anticoagulants with a focus on the status of the development of antidotes for the oral direct FXa and FIIa inhibitors.

Direct-acting oral anticoagulants: pharmacology, indications, management, and future perspectives.

In recent years, several direct-acting oral anticoagulants (DOAC) have become available for use in Europe and other regions in indications related to prophylaxis and treatment of venous and arterial thromboembolism. They include the oral direct thrombin inhibitor dabigatran etexilate (Pradaxa, Boehringer Ingelheim) and the oral direct FXa inhibitors rivaroxaban (Xarelto, Bayer HealthCare), apixaban (Eliquis, Bristol-Myers Squibb), and edoxaban (Lixiana/Savaysa, Daiichi-Sankyo). The new compounds have a predictable dose response and few drug-drug interactions (unlike vitamin k antagonists), and they do not require parenteral administration (unlike heparins). However, they accumulate in patients with renal impairment, lack widely available monitoring tests for measuring its anticoagulant activity, and no specific antidotes for neutralization in case of overdose and/or severe bleeding are currently available. In this review, we describe the pharmacology of the DOAC, the efficacy, and safety data from pivotal studies that support their currently approved indications and discuss the postmarketing experience available. We also summarize practical recommendations to ensure an appropriate use of the DOAC according to existing data. Finally, we discuss relevant ongoing studies and future perspectives.

Anticoagulant dosing in renal impairment

SummaryAnticoagulants are widely used for prophylaxis and treatment of venous thromboembolism in the elderly, who commonly have renal impairment and other comorbidities. Renal impairment is a risk factor for bleeding and thrombosis during anticoagulant therapy and can influence the balance between the safety and efficacy of such agents. Some anticoagulants, such as fondaparinux and the direct acting oral thrombin inhibitor dabigatran etexilate are contraindicated for use in patients with severe renal impairment (eGFR <30 ml/min). However, also the direct acting oral FXa-inhibitors rivaroxaban, edoxaban and apixaban need caution regarding dosing advice or contraindications when used in patients with renal impairment.

Venous thromboembolism rates in patients undergoing major hip and knee joint surgery at Waitemata District Health Board: a retrospective audit.

Symptomatic venous thromboembolism (VTE) complicates approximately 4% of major orthopaedic surgical procedures performed without thromboprophylaxis. Randomised clinical trials demonstrate primary thromboprophylaxis reduces VTE rates to <1%, with low rates of clinically important bleeding, using low molecular weight heparin (LMWH), oral FXa inhibitors or thrombin inhibitors. We reviewed the rates of VTE in patients undergoing major hip/knee joint surgery at Waitemata District Health Board (WDHB). Cases of VTE within 90 days of orthopaedic surgery were identified by retrospective audit of data from the haematology VTE database. The number of major hip/knee joint surgeries at WDHB from January 2006 to December 2010 was obtained from clinical coding data. The cumulative incidence of VTE within 90 days of surgery was 3.29%. The median time from surgery to diagnosis was 7 days. Deep vein thrombosis comprised 75% of cases, 77.6% distal and 23.2% proximal. Pulmonary embolism comprised 26.5% of VTE; 47.7% had right heart strain on computed tomography/echocardiography. Hip fracture surgery comprised one-third of patients. Of patients developing VTE, 85.5% had chemical thromboprophylaxis - aspirin 73%, LMWH 20 mg 16%, LMWH 40 mg 16%, therapeutic LMWH 3%, unfractionated heparin twice daily 1%, and warfarin 4%; 75.6% received mechanical prophylaxis, while 4% of patients received no prophylaxis. VTE incidence after major hip/knee joint surgery at WDHB is high, with pulmonary embolism comprising almost one-third of all VTE in this study, indicating the prophylaxis given is suboptimal. Implementation of appropriate, extended duration prophylaxis as per evidence-based guidelines is required to reduce these rates.

The effects of prevention of VTE using oral direct FXa inhibitor after total hip arthroplasty

The effects of prevention of VTE using oral direct FXa inhibitor after total hip arthroplasty

Abstract 18218: In Vitro Characterization of Andexanet Alfa (PRT064445), a Specific fXa Inhibitor Antidote versus Aripazine (PER977), a Non-specific Reversal Agent

New oral fXa inhibitors have been increasingly adopted for VTE or AF treatment in the outpatient setting instead of warfarin. Andexanet alfa (AnXa) is a modified, recombinant human fXa molecule developed as a specific antidote to reverse anticoagulant activity of fXa inhibitors during episodes of serious bleeding or before urgent surgery. PER977, a small molecule under development by Perosphere, Inc., is reported to reverse the effect of a broad range of anticoagulants (fXa and thrombin inhibitors, LMWH). In order to compare AnXa and PER977 mechanisms of action, we studied the in vitro activity of both agents in the presence or absence of fXa inhibitors rivaroxaban, apixaban, edoxaban or enoxaparin. In a buffer system containing purified human fXa with physiologic Ca 2+ (5 mM), AnXa dose-dependently reversed oral fXa inhibitor activity. Reversal activity was not observed for any fXa inhibitor with PER977 over a wide concentration range (≤2 mM). In human plasma, AnXa reversed both direct and ATIII-dependent fXa inhibitors, whereas no reversal effect by PER977 was detected. In the absence of a fXa inhibitor, PER977 potentiated fX activation by fIXa in a buffer system (purified human fX, fIXa and 5mM Ca 2+ ). Similar cofactor activity was observed with polylysine, indicating that PER977 may have properties similar to poly-cationic molecules. Procoagulant activity was observed in human plasma with PER977 at low concentrations (≤100 μM) as measured by clotting (aPTT) or thrombin generation, but inhibition in these assays was seen at higher concentrations ( ~1 mM). PER977 also potentiated human platelet activation as determined by P-selectin expression induced by 10 μM ADP. Platelet aggregation, whole blood hemolysis, or complement activation testing showed no effect with either AnXa or PER977. These data indicate that PER977 does not reverse the anticoagulant activity of a direct or indirect fXa inhibitor in vitro. Its observed in vivo effect on blood loss in animals may not be mediated by direct interaction of PER977 with the inhibitor, but may be in part attributed to an off-target effect, suggested by its potential procoagulant activity. In contrast, AnXa acts as a specific antidote to fXa inhibitors by sequestering them with a defined stoichiometry (1:1 molar ratio).

Abstract 14657: Andexanet-alfa and PER977 (Arapazine) Correct Blood Loss in a Rabbit Liver Laceration Model - Only Andexanet Reverses Markers of fXa-mediated Anticoagulation

As use of oral fXa inhibitors has increased, so has the need for an antidote to treat the major side effect of these drugs - bleeding. Andexanet alfa (AnXa) (PRT064445), a recombinant fXa derivative, reverses fXa inhibitor-mediated anticoagulation in animals and humans by sequestering the fXa inhibitor in a 1:1 molar ratio, reducing both anti-fXa activity and plasma free fraction of fXa inhibitor, and restoring normal thrombin generation, PT, and aPTT. These parameters correlate closely with reduced blood loss in a rivaroxaban-treated rabbit liver laceration model. Aripazine (PER) (PER977; Perosphere, Inc.) is a small molecule that has been reported to reverse the anticoagulant effects of a broad range of anticoagulants (fXa and thrombin inhibitors; heparins) by direct binding. We synthesized PER and compared it to AnXa in this same liver laceration model at PER doses of 0.3, 3 and 30 mg/kg, and observed reductions in blood loss of 27%, 16% and 76%, respectively ( p &lt;0.02 for 30 mg/kg dose only). There was an unexpected dose-responsive, but non-significant, trend toward decreased blood loss in non-anticoagulated rabbits with PER alone, with the 30 mg/kg dose showing a 23% decrease. The reduction in blood loss between the two agents was similar at the highest dose studied (76% decrease for 75 mg AnXa/rabbit [ASH 2012] and 76% decrease for 30 mg/kg PER in this study). PER dose groups, however, did not show reduction in riva-induced increases in PT, aPTT or anti-fXa activity. Moreover, there was no change in total plasma concentration of riva after PER administration compared to the 6-fold increase seen with AnXa due to its high affinity for riva and redistribution of riva from the extravascular to the intravascular compartment. Unlike the ~1:1 molar ratio needed for AnXa to sequester riva, a 30:1 PER:Riva molar ratio was needed to reduce blood loss. While the mechanism of action (MOA) for AnXa has been well characterized in preclinical and clinical studies, the mechanism of prevention of blood loss with PER in this model is unclear. In summary, the MOA of PER differs from that of AnXa, where direct (1:1) binding of AnXa to the fXa inhibitor decreases the free fraction of the anticoagulant, reverses PD markers and restores normal hemostasis. Further investigation of PER is required.

Comparison of the effect of edoxaban, a direct factor Xa inhibitor, with a direct thrombin inhibitor, melagatran, and heparin on intracerebral hemorrhage induced by collagenase in rats

Intracerebral hemorrhage (ICH) is a major clinical concern with anticoagulation therapy. The effect of a new oral direct FXa inhibitor, edoxaban, was determined in a rat model of ICH and compared with a direct thrombin inhibitor, melagatran, and heparin. To induce ICH, 0.1 U collagenase type VII was injected into the striatum of male Wistar rats under anesthesia with thiopental or halothane. Immediately after ICH induction, edoxaban, melagatran, or heparin were infused intravenously. Five hours after ICH induction, the brain was removed and ICH size was measured. To estimate the margin of safety, antithrombotic effects were evaluated in a rat venous thrombosis model. Edoxaban at 6mg/kg/h significantly increased ICH volume (1.8-fold) and prolonged prothrombin time (PT) 2.8-fold compared to the vehicle group. No deaths were observed with edoxaban. Melagatran at 1mg/kg/h increased ICH volume at 1mg/kg/h (2.8-fold) with 6.1-fold PT prolongation. At 3mg/kg/h, all rats died due to severe ICH (3.9-fold). Heparin at both 100 and 500U/kg/h significantly increased ICH. At 500U/kg/h, 5 out of 8 rats died. The doses required for 50% inhibition of thrombosis of edoxaban, melagatran, and heparin were 0.045mg/kg/h, 0.14mg/kg/h, and 55U/kg/h, respectively. The safety margins between antithrombotic and ICH exacerbation effects of these anticoagulants were 133, 7.1, and 1.8, respectively. The safety margin of edoxaban was wider than that of melagatran or heparin. These results suggest that edoxaban may be preferable from the perspective of ICH exacerbation risk.

Evaluation of edoxaban in patients with atrial fibrillation and severe renal impairment

Purpose: Compare safety and pharmacokinetics of edoxaban (EDX) 15 mg, an oral selective FXa inhibitor, in patients with nonvalvular atrial fibrillation (NVAF) and severe renal impairment (SRI; creatinine clearance [CLCR] ≥15 to <30 mL/min) to that of EDX 60 or 30 mg in NVAF patients with normal renal function or mild renal impairment (N/MiRI; CLCR ≥50 mL/min). Methods: Patients with NVAF and SRI or N/MiRI were enrolled in a randomized, open-label study in Japan. Patients requiring hemodialysis, at high risk for bleeding, or already receiving an anticoagulant (except warfarin, rivaroxaban, or dabigatran) were excluded. SRI patients received EDX 15 mg. N/MiRI patients were randomized to either EDX 60 or 30 mg. N/MiRI patients weighing ≤60 kg or receiving concomitant treatment with quinidine or verapamil had a 50% dose reduction. EDX was administered once daily for 12 wks. Blood samples to assess plasma EDX concentrations and prothrombin time (PT) were taken at wk 2 and 8. Bleeding and adverse events (AEs) were monitored and recorded throughout the study. Results: Of 93 patients enrolled, 50 had SRI and 43 N/MiRI (21 EDX 60 mg and 22 EDX 30 mg). In the EDX 15, 60, and 30 mg groups, median CLCR was 26.3, 62.5, and 64.4 mL/min, respectively. Safety outcomes and plasma concentrations at wk 8 are provided in the Table. No major bleeding events occurred in any treatment group. No serious AE was considered related to study drug. Median PT ratios were similar between the SRI and N/MiRI 30mg groups at all time points. View this table: Bleeding, AEs, and plasma concentrations Conclusions: These results indicate that EDX 15 mg may be an appropriate therapeutic option in SRI patients with AF.

The direct factor Xa inhibitor Rivaroxaban reduces platelet activation in congestive heart failure

BackgroundPlatelet activation in congestive heart failure (CHF) contributes to an increased risk for thromboembolic complications. Rivaroxaban, the first oral direct FXa inhibitor is approved in Europe for prevention and treatment of venous thrombosis, pulmonary embolism, and prevention of thromboembolic events in atrial fibrillation. As heart failure is an important risk factor for thromboembolism and increased platelet activation is common in heart failure, we investigated the potential effect of Rivaroxaban treatment on platelets in an experimental CHF model. Methods and resultsChronic myocardial infarction was induced in male Wistar rats by coronary ligation. Rats were randomized to placebo or Rivaroxaban (3 and 10mg/kg once daily). After 10 weeks platelet activation was assessed. Platelet-bound fibrinogen, detected by flow-cytometry, was significantly increased in CHF-Placebo (p<0.05) and reduced following treatment with Rivaroxaban (p<0.05 vs. CHF-Placebo). ADP-induced aggregation was significantly enhanced in CHF-Placebo vs. sham-operated animals (p<0.05) and normalized following chronic FXa inhibition (p<0.05 vs. CHF-Placebo). In separate in vitro experiments, attenuated platelet aggregation was present after incubating whole blood directly with Rivaroxaban but absent when the experiment was performed in platelet-rich plasma only. Thus, a direct effect on platelets could be excluded. ConclusionChronic direct factor Xa inhibition using Rivaroxaban reduces platelet activation in CHF rats by attenuating the secondary phase of ADP-induced platelet aggregation. Thus, Rivaroxaban may constitute a useful approach to prevent thromboembolic complications and reduce platelet activation in CHF at the same time.

Validation of the Use of Apixaban As an Alternate Anticoagulant for the Management of Patients with Heparin-Induced Thrombocytopenia.

Abstract 2265 Background:Heparin-induced thrombocytopenia (HIT), an immune mediated disorder due to antibodies generated against platelet factor 4 (PF4) complexed with heparin, is associated with a pronounced hypercoagulable state, thrombin generation, endothelial cell damage, and upregulation of an inflammatory state. Alternative anticoagulants that do not interact with HIT antibodies are needed for the anticoagulant management of these heparin compromised patients. Intravenous use direct thrombin inhibitors (DTIs) such as argatroban and lepirudin became the first effective non-heparin anticoagulant drug treatments for patients with HIT. Although DTIs are effective, their use is associated with a bleeding risk and drug-specific limitations. For the long-term anticoagulation, patients are switched from the intravenous DTI to oral warfarin. Aside from the unpredictable pharmacokinetics and need for routine monitoring, the slow onset of action and potential to precipitate venous limb gangrene/skin necrosis due to inhibition of protein C are concerning aspects of warfarin treatment of patients with HIT. Apixaban is a new small molecule, direct acting oral FXa inhibitor that may be considered for the anticoagulant management of patients with HIT. Methods:In order to determine if there is a lack of functional platelet activation and platelet aggregation for apixaban in the presence of HIT antibodies, the two traditional and widely used clinical laboratory tests for the diagnosis of HIT were utilized: the gold standard 14C-Serotonin Release Assay (14C-SRA; using washed platelets) and the heparin-induced platelet aggregation assay (PA-HIT; using platelet rich plasma). Based on different methodologies, these assays have different specificities and sensitivities to HIT antibodies and provide different yet complimentary information. This study employed HIT antibodies from multiple patients and platelets from different donors to assure the robustness of the data outcome. The response to apixaban concentrations covering the clinical dose range (0.05 to 50 mg/mL) was compared to the response obtained with clinically relevant concentrations of unfractionated heparin (UFH; 0.1 and 100 U/mL). Results:In the 14C-SRA (n=35) and in the PA-HIT (n=37), only baseline negative platelet activation and aggregation responses with all of the HIT specimens were observed at all apixaban concentrations (average across all concentrations: 11 ± 4 % serotonin release and 8 ± 3 % aggregation, respectively; mean ± SEM; positive responses are >20%). In comparison, UFH gave strong positive responses to each of the same HIT antibody specimen/platelet donor combinations (82 ± 3 % release and 78 ± 6 % aggregation at 0.1 U/mL; p<0.01 vs apixaban). Comparative studies demonstrated strong responses for enoxaparin (n=10; 73 ± 5 % release and 62 ± 7 % aggregation at 10 mg/mL) equal to UFH and no positive response for fondaparinux (n=20) in both test systems. Conclusions:This study confirms a consistent absence of platelet activation and platelet aggregation with apixaban in the presence of HIT antibodies across a wide range of concentrations. Based on the inert response of apixaban in these in vitro studies and because it is structurally unrelated to heparin, apixaban is not expected to contribute to the propagation of the HIT syndrome and could potentially be used for the anticoagulant management of patients with HIT. Since apixaban is a potent inhibitor of thrombin generation it is expected to have an additional benefit in blunting the hypercoagulable state which is observed in the HIT syndrome. Apixaban may provide an option for oral anticoagulation in patients with HIT both for in-hospital and out-patient settings, for the extended management of heparin compromised patients, and for the prevention of HIT. Clinical trials to determine dosing regimens that provide safe and effective anticoagulation during the various clinical phases of HIT are warranted. Disclosures:Walenga:Bristol-Myer Squibb: Research Funding. Prechel:Bristol-Myer Suibb: Research Funding. Hoppensteadt:Bristol-Myer Squibb: Research Funding. Escalante:Bristol-Myer Squibb: Research Funding. Chaudhry:Bristol-Myer Squibb: Research Funding. Jeske:Bristol-Myer Squibb: Research Funding. Bakhos:Bristol-Myer Squibb: Research Funding.

Comparison of antithrombotic and haemorrhagic effects of edoxaban, an oral direct factor Xa inhibitor, with warfarin and enoxaparin in rats

IntroductionFactor Xa (FXa) is a key serine protease in the coagulation cascade and a promising target for a new antithrombotic agent. Edoxaban is an oral, selective and direct FXa inhibitor. The objective of this study was to compare the antithrombotic and haemorrhagic effects of edoxaban with clinically available anticoagulants, warfarin and enoxaparin, in rat models of thrombosis and haemorrhage. MethodsRats were treated with single oral administration of edoxaban, repeated oral dosing of warfarin for 4days and single subcutaneous administration of enoxaparin before thrombosis or haemorrhage induction. Thrombosis was induced by the insertion of a platinum wire into the inferior vena cava for 60min. Tail template bleeding time was measured after making an incision on the tail. ResultsEdoxaban at 0.3, 1 and 3mg/kg exerted dose-dependent and significant inhibition of venous thrombus formation. The 50% thrombus inhibition dose (ED50) was 1.9mg/kg. At supra-therapeutic doses (10 and 20mg/kg), edoxaban significantly but moderately (less than 2-fold) prolonged bleeding time. Warfarin and enoxaparin also dose-dependently inhibited venous thrombosis and prolonged bleeding time. The ED50 values of warfarin and enoxaparin were 0.12mg/kg and 500IU/kg, and the 2-fold bleeding time prolongation doses (BT2) were 0.16mg/kg and 1700IU/kg, respectively. The safety margin (ratio of BT2 to ED50) of edoxaban (>10.5) was greater than those of warfarin (1.3) and enoxaparin (3.4). ConclusionsEdoxaban inhibited venous thrombosis comparably to warfarin and enoxaparin, and the attendant bleeding risk of edoxaban was lower than that of warfarin and enoxaparin in rats.

Prevention of Venous Thromboembolism with New Oral Anticoagulants versus Standard Pharmacological Treatment in Acute Medically Ill Patients

Venous thromboembolism (VTE) is a common and potentially avoidable cause of morbidity and mortality in patients hospitalized for acute medical illness. Our objective was to conduct a systematic review of studies that assessed the efficacy and safety of new oral anticoagulant (OAC) drugs versus standard pharmacological drugs and/or placebo in prevention of VTE in acute medically ill patients. PubMed.org and ClinicalTrials.gov databases were searched to identify studies that evaluated the efficacy and safety of a new OAC versus the standard pharmacological treatment and/or placebo in the prevention of VTE in medically ill patients. Relative risks (RR), weighted means and 95% CIs were calculated. Statistical heterogeneity was evaluated using Chi2 and I2 statistics. Two studies were included in the meta-analysis. The primary outcome in both studies was the composite of VTE-related death, symptomatic non-fatal pulmonary embolism (PE), symptomatic deep venous thrombosis (DVT) and asymptomatic proximal DVT. Both studies compared a factor (F)Xa inhibitor with enoxaparin in standard short-term thromboprophylaxis followed by a period where the FXa inhibitor was compared with placebo as prolonged thromboprophylaxis in medically ill patients. The primary major safety outcome in both studies was a composite of treatment-related major bleeding and clinically relevant non-major bleeding. A total of 14 629 patients were randomized. Compared with subjects treated with enoxaparin followed by placebo, the RR of the primary outcome during the prolonged treatment period was 0.79 (95% CI 0.66, 0.94), the RR for the primary outcome during the first short-term treatment period was 1.03 (95% CI 0.81, 1.31). For major bleeding during the prolonged treatment period, the RR was 2.69 (95% CI 1.65, 4.39) for patients treated with an FXa inhibitor compared with enoxaparin/placebo. For major bleeding during the shorter treatment period, the RR was 2.01 (95% CI 1.10, 3.65) in favour of enoxaparin. In acute medically ill patients, prolonged thromboprophylaxis with an oral FXa inhibitor is more protective than regular short-term treatment with enoxaparin. However, treatment with FXa inhibitors is significantly associated with major bleeding, both in long- and short-term treatment compared with enoxaparin.

Novel Oral Factor Xa and Thrombin Inhibitors in the Management of Thromboembolism

The last decade has seen the evaluation of several new oral anticoagulants that directly target thrombin or activated factor X (FXa). All demonstrate a rapid onset of action, a low potential for food and drug interactions, and a predictable anticoagulant effect that obviates the need for routine coagulation monitoring. Those agents at the most advanced stages of clinical development are a direct thrombin inhibitor, dabigatran, and direct FXa inhibitors, rivaroxaban and apixaban. Dabigatran and rivaroxaban are approved in more than 70 countries for prevention of venous thromboembolism in patients undergoing elective hip or knee arthroplasty, and apixaban is being considered for approval by regulatory agencies for this indication. Dabigatran was shown in a large phase III trial to be more effective and safer than warfarin for the prevention of stroke or systemic embolism in patients with atrial fibrillation and has recently been approved for this indication. Edoxaban, an oral FXa inhibitor, is also being evaluated in phase III clinical trials. This review summarizes the pharmacology, clinical trial results, and future role of the new oral anticoagulants in clinical practice.

E0461 The application prospects of oral FXa inhibitors in acute coronary syndrome

Acute coronary syndrome (ACS) patients would still have twice heart attack, even after they had received the standard regimen, such as vascular reconstruction, oral anti-platelet therapy, and other evidence based...

Prevention of venous thromboembolism with an oral factor Xa inhibitor, YM150, after total hip arthroplasty. A dose finding study (ONYX-2).

Anticoagulant prophylaxis substantially reduces the risk of venous thromboembolism (VTE) after major orthopedic surgery. The direct factor Xa inhibitor YM150 is currently under investigation for the prevention of VTE, stroke and ischemic vascular events in patients after orthopedic surgery, with atrial fibrillation and with acute coronary syndrome, respectively. To investigate the efficacy and safety of YM150 for the prevention of VTE following elective total hip arthroplasty. Patients were randomized to postoperative, once-daily, oral YM150 (5, 10, 30, 60 or 120 mg) (double-blind) or preoperative subcutaneous (open label) enoxaparin (40 mg) for 5 weeks. The primary efficacy endpoint comprised VTE diagnosed by mandatory bilateral venography or verified symptomatic deep vein thrombosis (DVT) plus all deaths up to 9 days after surgery. The primary safety outcome was major bleeding up to 9 days after surgery. Primary efficacy endpoint: of 1017 patients randomized, 960 patients were evaluable for safety and 729 patients for efficacy. A dose-related decrease in VTE incidence from YM150 5 to 60 mg (P = 0.0005) and from 5 to 120 mg (P = 0.0002) was found. The VTE incidence was 27.4%, 31.7%, 19.3%, 13.3% and 14.5% for 5, 10, 30, 60 and 120 mg YM150, respectively, and 18.9% for enoxaparin. Primary safety endpoint: there was one major bleed with YM150 (60 mg) and one with enoxaparin. The oral direct FXa inhibitor YM150 demonstrated a significant dose response regarding efficacy. Doses from 30 to 120 mg had comparable efficacy to enoxaparin, without compromising safety regarding major bleeding events.

Study of Coagulation and Platelets Response in An Open Label Randomised Non Treatment and Active Controlled (Dalteparin, Ximelagatran and DU- 176b) in 4 Groups of Elderly Healthy Subjects

Background: Several oral direct FXa inhibitors are being developed for the prevention and treatment of venous thrombosis. Their efficacy-safety ratio must be compared to that of LMWHs and oral direct FIIa inhibitors. Both families of drugs induce an alteration of blood coagulation through different mechanisms and after their interruption a possible coagulation rebound has been hypothesized.Patients and Method: In an open-label, randomised, non-treatment and active-controlled (Dalteparin, Ximelagatran), multiple dose study, 4 groups of 10 fed, elderly (64 to 75 years), healthy subjects were included to assess the pharmacodynamics of the oral direct anti-Xa DU-176b with a panel of biomarkers of blood coagulation, platelet and endothelium activation. Dalteparin 5000 anti-FXa (AFXa) IU SQ once a day, Ximelagatran direct anti-FIIa (AFIIa) 24 mg orally twice a day, and DU-176b AFXa 60 mg orally twice a day were the doses given while the 4th group received no treatment. Blood samples were collected before dosing and 1.5, 4, 12, and 24 hours after dosing on day one then on day 4 predose 12, 24, 48, and 72 hours after last drug administration.Results: Ximelagatran and DU-176b induced a consistent reduction of Thrombin-anti-Thrombin complexes (TAT) which was more marked than with Dalteparin. All study medications had a similar but moderate effect in reducing F1+2. Some clear differences were identified in the response of Thrombin Generation Test (TGT), Prothrombin Time (PT), and activated Partial Thromboplastin Time (aPTT). PT was more prolonged than aPTT with DU-176b. It was only slightly modified with Ximelagatran. These results deserve interest since all the study medications were used at doses considered as efficacious in the prevention of post operative thrombosis. No significant modification of platelet and endothelium biomarkers, soluble P selectin and CD 40L were evident. Interestingly anti-FXa amidolytic activity at peak levels were about 10 times higher with DU-176b than with Dalteparin when a LMWH reference preparation was used. Higher activity was maintained even at the trough level. Plasma drug concentrations were in good correlation with blood test results. No significant adverse events were observed and all subjects received all doses of study medication as planned. Most of the liver enzymes elevation were reported on day 7 after Dalteparin administration. Overall, there was no evidence of hypercoagulation or rebound effect following cessation of these three agents.Conclusion: Both PT and TGT would be the most preferable biomarker to evaluate efficacy of direct FXa inhibitor. And, no rebound phenomenon by any kind of anticoagulant was observed after short term treatment.DU-176bDalteparinXimelagatranNo TreatmentPT Peak ratio Trough 4th day1.5 1.11.05 1.01.1 1.01.05 1.0aPTT peak ratio1.301.241.440.98Trough at 4th day1.071.051.100.98D D-Dimer peak/Trough↓45%↓33%↓40%↓10%D F1–2 nmol/L>↓40%↓30%↓40%↓5%AFXa IU/ml peak3.960.31–0.400.010.01Trough1.460.020.020.02TGT Peak D MAX Trough↓65 % ↓50 %↓33 % ↓2 %↓6 % ↓6 %↓0 ↓3%TGT-Lag Time Peak Troughx 5 x 2.8x 1.5 x 1x 1.6 x 1.3x 1.04 x 1Remark: TFPI Variation (increase) only with Dalteparin

Recombinant Antidote for Reversal of Anticoagulation by Factor Xa Inhibitors.

Individuals anticoagulated with warfarin or heparin are typically treated with specific antidotes such as vitamin K or protamine, respectively, if they bleed or require surgery. In contrast, specific and effective antidotes are not available for the reversal of the anticoagulant effects of the low molecular weight heparins (LMWH) or the new oral anticoagulants targeting factor Xa (fXa) which are predictably only marginally affected by standard treatments using rfVIIa or fresh frozen plasma. The present study was designed to test the hypothesis that plasma-derived or recombinant fXa, modified to lack catalytic and membrane binding activities, could neutralize the anticoagulant activities of small molecule fXa inhibitors and LMWH. Plasma derived antidote (pd-Antidote) was prepared by chemical modification of the active site serine of fXa followed by chymotryptic removal of the Gla domain. Preliminary experiments showed that pd-Antidote dose dependently reversed the activity of rivaroxaban, apixaban or betrixaban, three small molecule fXa inhibitors currently in clinical trials. In a fXa amidolytic assay with 3nM enzyme, half maximal reversal of inhibitory activities (EC50) was attained at the following pd-Antidote concentrations: rivaroxaban =49 nM, apixaban = 122 nM, betrixaban = 41 nM. The pd-Antidote had no effect on the activity of fXa in the absence of inhibitors. Thus, active site inactivated pd-Antidote retained the ability to bind small molecule inhibitors of fXa. The activity of pd-Antidote was not limited to reversal of purified fXa catalytic activity. In a tissue factor-initiated thrombin generation assay in plasma, while pd-Antidote did not interfere with the normal function of prothrombinase complexes, it dose-dependently and completely reversed the inhibition produced by the small molecule fXa inhibitors. Pd-Antidote also demonstrated reversal of the in-vitro anticoagulant activity of the LMWH, enoxaparin. Addition of pd-Antidote (515nM) produced a 37% reduction of the clotting activity of enoxaparin (1.25Units/ml). In an activated partial thromboplastin time assay (aPTT), pd-Antidote also dose-dependently reversed the inhibitory effects of the fXa inhibitors. For example, aPTT prolongation by betrixaban (400nM) was reversed with an estimated EC50=650nM. Baseline aPTT was not altered upon addition of pd-Antidote up to a concentration of 2.5 μM, the highest concentration studied. Recombinant antidote (r-Antidote) was expressed in mammalian cells (Chinese hamster ovary) using a construct for human fXa with the S195A mutation and lacking the Gla-domain. The EC50's for purified r-Antidote in reversing 7.5 nM fXa inhibitors in the fXa catalytic activity assay were: rivaroxaban =17 nM, apixaban = 45 nM, betrixaban = 15 nM. As expected, there was no inhibition of fXa activity by r-Antidote alone. Purified r-Antidote also reversed anticoagulant activity in plasma clotting assays. Prothrombin time (PT) extensions achieved by supratherapeutic concentrations of rivaroxaban (1 μM) or apixaban (1 μM) were completely normalized upon addition of r-Antidote (1.5 μM). Ex vivo clotting prolongation by an excess of betrixaban (300 nM) was essentially reversed (88% correction) by pre-incubation with r-Antidote (570 nM) prior to addition of aPTT reagents. The concentration of r-Antidote required for corrective activity was related to the inhibitory potency of the oral fXa inhibitors. PT or aPTT baselines did not change upon addition of r-Antidote alone at 1.9 μM, the highest concentration tested. We also examined the ability of both pd-Antidote and r-Antidote to alleviate inhibitory activities produced upon oral dosing of fXa inhibitors in mice. Perturbation of whole blood PT/INR in dosed animals was followed as a marker of anticoagulation. The effect following dosing of a fXa inhibitor could be reversed by a single intravenous injection of antidote. INR measurements in blood samples of treated animals showed a >50% reduction of observed inhibitory activity compared to control animals. Our results suggest that these plasma derived or recombinant proteins have the potential to act as universal antidotes for reversal of anticoagulation of all current fXa inhibitors, both small molecule and antithrombin dependent, in patients with bleeding related medical emergencies or those requiring cessation of anticoagulation prior to surgery.