A randomized, repeat-dose, pharmacodynamic and safety study of an antidote-controlled factor IXa inhibitor

Summary. Background: Active and safe reversibility of anticoagulation is an unmet need in clinical care. Factor IXa, required for rapid thrombin generation on platelet surfaces, is a novel target for modulating coagulation. REG1 comprises RB006 (drug) and RB007 (antidote). RB006, a ribonucleic acid aptamer, exerts its anticoagulant effect by selectively binding FIXa. RB007, the complementary oligonucleotide antidote, binds to RB006 by Watson–Crick base pairing, neutralizing its anti‐FIXa activity. Objective: To test the multiple repeat‐dose safety, intraindividual pharmacodynamic reproducibility and graded active reversibility of REG1. Methods: We randomized 39 healthy volunteers to receive either three consecutive weight‐adjusted, drug–antidote treatment cycles, or double placebo. Each treatment cycle included an intravenous bolus of 0.75 mg kg^?1 RB006, followed 60 min later by a descending dose of RB007, ranging from a 2 : 1 to 0.125 : 1 antidote/drug ratio (1.5 mg kg^?1 to 0.094 mg kg^?1 RB007). Serial clinical assessments and coagulation measurements were performed through 14 days postrandomization. Results: Repeat doses of RB006 achieved highly reproducible activated partial thromboplastin time (APTT) levels with low intrasubject variability (coefficient of variation 5.5%, intraclass correlation coefficient 5.8 at 15 min postdose), while repeat doses of RB007 reversed the APTT levels dose‐dependently and reproducibly. There was no major bleeding and there were no other serious adverse events. Conclusions: This is the first human study demonstrating multiple repeat‐dose safety, intraindividual pharmacodynamic reproducibility and graded active reversibility of an RNA aptamer–oligonucleotide antidote pair. The results lay the foundation for studying the translation of this novel anticoagulation platform to a wide variety of clinical applications.     

Low molecular weight heparin inhibits plasma thrombin generation via direct targeting of factor IXa: contribution of the serpin‐independent mechanism

Summary. Background: Although heparin possesses multiple mechanisms of action, enhanced factor Xa inhibition by antithrombin is accepted as the predominant therapeutic mechanism. The contribution of FIXa inhibition to heparin activity in human plasma remains incompletely defined. Objectives: To determine the relevance of FIXa as a therapeutic target for heparins, particularly serpin‐independent inhibition of intrinsic tenase (FIXa–FVIIIa) activity. Patients/Methods: Thrombin generation was detected by fluorogenic substrate cleavage. The inhibitory potencies (EC₅₀s) of low molecular weight heparin (LMWH), super‐sulfated LMWH (ssLMWH), fondaparinux and unfractionated heparin (UFH) were determined by plotting concentration vs. relative velocity index (ratio ± heparin). Inhibition was compared under FIX‐dependent and FIX‐independent conditions (0.2 or 4 pm tissue factor [TF], respectively) in normal plasma, and in mock‐depleted or antithrombin/FIX‐depleted plasma supplemented with recombinant FIX. Results: UFH and fondaparinux demonstrated similar potency under FIX‐dependent and FIX‐independent conditions, whereas LMWH (2.9‐fold) and ssLMWH (5.1‐fold) demonstrated increased potency with limiting TF. UFH (62‐fold) and fondaparinux (42‐fold) demonstrated markedly increased EC₅₀ values in antithrombin‐depleted plasma, whereas LMWH (9.4‐fold) and ssLMWH (two‐fold) were less affected, with an EC₅₀ within the therapeutic range for LMWH. The molecular target for LMWH/ssLMWH was confirmed by supplementing FIX/antithrombin‐depleted plasma with 90 nm recombinant FIX possessing mutations in the heparin‐binding exosite. Mutated FIX demonstrated resistance to inhibition of thrombin generation by LMWH and ssLMWH that paralleled the effect of these mutations on intrinsic tenase inhibition. Conclusions: Therapeutic LMWH concentrations inhibit plasma thrombin generation via antithrombin‐independent interaction with the FIXa heparin‐binding exosite.     

Tissue factor pathway inhibitor is an inhibitor of factor VII‐activating protease

Background:  Factor VII‐activating protease (FSAP) is a serine protease that circulates in plasma in its inactive single‐chain form and can be activated upon contact with dead cells. When activated by apoptotic cells, FSAP leads to the release of nucleosomes. The serpins C1‐inhibitor and α₂‐antiplasmin are reported to be the major inhibitors of FSAP. However, regulation of FSAP activity by Kunitz‐type inhibitors is not well studied. Objectives:  To compare the inhibition of FSAP activity and FSAP‐induced nucleosome release from apoptotic cells by tissue factor pathway inhibitor (TFPI) with that of C1‐inhibitor and α₂‐antiplasmin. Methods:  Apoptotic cells were incubated with plasma or FSAP in presence of the inhibitor, and nucleosome release was analyzed with flow cytometry. Monoclonal antibodies against TFPI and altered forms of TFPI were used to investigate which domains of TFPI contribute to FSAP inhibition. Results and Conclusions:  We show that TFPI abrogates FSAP activity and nucleosome release from apoptotic cells. TFPI is a much more efficient inhibitor than C1‐inhibitor or α₂‐antiplasmin. The active site of K2 is required for inhibition of FSAP. A direct binding interaction between FSAP and the C‐terminal domain of TFPI is also required for efficient inhibition. Inhibition of FSAP‐induced nucleosome release by recombinant TFPI might, in part, explain the anti‐inflammatory effects of recombinant TFPI infusion observed in animal and human sepsis.     

Bortezomib delays the onset of factor VIII inhibitors in experimental hemophilia A,but fails to eliminate established anti‐factor VIII IgG‐producing cells

Summary. Background: Replacement therapy with exogenous factor VIII to treat hemorrhages induces inhibitory anti‐FVIII antibodies in up to 30% of patients with hemophilia A. Current approaches to eradicate FVIII inhibitors using high‐dose FVIII injection protocols (immune tolerance induction) or anti‐CD20 depleting antibodies (Rituximab) demonstrate limited efficacy; they are extremely expensive and/or require stringent compliance from the patients. Objectives: To investigate whether the proteasome inhibitor bortezomib, which depletes plasmocytes, modulates the anti‐FVIII immune response in FVIII‐deficient mice. Methods and results: Preventive 4‐week treatment of naïve mice with bortezomib at the time of FVIII administration delayed the development of inhibitory anti‐FVIII IgG, and depleted plasma cells as well as different lymphoid cell subsets. Conversely, curative treatment of inhibitor‐positive mice for 10 weeks, along with FVIII administration, failed to eradicate FVIII inhibitors to extents that would be clinically relevant if achieved in patients. Accordingly, bortezomib did not eradicate anti‐FVIII IgG‐secreting plasmocytes that had homed to survival niches in the bone marrow, despite significant elimination of total plasma cells. Conclusions: The data suggest that strategies for the efficient reduction of anti‐FVIII IgG titers in patients with hemophilia A should rely on competition for survival niches for plasmocytes in the bone marrow rather than the mere use of proteasome inhibitors.     

Phase I,randomized, double‐blind,placebo‐controlled,single‐dose escalation study of the recombinant factor VIIa variant BAY 86‐6150 in hemophilia

Summary. Background: BAY 86‐6150 is a new human recombinant factor VIIa variant developed for high procoagulant activity and longer action in people with hemophilia with inhibitors. Objectives: To investigate the safety, tolerability, pharmacodynamics, pharmacokinetics and immunogenicity of BAY 86‐6150 in non‐bleeding hemophilia subjects. Methods: The study included non‐bleeding men (18–65 years of age) with moderate or severe hemophilia A or B with or without inhibitors. Sixteen subjects were randomized 3 : 1 to four cohorts of escalating doses of BAY 86‐6150 (6.5, 20, 50 or 90 μg kg^?1 [n = 3 per cohort]) or placebo (n = 1 per cohort); an independent data‐monitoring committee reviewed previous cohort data before the next dose escalation. Blood sampling was performed predose and postdose; subjects were monitored for 50 days postdose. Results: At the tested doses, BAY 86‐6150 was not associated with clinically significant adverse events or dose‐limiting toxicities. BAY 86‐6150 pharmacokinetics exhibited a linear dose response, with a half‐life of 5–7 h. Subjects demonstrated consistent, dose‐dependent thrombin generation ex vivo in platelet‐poor plasma (PPP) (mean peak effect, 26–237 nm thrombin from 6.5 to 90 μg kg^?1). Peak thrombin levels over time paralleled BAY 86‐6150, with thrombin kinetics appearing to be slightly shorter; thus, circulating BAY 86‐6150 retained activity. There were corresponding decreases in activated partial thromboplastin and prothrombin times. No subject developed de novo anti‐BAY 86‐6150 neutralizing antibodies during the 50‐day follow‐up. Conclusions: In this first‐in‐human, multicenter, randomized, double‐blind, placebo‐controlled, single‐dose escalation study, BAY 86‐6150 was tolerated at the highest dose (90 μg kg^?1), with no safety concerns. Safety and efficacy will be further evaluated in phase II/III studies.     

Risk of venous thrombosis in pregnancy among carriers of the factor V Leiden and the prothrombin gene G20210A polymorphisms

Background: Pregnancy is associated with a 10‐fold increased risk of venous thrombosis (VT), with different risk profiles for the antenatal and postnatal periods. The purpose of this study was to assess the risk of pregnancy‐related VT associated with the factor (F)V Leiden and prothrombin gene G20210A polymorphisms. Materials and Methods: The study comprised 377 155 women with 613 232 pregnancies at 18 Norwegian hospitals from 1 January 1990 to 31 December 2003. Of a total 559 cases with a validated first lifetime diagnosis of VT in pregnancy or within 14 weeks postpartum, and 1229 controls naive for VT, 313 cases and 353 controls donated biological material. Results: The odds ratios for VT during pregnancy or puerperium were 5.0 [95% confidence interval (CI) 3.1–8.3] and 9.4 (95% CI 2.1–42.4) for heterozygous carriers of the FV Leiden and the prothrombin gene polymorphisms, respectively. All homozygous carriers of the FV Leiden polymorphism (n = 8) and the prothrombin polymorphism (n = 1) developed VT, indicating a very high risk of VT. We estimated that pregnancy‐related VT occurred in 1.1/1000 non‐carriers, in 5.4/1000 heterozygous carriers of the FV Leiden polymorphism, and in 9.4/1000 heterozygous carriers of the prothrombin polymorphism. To avoid one VT, the number of pregnant women needed to be screened for these two polymorphisms and the number needed to be given thromboprophylaxis were 2015 and 157, respectively. Conclusions: Although the relative risk for VT during pregnancy and after delivery was increased among carriers of the FV Leiden and the prothrombin polymorphisms, the overall probability for pregnancy‐related VT was low.     

Efficacy and safety of the oral direct factor Xa inhibitor apixaban for symptomatic deep vein thrombosis. The Botticelli DVT dose-ranging study

Summary.  Background:  Apixaban, an oral potent reversible direct inhibitor of activated factor X, has shown promise in the prevention of venous thromboembolism following major orthopedic surgery. We conducted a dose-ranging study in patients with deep vein thrombosis. Methods:  Consecutive patients with symptomatic deep vein thrombosis were included and randomized to receive 84–91 days of apixaban 5 mg twice-daily, 10 mg twice-daily, or 20 mg once-daily, or low molecular weight heparin (LMWH) followed by a vitamin K antagonist (VKA). The primary efficacy outcome was the composite of symptomatic recurrent venous thromboembolism and asymptomatic deterioration of bilateral compression ultrasound or perfusion lung scan. The principal safety outcome was the composite of major and clinically relevant, non-major bleeding. Results:  The mean age of the 520 included patients was 59 years, and 62% were male. The primary outcome occurred in 17 of the 358 apixaban-treated patients [4.7%, 95% confidence interval (CI) 2.8–7.5%] and in five of the 118 LMWH/VKA-treated patients (4.2%, 95% CI  1.4–9.6%) who were evaluable. The incidence in all three apixaban groups was low and comparable without evidence of a dose response. The principal safety outcome occurred in 28 (7.3%) of the 385 apixaban-treated patients and in 10 (7.9%) of the 126 LMWH/VKA-treated patients. No dose response for apixaban was observed. Conclusion:  These observations warrant further evaluation of apixaban in phase III studies. The attractive fixed-dose regimen of this compound may meet the demand to simplify anticoagulant treatment in patients with established venous thromboembolism.     

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

Background: 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. Objectives: To investigate the efficacy and safety of YM150 for the prevention of VTE following elective total hip arthroplasty. Patients/methods: 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. Results: 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 to120 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. Conclusions: 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.