Joanne van Ryn

Hemostatic Therapy in Experimental Intracerebral Hemorrhage Associated With the Direct Thrombin Inhibitor Dabigatran

Background and Purpose— Dabigatran-etexilate (DE) recently has been approved for stroke prevention in atrial fibrillation. However, lack of effective antagonists represents a major concern in the event of intracerebral hemorrhage (ICH). The aims of the present study were to establish a murine model of ICH associated with dabigatran, and to test the efficacy of different hemostatic factors in preventing hematoma growth. Methods— In C57BL/6 mice receiving DE (4.5 or 9.0 mg/kg), in vivo and in vitro coagulation assays and dabigatran plasma levels were measured repeatedly. Thirty minutes after inducing ICH by striatal collagenase injection, mice received an intravenous injection of saline, prothrombin complex concentrate (PCC; 100 U/kg), murine fresh-frozen plasma (200 &mgr;L), or recombinant human factor VIIa (8.0 mg/kg). ICH volume was quantified on brain cryosections 24 hours later. Results— DE substantially prolonged tail vein bleeding time and ecarin clotting time for 4 hours corresponding to dabigatran plasma levels. Intracerebral hematoma expansion was observed mainly during the first 3 hours on serial T2* MRI. Anticoagulation with high doses of DE increased the hematoma volume significantly. PCC and, less consistently, fresh-frozen plasma prevented excess hematoma expansion caused by DE, whereas recombinant human factor VIIa was ineffective. Prevention of hematoma growth and reversal of tail vein bleeding time by PCC were dose-dependent. Conclusions— The study provides strong evidence that PCC and, less consistently, fresh-frozen plasma prevent excess intracerebral hematoma expansion in a murine ICH model associated with dabigatran. The efficacy and safety of this strategy must be further evaluated in clinical studies.

Safety, tolerability, and efficacy of idarucizumab for the reversal of the anticoagulant effect of dabigatran in healthy male volunteers: a randomised, placebo-controlled, double-blind phase 1 trial

BACKGROUND Idarucizumab is a monoclonal antibody fragment that binds dabigatran with high affinity in a 1:1 molar ratio. We investigated the safety, tolerability, and efficacy of increasing doses of idarucizumab for the reversal of anticoagulant effects of dabigatran in a two-part phase 1 study (rising-dose assessment and dose-finding, proof-of-concept investigation). Here we present the results of the proof-of-concept part of the study. METHODS In this randomised, placebo-controlled, double-blind, proof-of-concept phase 1 study, we enrolled healthy volunteers (aged 18-45 years) with a body-mass index of 18·5-29·9 kg/m(2) into one of four dose groups at SGS Life Sciences Clinical Research Services, Belgium. Participants were randomly assigned within groups in a 3:1 ratio to idarucizumab or placebo using a pseudorandom number generator and a supplied seed number. Participants and care providers were masked to treatment assignment. All participants received oral dabigatran etexilate 220 mg twice daily for 3 days and a final dose on day 4. Idarucizumab (1 g, 2 g, or 4 g 5-min infusion, or 5 g plus 2·5 g in two 5-min infusions given 1 h apart) was administered about 2 h after the final dabigatran etexilate dose. The primary endpoint was incidence of drug-related adverse events, analysed in all randomly assigned participants who received at least one dose of dabigatran etexilate. Reversal of diluted thrombin time (dTT), ecarin clotting time (ECT), activated partial thromboplastin time (aPTT), and thrombin time (TT) were secondary endpoints assessed by measuring the area under the effect curve from 2 h to 12 h (AUEC2-12) after dabigatran etexilate ingestion on days 3 and 4. This trial is registered with ClinicalTrials.gov, number NCT01688830. FINDINGS Between Feb 23, and Nov 29, 2013, 47 men completed this part of the study. 12 were enrolled into each of the 1 g, 2 g, or 5 g plus 2·5 g idarucizumab groups (nine to idarucizumab and three to placebo in each group), and 11 were enrolled into the 4 g idarucizumab group (eight to idarucizumab and three to placebo). Drug-related adverse events were all of mild intensity and reported in seven participants: one in the 1 g idarucizumab group (infusion site erythema and hot flushes), one in the 5 g plus 2·5 g idarucizumab group (epistaxis); one receiving placebo (infusion site haematoma), and four during dabigatran etexilate pretreatment (three haematuria and one epistaxis). Idarucizumab immediately and completely reversed dabigatran-induced anticoagulation in a dose-dependent manner; the mean ratio of day 4 AUEC2-12 to day 3 AUEC2-12 for dTT was 1·01 with placebo, 0·26 with 1 g idarucizumab (74% reduction), 0·06 with 2 g idarucizumab (94% reduction), 0·02 with 4 g idarucizumab (98% reduction), and 0·01 with 5 g plus 2·5 g idarucizumab (99% reduction). No serious or severe adverse events were reported, no adverse event led to discontinuation of treatment, and no clinically relevant difference in incidence of adverse events was noted between treatment groups. INTERPRETATION These phase 1 results show that idarucizumab was associated with immediate, complete, and sustained reversal of dabigatran-induced anticoagulation in healthy men, and was well tolerated with no unexpected or clinically relevant safety concerns, supporting further testing. Further clinical studies are in progress. FUNDING Boehringer Ingelheim Pharma GmbH & Co KG.

Dabigatran: An Oral Novel Potent Reversible Nonpeptide Inhibitor of Thrombin

Dabigatran is a highly selective, reversible, and potent thrombin inhibitor and is orally available as the prodrug, dabigatran etexilate. It has shown antithrombotic efficacy in animal models of thrombosis, with a rapid onset of action and predictable pharmacodynamic response. Peak plasma concentrations of dabigatran occur 1 to 2 hours after ingestion of the prodrug. The terminal half-life of dabigatran is 12 to 14 hours in elderly volunteers. Dabigatran is not metabolized by cytochrome P450 isoenzymes and does not interact with food. Dabigatran has a low potential for drug-drug interactions and is predominantly renally excreted. Dabigatran etexilate as chronic therapy effectively prevents the recurrence of venous thromboembolism and cardioembolic stroke. For the first time, it has been demonstrated clinically that there may be an effective and safe alternative to warfarin.

Idarucizumab for Dabigatran Reversal — Full Cohort Analysis

Background Idarucizumab, a monoclonal antibody fragment, was developed to reverse the anticoagulant effect of dabigatran. Methods We performed a multicenter, prospective, open‐label study to determine whether 5 g of intravenous idarucizumab would be able to reverse the anticoagulant effect of dabigatran in patients who had uncontrolled bleeding (group A) or were about to undergo an urgent procedure (group B). The primary end point was the maximum percentage reversal of the anticoagulant effect of dabigatran within 4 hours after the administration of idarucizumab, on the basis of the diluted thrombin time or ecarin clotting time. Secondary end points included the restoration of hemostasis and safety measures. Results A total of 503 patients were enrolled: 301 in group A, and 202 in group B. The median maximum percentage reversal of dabigatran was 100% (95% confidence interval, 100 to 100), on the basis of either the diluted thrombin time or the ecarin clotting time. In group A, 137 patients (45.5%) presented with gastrointestinal bleeding and 98 (32.6%) presented with intracranial hemorrhage; among the patients who could be assessed, the median time to the cessation of bleeding was 2.5 hours. In group B, the median time to the initiation of the intended procedure was 1.6 hours; periprocedural hemostasis was assessed as normal in 93.4% of the patients, mildly abnormal in 5.1%, and moderately abnormal in 1.5%. At 90 days, thrombotic events had occurred in 6.3% of the patients in group A and in 7.4% in group B, and the mortality rate was 18.8% and 18.9%, respectively. There were no serious adverse safety signals. Conclusions In emergency situations, idarucizumab rapidly, durably, and safely reversed the anticoagulant effect of dabigatran. (Funded by Boehringer Ingelheim; RE‐VERSE AD ClinicalTrials.gov number, NCT02104947.)

COX-2 Selectivity and Inflammatory Processes

Increasing amounts of experimental and clinical data support the role of selective cyclooxygenase (COX)-2 inhibition in anti-inflammatory processes and the involvement of COX-1 inhibition in the side effects associated with non steroidal anti-inflammatory drug use. This review will focus on the differences in the structure of the COX-1 and COX-2 molecules, particularly the active site and how they are bound by various NSAIDs to achieve COX-2 selectivity. This COX-2 selectivity will then be characterized in pharmacological assays in vitro and in animal models in vivo. Finally, clinical information available for this new class of selective inhibitors will be discussed.

Idarucizumab The Antidote for Reversal of Dabigatran

Four non–vitamin K antagonist oral anticoagulants (NOACs), including the direct thrombin inhibitor dabigatran and the direct factor Xa inhibitors rivaroxaban, apixaban, and edoxaban, are currently licensed as alternatives to heparins and vitamin K antagonists for the prevention and treatment of venous thromboembolism and for the prevention of stroke in patients with nonvalvular atrial fibrillation. Dabigatran is the only NOAC that inhibits thrombin; the others inhibit factor Xa. All of the NOACs are at least as effective as vitamin K antagonists for the prevention of stroke in patients with atrial fibrillation and for the treatment of venous thromboembolism, and they are associated with less life-threatening bleeding, in particular less intracranial hemorrhage.1,2 Nonetheless, serious bleeding can occur with NOACs. In addition, patients taking NOACs may sustain trauma and may require urgent surgery or interventions. Consequently, the availability of specific reversal agents for NOACs could improve patient management during these emergency situations. Idarucizumab is a humanized monoclonal antibody fragment that has been developed as a specific reversal agent for dabigatran. In vitro and ex vivo studies have demonstrated that idarucizumab promptly restores dabigatran-prolonged coagulation parameters to baseline values,3–5 and studies in healthy volunteers and patients with life-threatening bleeding or requiring emergency surgery or invasive procedures show that it completely reverses the anticoagulant effects of dabigatran within minutes in the majority of patients. Idarucizumab was recently licensed in the United States and Europe. This article summarizes the pharmacology and clinical data on the use of idarucizumab to reverse dabigatran. The first step in the development of idarucizumab was to immunize mice with dabigatran-derived haptens coupled to carrier proteins to produce antibodies against dabigatran.3 Monoclonal antibodies exhibiting the highest affinity for dabigatran were selected, and the antigen-binding fragment (Fab) was isolated (Figure 1). Murine protein sequences were replaced …

Interpretation of Point-of-care INR Results in Patients Treated with Dabigatran

BACKGROUND Point-of-care devices for measurement of the international normalized ratio (INR) are commonly used to monitor therapy and maintain therapeutic levels of anticoagulation in patients treated with vitamin K antagonists. Dabigatran, a new oral, reversible direct thrombin inhibitor approved for stroke prevention in patients with atrial fibrillation does not require routine coagulation monitoring. However, case reports have identified falsely elevated point-of-care INR levels in patients treated with dabigatran using one of these devices (Hemochron). This in vitro study was designed to verify this issue. METHODS We compared INR levels in whole blood and plasma using a Hemochron Jr. Signature+ point-of-care device (International Technidyne Corporation, Edison, NJ) with routine laboratory monitoring, using blood from healthy volunteers that was spiked with increasing concentrations of dabigatran. RESULTS Prothrombin time and INR levels were increased about 2- to 4-fold with the point-of-care device compared with laboratory measures across the plasma dabigatran concentration range 50-1400 ng/mL. At plasma concentrations of dabigatran likely to be observed in patients, at a dose of 150 mg twice daily (60-275 ng/mL), whole blood point-of-care INR values increased from 1.7 to 4.0, versus 1.1 to 1.5 measured with the laboratory coagulometer. Similar differences in prothrombin time were observed in plasma samples. CONCLUSIONS INR levels in patients taking dabigatran are substantially higher using a Hemochron Jr. point-of-care device compared with laboratory values. We discourage the use of these devices specifically, as well as the use of the INR in general, for measuring the anticoagulant effect of dabigatran.

Genetic and pharmacological modifications of thrombin formation in apolipoprotein e-deficient mice determine atherosclerosis severity and atherothrombosis onset in a neutrophil-dependent manner.

Background Variations in the blood coagulation activity, determined genetically or by medication, may alter atherosclerotic plaque progression, by influencing pleiotropic effects of coagulation proteases. Published experimental studies have yielded contradictory findings on the role of hypercoagulability in atherogenesis. We therefore sought to address this matter by extensively investigating the in vivo significance of genetic alterations and pharmacologic inhibition of thrombin formation for the onset and progression of atherosclerosis, and plaque phenotype determination. Methodology/Principal Findings We generated transgenic atherosclerosis-prone mice with diminished coagulant or hypercoagulable phenotype and employed two distinct models of atherosclerosis. Gene-targeted 50% reduction in prothrombin (FII−/WT:ApoE−/−) was remarkably effective in limiting disease compared to control ApoE−/− mice, associated with significant qualitative benefits, including diminished leukocyte infiltration, altered collagen and vascular smooth muscle cell content. Genetically-imposed hypercoagulability in TMPro/Pro:ApoE−/− mice resulted in severe atherosclerosis, plaque vulnerability and spontaneous atherothrombosis. Hypercoagulability was associated with a pronounced neutrophilia, neutrophil hyper-reactivity, markedly increased oxidative stress, neutrophil intraplaque infiltration and apoptosis. Administration of either the synthetic specific thrombin inhibitor Dabigatran etexilate, or recombinant activated protein C (APC), counteracted the pro-inflammatory and pro-atherogenic phenotype of pro-thrombotic TMPro/Pro:ApoE−/− mice. Conclusions/Significance We provide new evidence highlighting the importance of neutrophils in the coagulation-inflammation interplay during atherogenesis. Our findings reveal that thrombin-mediated proteolysis is an unexpectedly powerful determinant of atherosclerosis in multiple distinct settings. These studies suggest that selective anticoagulants employed to prevent thrombotic events may also be remarkably effective in clinically impeding the onset and progression of cardiovascular disease.

Antiinflammatory and antifibrotic effects of the oral direct thrombin inhibitor dabigatran etexilate in a murine model of interstitial lung disease

OBJECTIVE Activation of the coagulation cascade leading to generation of thrombin has been documented extensively in various forms of lung injury, including that associated with systemic sclerosis. We previously demonstrated that the direct thrombin inhibitor dabigatran inhibits thrombin-induced profibrotic signaling in lung fibroblasts. This study was undertaken to test whether dabigatran etexilate attenuates lung injury in a murine model of interstitial lung disease. METHODS Lung injury was induced in female C57BL/6 mice by a single intratracheal instillation of bleomycin. Dabigatran etexilate was given as supplemented chow beginning on day 1 of bleomycin instillation (early treatment, study of antiinflammatory effect) or on day 8 following bleomycin instillation (late treatment, study of antifibrotic effect). Mice were killed 2 weeks or 3 weeks after bleomycin instillation, and lung tissue, bronchoalveolar lavage (BAL) fluid, and plasma were investigated. RESULTS Both early treatment and late treatment with dabigatran etexilate attenuated the development of bleomycin-induced pulmonary fibrosis. Dabigatran etexilate significantly reduced thrombin activity and levels of transforming growth factor β1 in BAL fluid, while simultaneously reducing the number of inflammatory cells and protein concentrations. Histologically evident lung inflammation and fibrosis were significantly decreased in dabigatran etexilate-treated mice. Additionally, dabigatran etexilate reduced collagen, connective tissue growth factor, and α-smooth muscle actin expression in mice with bleomycin-induced lung fibrosis, whereas it had no effect on basal levels of these proteins. CONCLUSION Inhibition of thrombin using the oral direct thrombin inhibitor dabigatran etexilate has marked antiinflammatory and antifibrotic effects in a bleomycin model of pulmonary fibrosis. Our data provide preclinical information about the feasibility and efficacy of dabigatran etexilate as a new therapeutic approach for the treatment of interstitial lung disease.

Differential contribution of FXa and thrombin to vascular inflammation in a mouse model of sickle cell disease

Activation of coagulation and vascular inflammation are prominent features of sickle cell disease (SCD). Previously, we have shown that inhibition of tissue factor (TF) attenuates activation of coagulation and vascular inflammation in mouse models of SCD. In this study, we examined the mechanism by which coagulation proteases enhance vascular inflammation in sickle BERK mice. To specifically investigate the contribution of FXa and thrombin, mice were fed chow containing either rivaroxaban or dabigatran, respectively. In addition, we used bone marrow transplantation to generate sickle mice deficient in either protease activated receptor-1 (PAR-1) or protease activated receptor-2 (PAR-2) on nonhematopoietic cells. FXa inhibition and PAR-2 deficiency in nonhematopoietic cells attenuated systemic inflammation, measured by plasma levels of interleukin-6 (IL-6). In contrast, neither thrombin inhibition nor PAR-1 deficiency in nonhematopoietic cells affected plasma levels of IL-6 in sickle mice. However, thrombin did contribute to neutrophil infiltration in the lung, independently of PAR-1 expressed by nonhematopoietic cells. Furthermore, the TF-dependent increase in plasma levels of soluble vascular cell adhesion molecule-1 in sickle mice was not mediated by FXa or thrombin. Our data indicate that TF, FXa, and thrombin differentially contribute to vascular inflammation in a mouse model of SCD.