J. van Ryn

Dabigatran etexilate - a novel, reversible, oral direct thrombin inhibitor: Interpretation of coagulation assays and reversal of anticoagulant activity

Dabigatran etexilate is an oral, reversible direct thrombin inhibitor that is approved in the EU and several other countries for the prevention of venous thromboembolism after elective hip and knee replacement, and is in advanced clinical development for other thromboembolic disorders. Dabigatran has a predictable pharmacokinetic profile, allowing for a fixed-dose regimen without the need for routine coagulation monitoring. In certain clinical situations such as serious bleeding into critical organs (e.g. intracerebral bleeding), potential overdose and emergency surgery, clinicians will need to make an assessment of the anticoagulant status of a patient receiving dabigatran before deciding on future management strategies. If available, thrombin clotting time (TT), ecarin clotting time (ECT) and TT determined by Hemoclot thrombin inhibitor assay are sensitive tests to evaluate the anticoagulant effects of dabigatran. Prothrombin time (INR) is less sensitive than other assays and cannot be recommended. The activated partial thromboplastin time (aPTT) can provide a useful qualitative assessment of anticoagulant activity but is less sensitive at supratherapeutic dabigatran levels. There are limited data for activated clotting time (ACT). Overall, the aPTT and TT are the most accessible qualitative methods for determining the presence or absence of anticoagulant effect. Although there is no specific antidote to antagonise the anticoagulant effect of dabigatran, due to its short duration of effect drug discontinuation is usually sufficient to reverse any excessive anticoagulant activity. In case of potential overdose, the feasibility of early administration of activated charcoal and subsequent charcoal filtration are undergoing preclinical evaluation. Dabigatran can also be dialysed in patients with renal impairment. In instances of life-threatening bleeding, where conventional measures have failed or are unavailable, other non-specific prohaemostatic agents such as recombinant activated factor VII and prothrombin complex concentrates can be considered.

A specific antidote for dabigatran: functional and structural characterization

Dabigatran etexilate is a direct thrombin inhibitor and used widely as an anticoagulant for the prevention of stroke in patients with atrial fibrillation. However, anticoagulation therapy can be associated with an increased risk of bleeding. Here, we present data on the identification, humanization, and in vitro pharmacology of an antidote for dabigatran (aDabi-Fab). The X-ray crystal structure of dabigatran in complex with the antidote reveals many structural similarities of dabigatran recognition compared with thrombin. By a tighter network of interactions, the antidote achieves an affinity for dabigatran that is ~350 times stronger than its affinity for thrombin. Despite the structural similarities in the mode of dabigatran binding, the antidote does not bind known thrombin substrates and has no activity in coagulation tests or platelet aggregation. In addition we demonstrate that the antidote rapidly reversed the anticoagulant activity of dabigatran in vivo in a rat model of anticoagulation. This is the first report of a specific antidote for a next-generation anticoagulant that may become a valuable tool in patients who require emergency procedures.

Dabigatran etexilate for stroke prevention in patients with atrial fibrillation: Resolving uncertainties in routine practice

Dabigatran etexilate is a new oral anticoagulant recently approved in Europe for the prevention of stroke or systemic embolism in adult patients with non-valvular atrial fibrillation (AF) and at least one risk factor for stroke. With a fast onset of action and a predictable anticoagulant effect obviating the need for coagulation monitoring, dabigatran etexilate offers practical advantages over vitamin K antagonists in clinical practice. However, clinicians may have questions about practical aspects of dabigatran etexilate use including monitoring anticoagulant efficacy, interruption for surgical or invasive procedures and management of bleeding. This review article aims to address these concerns and provide guidance on the use of dabigatran etexilate in special situations, such as acute coronary syndromes and cardiac revascularisation. In addition, cut-off values for different coagulation assay results associated with an increased risk of bleeding are given.

Reversal of dabigatran anticoagulation by prothrombin complex concentrate (Beriplex P/N) in a rabbit model.

Summary.  Background:  One limitation of the direct thrombin inhibitor dabigatran is the lack of specific antidotes that allow acute bleeding events to be managed or urgent interventional procedures performed. Prothrombin complex concentrates (PCCs) have served as a standard treatment for the reversal of coumarin anticoagulation.

A randomised study in healthy volunteers to investigate the safety, tolerability and pharmacokinetics of idarucizumab, a specific antidote to dabigatran

Idarucizumab, a monoclonal antibody fragment that binds dabigatran with high affinity, is in development as a specific antidote for dabigatran. In this first-in-human, single-rising-dose study, we investigated the pharmacokinetics, safety and tolerability of idarucizumab. Healthy male volunteers aged 18-45 years received between 20 mg and 8 g idarucizumab as a 1-hour intravenous infusion in 10 sequential dose groups, or 1, 2 or 4 g idarucizumab as a 5-minute infusion. Subjects within each dose group were randomised 3:1 to idarucizumab or placebo. A total of 110 randomised subjects received study drug (27 placebo, 83 idarucizumab). Peak and total exposure to idarucizumab increased proportionally with dose. Maximum plasma concentrations were achieved near the end of infusion, followed by a rapid decline, with an initial idarucizumab half-life of ~45 minutes. For the 5-minute infusions, this resulted in a reduction of plasma concentrations to less than 5 % of peak within 4 hours. Idarucizumab (in the absence of dabigatran) had no effect on coagulation parameters or endogenous thrombin potential. Overall adverse event (AE) frequency was similar for idarucizumab and placebo, and no relationship with idarucizumab dose was observed. Drug-related AEs (primary endpoint) were rare (occurring in 2 placebo and 3 idarucizumab subjects) and were mostly of mild intensity; none of them resulted in study discontinuation. In conclusion, the pharmacokinetic profile of idarucizumab meets the requirement for rapid peak exposure and rapid elimination, with no effect on pharmacodynamic parameters. Idarucizumab was safe and well tolerated in healthy males.

EXPERIMENTAL MODELS USED TO INVESTIGATE THE DIFFERENTIAL INHIBITION OF CYCLOOXYGENASE-1 AND CYCLOOXYGENASE-2 BY NON-STEROIDAL ANTI-INFLAMMATORY DRUGS

Abstract. Numerous in vitro assays have been developed for testing and comparing the relative inhibitory activities of non-steroidal anti-inflammatory drugs against cyclooxygenase (COX)-1 and COX-2. Despite variability among these systems, which precludes direct comparison of data, analysis of the ratio of inhibition of COX-1 to COX-2 by non-steroidal anti-inflammatory drugs, suggests inhibitors can be classified based on their COX selectivity. Standard non-steroidal anti-inflammatory drugs can be considered nonselective; compounds such as meloxicam and nimesulide can be classified as COX-2 preferential; and compounds such as SC 58125 and L-754,337 are selective for COX-2. Although in vitro systems are important for characterizing COX-1 and COX-2 inhibitory activity, the clinical relevance of these data should be considered carefully. The level of inhibition of COX-1 and COX-2, in vivo at a given dose in patients, cannot be predicted from in vitro data alone. The pharmacokinetic properties of each compound, including plasma levels, distribution and binding to plasma proteins, have to be taken into account. Human pharmacology studies concentrating on the inhibition of prostanoid synthesis in target tissues are of paramount importance in determining the clinical relevance of COX-2 selectivity.

Differential inhibition of cyclooxygenases-1 and -2 by meloxicam and its 4′-isomer

Abstract.Objective and Design: Two structurally related compounds, meloxicam (Mel) and its structural 4′-isomer (4′-Mel), were compared to examine the role of a slightly different chemical structure on cyclooxygenase (COX) selectivity in in vitro and in vivo experimental models.¶Material or Subjects: In vitro studies were performed using human whole blood obtained from healthy volunteers, in vivo studies were performed in rats.¶Treatment: A concentration-response curve was obtained in the whole blood assay for Mel, 4′-Mel, indomethacin, piroxicam and diclofenac. These were used to calculate the respective IC50 values of either prostaglandin E2 (PGE2) or thromboxane B2 (TxB2). Similarly, a dose-response curve was obtained for Mel, 4′-Mel and piroxicam when measuring in vivo prostaglandin production, anti-inflammatory activity and gastric tolerance to determine the dose resulting in a 50% reduction of the each parameter.¶Methods: COX selectivity was investigated in vitro using a human whole blood assay. PGE2 synthesis in vivo was measured in inflammatory exudate, in the stomach and kidneys of rats. Anti-inflammatory effects were measured in an adjuvant arthritis model and gastric tolerance was tested in an ulcerogenicity model in vivo in rats.¶Results: In the human whole blood assay, the ratio of IC50 values for COX-1 vs. COX-2 inhibition was 13 for Mel and 1.8 for 4′-Mel. In inflammatory exudate in rats, Mel and 4′-Mel inhibited PGE2 synthesis to a similar extent, ID50 values ∼ 0.3 mg/kg. In contrast, Mel was a weaker inhibitor of PG synthesis than 4′-Mel in the rat stomach and in the rat kidney. Paw swelling was reduced by 50% with 0.1 and 0.2 mg/kg for Mel and 4′-Mel, respectively, in the rat adjuvant arthritis model. Gastric tolerance (UD50) was 2.4 mg/kg for Mel and 0.4 mg/kg for 4′-Mel.¶Conclusions: These data demonstrate that the in vitro and in vivo pharmacological profile of meloxicam is structurally dependent and that minor structural changes can lead to significant differences in the selectivity for COX-1 and COX-2 in vitro and to different profiles in vivo suggesting different therapeutic potential.

Reversal of dabigatran anticoagulation ex vivo: Porcine study comparing prothrombin complex concentrates and idarucizumab

Urgent surgery or life-threatening bleeding requires prompt reversal of the anticoagulant effects of dabigatran. This study assessed the ability of three- and four-factor prothrombin complex concentrate (PCC) and idarucizumab (specific antidote for dabigatran) to reverse the anticoagulant effects of dabigatran in a porcine model of trauma. Twelve animals were given dabigatran etexilate (DE) orally and dabigatran intravenously, before infliction of trauma. Six animals received tranexamic acid plus fibrinogen concentrate 12 minutes post-injury. Six PCCs (each 30 and 60 U/kg) and idarucizumab (30 and 60 mg/kg) were added to blood samples ex vivo. Coagulation was assessed by several coagulation assays. All coagulation parameters were altered after dabigatran infusion (plasma level: 442 ± 138 ng/ml). Both three- and four-factor PCCs mostly or completely reversed the effects of dabigatran on thromboelastometry variables and PT but not on aPTT. Idarucizumab neutralised plasma concentrations of dabigatran, and reversed the effects of the drug on coagulation variables. Thrombin generation showed dose-dependent over-correction following the addition of PCC, implying that elevated levels of thrombin are required to overcome dabigatran-induced coagulopathy. In contrast, treatment with idarucizumab returned thrombin generation to baseline levels. Following trauma, therapy with tranexamic acid plus fibrinogen improved correction of coagulation parameters by PCC, and thromboelastometry parameters by idarucizumab. All investigated PCCs improved dabigatran- and trauma-induced coagulopathy to a similar degree. In conclusion, this study shows that three- and four-factor PCCs are similarly effective for dabigatran reversal. Idarucizumab also reversed the effects of dabigatran and, unlike PCCs, was not associated with over-correction of thrombin generation.

Clinical experience with cyclooxygenase-2 inhibitors

Increasing amounts of experimental and clinical data support the role of selective cyclooxygenase (COX)-2 inhibition in anti-inflammatory processes and the role of COX-1 inhibition in increasing the frequency of side effects. This article reviews the regulation of COX-2 in inflammatory processes based on in vitro and in vivo work. In addition, it summarizes the various in vitro assays used to classify the new generation of selective and highly selective inhibitors of COX-2, since prior categorization of NSAIDs does not satisfactorily encompass the COX-2 concept. Finally, the latest published clinical data of new selective and highly selective inhibitors of COX-2 (meloxicam, nimesulide, etodolac, celecoxib and MK966) are discussed.

Inhibition of staphylothrombin by dabigatran reduces Staphylococcus aureus virulence

Summary.  Background: Staphylocoagulase and von Willebrand binding protein (VWbp) bind to prothrombin to form the staphylothrombin complex that converts fibrinogen into fibrin. Objectives: To study the role of staphylothrombin and its inhibition by dabigatran on Staphylococcus aureus virulence. Methods: We studied the effect of staphylothrombin inhibition on bacterial attachment to polystyrene surfaces, leukocyte activation and bactericidal activity for S. aureus ATCC 25923, S. aureus Newman, and staphylocoagulase‐ and VWbp‐negative S. aureus Newman mutants in the presence or absence of prothrombin and fibrinogen. We measured the abscess size after subcutaneous (s.c.) injection of S. aureus ATCC 25923 and S. aureus Newman, as well as an S. aureus Newman mutant strain lacking staphylocoagulase and VWbp, in mice treated with either dabigatran or placebo. Results: Staphylothrombin‐mediated fibrin increased the association of S. aureus to polystyrene surfaces and reduced the bactericidal activity of leukocytes. The absence or inhibition of staphylothrombin decreased the bacterial association, enhanced leukocyte activation and reduced bacterial survival in vitro. Abscess size was smaller in mice treated with dabigatran or infected with a coagulase‐negative mutant. Conclusion: Inhibition or the absence of staphylothrombin reduced S. aureus virulence in in vitro and in vivo models.