Preclinical Study on a Novel Fluoroderivative of Dabigatran Etexilate in Animal Models.

ABSTRACT: Here, the fluorinated derivative, R1, was synthesized from the fluorinated dabigatran derivative (R0). The in vivo pharmacokinetic characteristics of orally administered R1, R0 injection, and dabigatran etexilate in rats were compared. Safety evaluation results showed no significant changes in the QRS wave or PR and QT intervals in rat lead II electrocardiograms. The possible toxicity of R1 was studied using the limit test method, and no obvious toxicity occurred in mice after the acute oral administration of R1. R1 inhibited thrombin-induced platelet aggregation in a dose-dependent manner, had an inhibitory effect on platelet aggregation induced by arachidonic acid and adenosine diphosphate, could significantly prolong prothrombin time and activated partial thromboplastin time, and increased fibrinogen levels. R1 is the optimal candidate compound from among more than 100 candidate compounds designed and synthesized by our research group. It was first selected through preliminary in vitro anticoagulant activity screening and further through in vivo mouse activity testing. A systematic pharmacodynamic study showed that R1 was superior to the raw material drug dabigatran ester; particularly, the absolute bioavailability of R1 increased by 206%, and this can overcome the low bioavailability defect associated with the marketed drug dabigatran ester. Another safety assessment of R1 indicated that there were no risks of acute poisoning in rats and cardiac toxicity in mice or rats. Therefore, R1 can be considered a new candidate anticoagulant compound with great potential and significance for further clinical research.

Vascular protease-activated receptor 4 upregulation, increased platelet aggregation, and coronary lipid deposits induced by long-term dabigatran administration - results from a diabetes animal model.

Essentials The impact of long-term thrombin inhibition outside the coagulation cascade is far from clear. We aimed to assess the impact of dabigatran etexilate (DE) in diabetic and control rats. In diabetic rats, DE increased platelet aggregation and lead to coronary lipid deposits. Long-term thrombin inhibition may increase atherosclerotic and atherothrombotic risk. SUMMARY: Background Besides its role in the coagulation cascade, thrombin contributes to platelet aggregation and to a plethora of non-hemostatic functions. Objectives To assess the impact of long-term thrombin inhibition with dabigatran etexilate (DE) on platelet aggregation and on extrahemostatic thrombin-related functions in diabetic and control rats. Methods Markers of inflammation, endothelial dysfunction, oxidative stress, angiogenesis and cell adhesion molecules were quantified in control rats (Control; n = 6), DE-treated control rats (Control-Dabi; n = 8), diabetic rats (Diabetes; n = 5), and DE-treated diabetic rats (Diabetes-Dabi; n = 8). Agonist-induced platelet aggregation, aortic and coronary lipid deposits and aortic protease-activated receptor 4 (PAR4) expression were also assessed. Results Control-Dabi rats showed significantly higher high-sensitivity C-reactive protein, von Willebrand factor (VWF), vascular endothelial growth factor (VEGF) and fibronectin levels, and significantly lower PAR4 agonist-induced aggregation, than Control rats. Control-Dabi rats also showed mild aortic lipid deposits, whereas no such changes were observed in Control rats. Diabetes-Dabi rats showed significantly higher VWF, VEGF and fibronectin levels than Diabetes rats, and similar PAR4 agonist-induced aggregation as Diabetes rats, and significantly higher ADP-induced aggregation than Diabetes rats. Coronary lipid deposits were observed in 75% of Diabetes-Dabi rats and in none of the Diabetes rats. PAR4 expression was 20.4% higher in Control-Dabi rats and 27.4% higher in Diabetes-Dabi rats than in their non-treated peers. Conclusions This study indicates that long-term thrombin inhibition increases vascular PAR4 expression, promotes atherosclerosis-related mechanisms, and, in diabetic rats, increases platelet aggregation and favors the occurrence of coronary lipid deposits. These experimental data suggest that long-term thrombin inhibition may increase atherosclerotic and atherothrombotic risk, particularly in the presence of diabetes.

Incomplete dabigatran reversal with idarucizumab.

CONTEXT: With increasing use of direct oral anticoagulants (DOACs), urgent reversal of these agents becomes a growing concern. Idarucizumab is a humanized monoclonal antibody fragment that specifically binds to dabigatran with higher affinity than thrombin, rapidly neutralizing its anticoagulant effect without increased risk of thrombosis. CASE DETAILS: We describe two cases in which the recommended dose of idarucizumab was unsuccessful in completely reversing the anticoagulant effects of dabigatran. Both of these patients were noted to have supratherapeutic international normalized ratios (INRs) and high dabigatran concentrations. In the first case, an 86-year-old male underwent an emergent procedure and experienced excessive hemorrhaging refractory to blood product repletion, idarucizumab, and factor eight inhibitor bypass activity (FEIBA). In the second case, a 62-year-old female in shock was found to have elevated dabigatran concentrations despite two doses of idarucizumab, continuous renal replacement therapy (CRRT), blood product repletion, and FEIBA. Both patients ultimately expired from their coagulopathies. DISCUSSION: These cases illustrate the potential for incomplete reversal of dabigatran with the recommended 5 g of idarucizumab and emphasize the importance of early detection of dabigatran toxicity. While direct dabigatran serum concentrations are not readily available, the INR may be a useful surrogate marker for supratherapeutic dabigatran concentrations.

Reversing Dabigatran Anticoagulation with Prothrombin Complex Concentrate versus Idarucizumab as Part of Multimodal Hemostatic Intervention in an Animal Model of Polytrauma.

BACKGROUND: Although idarucizumab is the preferred treatment for urgent dabigatran reversal, it is not always available. Prothrombin complex concentrate (PCC) may be an alternative and, with bleeding in trauma, additional hemostatic therapy may be required. The authors investigated multimodal treatment in a preclinical polytrauma model. METHODS: Dabigatran etexilate (30 mg/kg twice daily) was given orally to 45 male pigs for 3 days. On day 4, animals received a dabigatran infusion before blunt liver injury and bilateral femur fractures. After injury, animals were randomized 1:1:1:1:1 to receive placebo (control), tranexamic acid (TXA; 20 mg/kg) plus human fibrinogen concentrate (FCH; 80 mg/kg) (TXA-FCH group), PCC (25 U/kg or 50 U/kg) plus TXA plus FCH (PCC25 and PCC50 groups), or 60 mg/kg idarucizumab (IDA) plus TXA plus FCH (IDA group). Animals were monitored for 240 min after trauma, or until death. RESULTS: The degree of injury was similar in all animals before intervention. Control and TXA-FCH animals had the highest total postinjury blood loss (3,652 ± 601 and 3,497 ± 418 ml) and 100% mortality (mean survival time 96 and 109 min). Blood loss was significantly lower in the PCC50 (1,367 ± 273 ml) and IDA (986 ± 144 ml) groups, with 100% survival. Thrombin-antithrombin levels and thrombin generation were significantly elevated in the PCC50 group. CONCLUSIONS: Idarucizumab may be considered the optimal treatment for emergency reversal of dabigatran anticoagulation. However, this study suggests that PCC may be similarly effective as idarucizumab and could therefore be valuable when idarucizumab is unavailable. (Anesthesiology 2017; 127:852-61).

Dose requirements for idarucizumab reversal of dabigatran in a lethal porcine trauma model with continuous bleeding.

Idarucizumab is licensed for emergency reversal of dabigatran. A single 5 g dose is usually sufficient, but higher doses may sometimes be required and optimum dosing has not been defined. It was the aim of this study to investigate the effect of idarucizumab, given once or as a split dose, after double trauma in pigs anticoagulated with dabigatran. Dabigatran etexilate (30 mg/kg bid) was given to 18 male pigs orally for 3 days. On day 4, animals were randomised 1:1:1 to receive idarucizumab 60+0, 60+60 or 120+0 mg/kg. Doses were administered 15 and 75 minutes after initial liver trauma. At 60 minutes, a second liver injury was undertaken. Animals were monitored for 5 hours after initial trauma or until death. Blood loss during the first hour was 990 ± 109 ml, 988 ± 84 ml and 964 ± 75 ml in the 60+0, 60+60 and 120+0 groups, respectively. In the 120+0 and 60+60 groups, total blood loss was 1659 ± 346 and 1426 ± 106 ml, respectively, and survival at 5 hours was 100 %. However, in the 60+0 group, total blood loss was 3561 ± 770 ml and survival was 50 %. Analysis of dabigatran plasma concentrations showed that equimolar concentrations of idarucizumab are necessary to bind all dabigatran and achieve sufficient thrombin generation. At sufficient doses, idarucizumab rapidly reduced blood loss and improved survival in this lethal porcine model of double trauma with dabigatran anticoagulation. In clinical practice, should bleeding continue after initial treatment with the approved 5 g dose of idarucizumab, a second dose may potentially be effective to control bleeding caused by redistribution of unbound dabigatran.

Dabigatran aggravates topoisomerase I peptide-loaded dendritic cells-induced lung and skin fibrosis.

OBJECTIVES: Dysregulated coagulation cascade has been implicated in development of fibrosis in systemic sclerosis (SSc). Thrombin, a key mediator of the coagulation pathway, has both proinflammatory and procoagulant properties. Here, we evaluated the efficacy of oral dabigatran, a direct thrombin inhibitor, on topoisomerase I dendritic cells (TOPOIA DCs)-induced lung and skin fibrosis, an experimental model of SSc. METHODS: Mice were repeatedly immunized with TOPOIA DCs. Dabigatran was administered in food either during the onset of fibrotic (late treatment) or inflammatory (early treatment) phase. RESULTS: Early administration of dabigatran caused an aggravation of pulmonary fibrosis associated with signs of severe perivascular inflammation while late treatment was not protective when compared to the untreated TOPOIA DCs group. Thrombin was increased in lungs of TOPOIA DCs immunized group and, paradoxically, further augmented by administration of dabigatran to immunized mice. As in lungs, early and not late drug administration exacerbated skin fibrosis. Moreover, early dabigatran treatment induced a profibrotic and inflammatory skin gene expression signature with upregulated expression of Col5a1, Timp1, Tweakr, Vwf, Il6, Il33, Il4 and Ifng. CONCLUSIONS: Dabigatran aggravated lung and skin fibrosis in a TOPOIA DCs-induced model of SSc-like disease. Therefore, our results argue against the use of dabigatran to treat patients with SSc.

A novel hirudin derivative inhibiting thrombin without bleeding for subcutaneous injection.

Currently, anticoagulants would be used to prevent thrombosis. Thrombin is an effector enzyme for haemostasis and thrombosis. We designed a direct thrombin inhibitor peptide (DTIP) using molecular simulation and homology modelling and demonstrated that the C-terminus of DTIP interacts with exosite I, and N-terminus with the activity site of thrombin, respectively. DTIP interfered with thrombin-mediated coagulation in human, rat and mouse plasma (n=10 per group) and blocked clotting in human whole blood in vitro. When administered subcutaneously, DTIP showed potent and dose-dependent extension of aPTT, PT, TT and CT in rats (n=10 per group). The antithrombotic dose of DTIP induced significantly less bleeding than bivalirudin determined by transecting distal tail assay in rats. Furthermore, DTIP reached peak blood concentration in 0.5-1 hour and did not cause increased bleeding after five days of dosing compared to dabigatran etexilate. The antithrombotic effect of DTIP was evaluated in mice using lethal pulmonary thromboembolism model and FeCl3-induced mesenteric arteriole thrombus model. DTIP (1.0 mg/kg, sc) prevented deep venous thrombosis and increased the survival rate associated with pulmonary thromboembolism from 30 % to 80 %. Intravital microscopy showed that DTIP (1.0 mg/kg, sc) decelerated mesenteric arteriole thrombosis caused by FeCl3 injury. These data establish that DTIP is a novel antithrombotic agent that could be used to prevent thrombosis without conferring an increased bleeding risk.

Dabigatran overdose: a case report of acute hepatitis. Extracorporeal treatment.

Dabigatran is an oral, direct thrombin inhibitor approved by international regulatory agencies for stroke prevention in patients with paroxysmal or persistent non-rheumatic atrial fibrillation (AF). The benefits of dabigatran are widely described, but its use in the geriatric population is not without risk. Chronic kidney disease is a common comorbidity with AF, and thus frequent checks of renal function in elderly patients are recommended. We report a case of dabigatran intoxication in an elderly man affected by heart failure and worsening renal function, who developed acute hepatitis and coma, which was successfully treated with continuous veno-venous hemodiafiltration. Although extracorporeal therapy has been suggested as a strategy for clearing dabigatran during acute bleeding, this approach may be useful in other dabigatran-related, life-threatening conditions, such as that described in this report.

The discovery of dabigatran etexilate for the treatment of venous thrombosis.

INTRODUCTION: Venous thromboembolism (VTE) can be life-threatening and requires anticoagulant treatment; for many years, vitamin K antagonists, e.g. warfarin, were the only oral anticoagulants available for long-term treatment. Although highly effective, they have many limitations including a slow onset, a multitude of drug-drug and drug-food interactions, and a narrow therapeutic range. These limitations spurred the search for non-vitamin K antagonist oral anticoagulants (NOACs), such as dabigatran etexilate. AREAS COVERED: The authors illustrate the progression of preclinical and clinical studies leading to the development of dabigatran, the only approved NOAC to act by direct thrombin inhibition. They focus on molecule discovery, animal models of thrombosis, clinical trials and post-launch activities in VTE treatment. EXPERT OPINION: Dabigatran demonstrated comparable efficacy to the highly effective warfarin, and a more favourable safety profile in trials of VTE treatment. A favourable anticoagulant safety profile in addition to efficacy is essential for VTE treatment. Availability of the dabigatran-specific reversal agent, idarucizumab, provides a means of rapidly reversing the anticoagulant effect if required. Future investigations into the optimal duration of VTE treatment and an evaluation of the impact of idarucizumab, in real-world studies, could provide valuable information to help optimise treatment for selected patients.

Efficacy of prothrombin complex concentrates for the emergency reversal of dabigatran-induced anticoagulation.

Dabigatran is effective in decreasing the risk of ischaemic stroke in patients with atrial fibrillation. However, like all anticoagulants, it is associated with a risk of bleeding. In cases of trauma or emergency surgery, emergency reversal of dabigatran-induced anticoagulation may be required. A specific reversal agent for dabigatran, idarucizumab, has been approved by the US Food and Drug Administration. Alternative reversal agents are available, such as prothrombin complex concentrates (PCCs) and activated PCCs (aPCCs). In this review we evaluate the role of PCCs and aPCCs in the reversal of dabigatran anticoagulation and consider which tests are appropriate for monitoring coagulation in this setting. Pre-clinical studies, small clinical studies and case reports indicate that PCCs and aPCCs may be able to reverse dabigatran-induced anticoagulation in a dose-dependent manner. However, dosing based on coagulation parameters can be difficult because available assays may not provide adequate sensitivity and specificity for measuring anticoagulation induced by dabigatran or the countering effects of PCCs/aPCCs. In addition, PCCs or aPCCs can potentially provoke thromboembolic complications. Despite these limitations and the fact that PCCs and aPCCs are not yet licensed for dabigatran reversal, their use appears to be warranted in patients with life-threatening haemorrhage if idarucizumab is not available.

Reversal of lipophilic weak bases using pH gradient acidic centre liposomes: demonstration of effect in dabigatran-induced anticoagulation.

INTRODUCTION: Liposomes have recently emerged as rational vehicles for drug detoxification. Modification of the core pH may further enhance the ability of liposomes to sequester lipophilic toxins that are weak bases. Dabigatran, a reversible inhibitor of thrombin, has been widely promoted as a novel oral anticoagulant. As a lipophilic weak-base, it provides a rational target for reversal with acidic-centred liposomal preparations. The present study tests the hypothesis that acidic centre liposomes will reverse dabigatran induced anticoagulation. METHOD: Following enteric dabigatran dosing in vitro assessment of thrombin clotting times (TCT) was undertaken in rabbit plasma spiked with incremental liposome concentrations. Tail vein bleeding was assessed following intravenous liposome injection in rats after enteric dabigatran administration. RESULTS: Liposomes achieved reversal of TCT to baseline at low levels of thrombin inhibition, and partial reversal of TCT at higher levels. Liposomes completely reversed the effects of dabigatran on rat tail vein bleeding time (134.0 (6.7) s liposomes vs. 410 (37.8) s control; p < 0.01). CONCLUSION: Dabigatran-induced coagulopathy was reversed in vitro and in vivo by acidic-centred liposomes. pH-modified liposomes are a promising investigational entity in the antidotal treatment of pharmacologic weak bases that are lipid soluble at physiologic pH.

Differential Effects of Dabigatran and Warfarin on Bone Volume and Structure in Rats with Normal Renal Function.

BACKGROUND: Warfarin, a widely used anticoagulant, is a vitamin K antagonist impairing the activity of vitamin K-dependent Bone Gla Protein (BGP or Osteocalcin) and Matrix Gla Protein (MGP). Because dabigatran, a new anticoagulant, has no effect on vitamin K metabolism, the aim of this study was to compare the impact of warfarin and dabigatran administration on bone structure and vascular calcification. METHODS: Rats with normal renal function received for 6 weeks warfarin, dabigatran or placebo. Bone was evaluated immuno-histochemically and hystomorphometrically after double labelling with declomycin and calcein. Aorta and iliac arteries were examined histologically. RESULTS: Histomorphometric analysis of femur and vertebrae showed significantly decreased bone volume and increased trabecular separation in rats treated with warfarin. Vertebra analysis showed that the trabecular number was higher in dabigatran treated rats. Osteoblast activity and resorption parameters were similar among groups, except for maximum erosion depth, which was higher in warfarin treated rats, suggesting a higher osteoclastic activity. Therefore, warfarin treatment was also associated with higher bone formation rate/bone surface and activation frequency. Warfarin treatment may cause an increased bone turnover characterized by increased remodelling cycles, with stronger osteoclast activity compared to the other groups. There were no differences among experimental groups in calcium deposition either in aortic or iliac arteries. CONCLUSIONS: These findings suggest for the first time that dabigatran has a better bone safety profile than warfarin, as warfarin treatment affects bone by reducing trabecular size and structure, increasing turnover and reducing mineralization. These differences could potentially result in a lower incidence of fractures in dabigatran treated patients.

Design, Synthesis, and Biological Evaluation of Dabigatran Etexilate Mimics, a Novel Class of Thrombin Inhibitors.

Human α-thrombin is a particularly promising target for anticoagulant therapy, and identification of oral small-molecular inhibitors of thrombin remains a research focus. On the basis of the X-ray crystal structure of human α-thrombin and its inhibitor dabigatran, we designed and synthesized a series of dabigatran etexilate mimics containing a novel tricyclic fused scaffold. The biological evaluations reveal that all of the compounds possess moderate activity of antiplatelet aggregation induced by thrombin in vitro. Moreover, compound I-8, which contains 2-hydroxymethyl-3,5,6-trimethylpyrazine (HTMP), a cleavable moiety with antiplatelet activity, shows the best anticoagulant effect among the tested compounds in vivo. Those synthesized compounds that have better in vitro activity were subjected to bleeding complication tests, and the results demonstrate that the novel compounds are less likely to have bleeding risk than dabigatran etexilate.