Dabigatran Concentrations Research Articles

Idarucizumab for Dabigatran Reversal

BackgroundSpecific reversal agents for non–vitamin K antagonist oral anticoagulants are lacking. Idarucizumab, an antibody fragment, was developed to reverse the anticoagulant effects of dabigatran.MethodsWe undertook this prospective cohort study to determine the safety of 5 g of intravenous idarucizumab and its capacity to reverse the anticoagulant effects of dabigatran in patients who had serious bleeding (group A) or required 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 determination at a central laboratory of the dilute thrombin time or ecarin clotting time. A key secondary end point was the restoration of hemostasis.ResultsThis interim analysis included 90 patients who received idarucizumab (51 patients in group A and 39 in group B). Among 68 patients with an elevated dilute thrombin time and 81 with an elevated ecarin clotting time at baseline, the median maximum percentage reversal was 100% (95% confidence interval, 100 to 100). Idarucizumab normalized the test results in 88 to 98% of the patients, an effect that was evident within minutes. Concentrations of unbound dabigatran remained below 20 ng per milliliter at 24 hours in 79% of the patients. Among 35 patients in group A who could be assessed, hemostasis, as determined by local investigators, was restored at a median of 11.4 hours. Among 36 patients in group B who underwent a procedure, normal intraoperative hemostasis was reported in 33, and mildly or moderately abnormal hemostasis was reported in 2 patients and 1 patient, respectively. One thrombotic event occurred within 72 hours after idarucizumab administration in a patient in whom anticoagulants had not been reinitiated.ConclusionsIdarucizumab completely reversed the anticoagulant effect of dabigatran within minutes. (Funded by Boehringer Ingelheim; RE-VERSE AD ClinicalTrials.gov number, NCT02104947.)

Dabigatran Concentration: Variability and Potential Bleeding Prediction In "Real-Life" Patients With Atrial Fibrillation.

Routine laboratory monitoring is currently not recommended in patients receiving dabigatran despite its considerable variation in plasma concentration. However, in certain clinical situations, measurements of the dabigatran effect may be desirable. We aimed to assess the variability of dabigatran trough and peak concentration and explore the potential relationship between dabigatran concentration and adverse events. We included 44 patients with atrial fibrillation who started treatment with dabigatran 150 mg (D150) or 110 mg (D110) twice daily. They contributed 170 trough and peak blood samples that were collected 2-4 and 6-8 weeks after dabigatran initiation. Plasma dabigatran concentration was measured by LC-MS/MS and indirectly, by selected coagulation tests. D110 patients were older (74 ± 7 versus 68 ± 6 years), had lower creatinine clearance (68 ± 21 versus 92 ± 24 mL/min) and higher CHA2 DS2 -VASc score (3.1 ± 1.3 versus 2.3 ± 0.9) compared to D150 patients (all p < 0.05), but both had similar dabigatran concentrations in both trough and peak samples. Dabigatran concentrations varied less in trough than in peak samples (17.0 ± 13.6 versus 26.6 ± 19.2%, p = 0.02). During the 12-month follow-up, 4 patients on D150 and 6 on D110 suffered minor bleeding. There was no major bleeding or thromboembolic event. Patients with bleeding had significantly higher average trough dabigatran concentrations (93 ± 36 versus 72 ± 62 μg/L, p = 0.02) than patients without bleeding, while peak dabigatran values had no predictive value. Dabigatran dose selection according to the guidelines resulted in appropriate trough concentrations with acceptable repeatability. High trough concentrations may predispose patients to the risk of minor bleeding.

Characterization of thrombin-bound dabigatran effects on protease-activated receptor-1 expression and signaling in vitro.

Thrombin, the key effector protease of the coagulation cascade, drives fibrin deposition and activates human platelets through protease-activated receptor-1 (PAR1). These processes are critical to the progression of thrombotic diseases. Thrombin is the main target of anticoagulant therapy, and major efforts have led to the discovery of new oral direct inhibitors of thrombin. Dabigatran is the first oral anticoagulant licensed for the prevention of thromboembolisms associated with orthopedic surgery and stroke prevention in atrial fibrillation. Dabigatran is a direct thrombin inhibitor that effectively blocks thrombin's catalytic activity but does not preclude thrombin's exosites and binding to fibrinogen. Thus, we hypothesized that catalytically inactive thrombin retains the capacity to bind to PAR1 through exosite-I and may modulate its function independent of receptor cleavage and activation. Here, we report that dabigatran at clinically relevant concentrations is an effective and acute inhibitor of thrombin-induced PAR1 cleavage, activation, internalization, and β-arrestin recruitment in vitro. Interestingly, prolonged exposure to catalytic inactive thrombin incubated with dabigatran at 20-fold higher therapeutic concentration resulted in increased PAR1 cell-surface expression, which correlated with higher detectable levels of ubiquitinated receptor. These findings are consistent with ubiquitin function as a negative regulator of PAR1 constitutive internalization. Increased PAR1 expression also enhanced agonist-induced phosphoinositide hydrolysis and endothelial barrier permeability. Thus, catalytically inactive thrombin appears to modulate PAR1 function in vitro by stabilizing receptor cell-surface expression; but given the high clearance rate of thrombin, the high concentration of dabigatran required to achieve this effect the in vivo physiologic relevance is unknown.

Quantification of dabigatran and indirect quantification of dabigatran acylglucuronides in human plasma by LC-MS/MS.

An assay for the quantification of dabigatran and its active metabolites, dabigatran acylglucuronides, has not previously been described in detail. For the quantification of total dabigatran concentration (free dabigatran and acylglucuronides), samples were subjected to alkaline hydrolysis. For the quantification of free dabigatran, samples were acidified with ammonium formate. Following acetonitrile protein precipitation, the samples were analyzed by LC-MS/MS using gradient elution to ensure separation of dabigatran from dabigatran acylglucuronides. Mean recoveries ≥98% were achieved. The assay was validated over the range 2.5-1000 ng/ml dabigatran, imprecision was <9% CV (<15% at LLOQ) and accuracy was 101-114%. An assay for dabigatran with indirect quantification of dabigatran acylglucuronides in plasma was developed, validated and applied.

Relation Between Dabigatran Concentration, as Assessed Using the Direct Thrombin Inhibitor Assay, and Activated Clotting Time/Activated Partial Thromboplastin Time in Patients With Atrial Fibrillation

Dabigatran is a direct thrombin inhibitor that has been approved for preventing stroke in patients with atrial fibrillation. In this study, we aimed to assess the associations between the dabigatran concentration (calculated through plasma-diluted thrombin time, as assessed using the Hemoclot assay) and the activated partial thromboplastin time (aPTT) and activated clotting time (ACT). We recruited 137 patients with atrial fibrillation who were receiving a normal dose of dabigatran (300mg/d) or a reduced dose of dabigatran (220mg/d, usually administered to patients who were elderly, had moderate renal dysfunction, or who were also receiving verapamil). We then assessed the aPTT, ACT, and Hemoclot results of the patients and calculated the plasma dabigatran concentration. The mean plasma concentration of dabigatran was 127 ± 88ng/ml, although no significant differences in dabigatran concentration, ACT, or aPTT were observed when we compared the 2 doses of dabigatran (300 or 220mg/d). The dabigatran concentration was within the therapeutic levels in most patients, although a high value (>300ng/ml) was observed in several patients, which indicated a high risk of bleeding. The dabigatran concentration was strongly and positively correlated with ACT and aPTT (r= 0.87, p <0.001; and r= 0.76, p <0.001; respectively). Multivariate analysis revealed that verapamil use was independently associated with elevated dabigatran concentrations (p <0.001). Therefore, ACT and aPTT may be useful for bedside assessment of the anticoagulant activity of dabigatran, and verapamil use may be a risk factor for elevated dabigatran concentrations.

Concomitant use of dronedarone with dabigatran in patients with atrial fibrillation in clinical practice

IntroductionDronedarone is a strong P-glycoprotein inhibitor with a potential to increase bioavailability of dabigatran. We sought to measure and report plasma concentrations of dabigatran in patients with atrial fibrillation (AF) on concomitant dronedarone treatment. Materials and methodsA cohort of 33 patients (mean age 64years, 16 men) concomitantly treated with dabigatran at a dose of 110mg twice a day (bid) and dronedarone at a dose of 400mg bid at the discretion of the patient’s cardiologist were followed prospectively. ResultsMedian trough plasma concentration of dabigatran at one week and one month after the concomitant treatment start was 102.0 (range 8-251) ng/ml and 84 (range 27-302) ng/ml respectively. Median treatment length was 13 (range 1-21) months. There was one major bleeding event (2,8% per patient-year) and no thrombotic events during a total of 35.5 patient-years. ConclusionsMedian trough plasma concentration of dabigatran in our study was observed to be similar to median trough plasma concentration of dabigatran at a dose of 150mg bid without concomitant dronedarone in earlier studies with low reported rate of bleeding and thrombosis. Since concomitant treatment offers potential benefits to patients with AF, larger future trials that might refute the current contraindication are warranted.

Comparison of intermittent and continuous extracorporeal treatments for the enhanced elimination of dabigatran

Context. Severe bleeding associated with dabigatran frequently requires intensive care management. An antidote is currently unavailable and data reporting the effect of dialysis on elimination of dabigatran are encouraging, but limited. Objective. To report the effect of intermittent hemodialysis (IHD) and continuous renal replacement therapy (CRRT) at enhancing elimination of dabigatran. Materials and methods. Patients were identified by existing collaborative networks. Pre-filter dabigatran plasma concentrations were measured in all patients, and in dialysate of three patients. Results. Seven patients received dialysis, five with active bleeding and two requiring emergent surgery. Five received IHD and two received CRRT. The plasma elimination half-life of dabigatran was 1.5–4.9 h during IHD, and 14.0–27.5 h during CRRT. Mean dabigatran plasma clearance during IHD was 85–169 mL/min in three patients. Time to obtain a subtherapeutic dabigatran concentration depended on the initial concentration, being 8–18 h for IHD in three patients while 4 h was insufficient in a supratherapeutic case. A 38% rebound in dabigatran levels occurred after one case during IHD, and thrombin time increased after IHD in another, but not after 144 h CRRT or 17 h IHD in two others; data were incomplete in three cases. The amount removed during IHD was proportional to the pre-IHD concentration and clearance, but was consistently low at 3.3–17.4 mg in three patients where this was determined. Moderate bleeding occurred while obtaining vascular access in one patient. Two patients died from intracerebral bleeding, and the influence of treatments could not be determined in these cases. Discussion and conclusions. IHD enhanced elimination of dabigatran more efficiently than CRRT, but their net effect remains poorly defined. Dialysis decisions, including modality and duration, must be individualized based on a risk–benefit assessment.

Venovenous haemodiafiltration for the management of dabigatran overdose in intensive care unit.

Dabigatran is a direct thrombin inhibitor indicated for thromboembolism prophylaxis in patients with non-valvular atrial fibrillation. The procedure to manage dabigatran-associated haemorrhages is not well formalized. Conventional haemodialysis has been evaluated with good results. Patients with dabigatran-associated bleeding may be unstable and convective techniques like venovenous haemodiafiltration (HDF) can be interesting. We report the case of a 74-year-old, critically ill patient with haemorrhagic shock and dabigatran overexposure due to acute kidney injury. He underwent HDF and dabigatran blood concentrations decreased from 325.3 ng/mL to 160.5 ng/mL. We report here key pharmacokinetics parameters (half-life, extraction coefficient, clearance).

Assessing the anticoagulant effect of dabigatran in children: An in vitro study

ObjectivesAge-related changes in the hemostatic system result in variation in response to anticoagulants and coagulation assays over childhood. This study used in vitro methods to determine i) optimum coagulation assays for dabigatran in children and ii) anticoagulant effect of dabigatran across pediatric age groups. Materials and MethodsPooled plasma samples from healthy children aged 0 to <1, 1 to <5, 5 to <10, 10 to <17years and adults were spiked with increasing concentrations of dabigatran and the effect was assessed in five coagulation assays. The samples were also assessed for overall hemostasis potential using a fibrin clot formation and lysis assay. ResultsIn all five coagulation assays, there were no differences in responses to dabigatran over all pediatric age groups. The international normalized ratio was the least sensitive measure. Activated partial thromboplastin time showed moderate sensitivity and a nonlinear response curve. Thrombin time (TT), dilute TT (dTT) and ecarin clotting time were linearly correlated with dabigatran concentrations; however, the ecarin time and TT were overly sensitive. In the overall hemostasis potential assay, increasing dabigatran concentrations delayed the initiation of clot formation and reduced the time to 50% clot lysis. The responses to initiation of clot formation and clot lysis were consistent across all pediatric groups and comparable to responses in adults. ConclusionThe dTT is the most suitable assay for measuring dabigatran concentrations in children. Fibrin clot generation and lysis assay responses to dabigatran in children over all ages were consistent and comparable to those of adults.

Measurement of dabigatran plasma concentrations by calibrated thrombin clotting time in comparison to LC-MS/MS in human volunteers on dialysis

IntroductionDabigatran etexilate is an oral direct thrombin inhibitor. Although routine anticoagulation monitoring with dabigatran is not usually required, a simple and precise laboratory test to measure dabigatran concentrations in patient plasma may be useful in certain clinical circumstances, such as emergency situations. The HEMOCLOT® Thrombin Inhibitors assay has demonstrated accurate and precise determination of dabigatran concentrations within a range of 50–500ng/ml. The objective of this study was to assess comparability of dabigatran concentrations determined by HEMOCLOT® and by liquid chromatography/tandem mass spectrometry (LC-MS/MS) in plasma samples from human volunteers with end-stage renal disease (ESRD) undergoing regular haemodialysis (HD) during a Phase I study. Materials and MethodsOverall, 304 plasma samples were obtained from seven ESRD patients in dabigatran steady-state for measurement by HEMOCLOT® (calibrated diluted thrombin time [dTT]) and by LC-MS/MS. Agreement of dabigatran concentrations was assessed by regression analysis and difference plots. ResultsThe measurements of calibration standards of the HEMOCLOT® assay showed excellent precision with coefficients of variation <5%. Accuracy determined by analysis of two quality control samples was 90% and 111%. HEMOCLOT®-derived dabigatran plasma concentrations paralleled those obtained by LC-MS/MS. The mean ratio of the LC-MS/MS and dTT-derived concentrations was 0.955 (67% limits of agreement: 0.771–1.18). ConclusionsThe HEMOCLOT® Thrombin Inhibitors assay is suitable for measuring dabigatran plasma concentrations in volunteers with ESRD undergoing haemodialysis. The agreement between dabigatran concentrations determined by the HEMOCLOT® assay and the LC-MS/MS reference method met bioanalytical acceptance criteria.

Relationship between plasma dabigatran concentration and activated partial thromboplastin time in Japanese patients with non-valvular atrial fibrillation

BackgroundActivated partial thromboplastin time (aPTT) is recommended for monitoring anticoagulant activity in dabigatran-treated patients; however, there are limited data in Japanese patients. To clarify the relationship between plasma dabigatran concentration and aPTT, we analyzed plasma dabigatran concentration and aPTT at various time points following administration of oral dabigatran in a Japanese hospital. MethodsWe enrolled 149 patients (316 blood samples) with non-valvular atrial fibrillation (NVAF) who were taking dabigatran. Patients had a mean age of 66.6±10.0 years (range: 35–84) and 66% were men. Plasma dabigatran concentrations and aPTT were measured using the Hemoclot® direct thrombin inhibitor assay and Thrombocheck aPTT-SLA®, respectively. Samples were classified into eight groups according to elapsed times in hours since oral administration of dabigatran. ResultsSignificantly higher dabigatran concentrations were observed in samples obtained from patients with low creatinine clearance (CLCr) (CLCr<50mL/min). Dabigatran concentrations and aPTT were highest in the 4-h post-administration range. Additionally, there was a significant correlation between plasma dabigatran concentrations and aPTT (y=0.063x+32.596, r2=0.648, p<0.001). However, when plasma dabigatran concentrations were 200ng/mL or higher, the correlation was lower (y=0.040x+38.034 and r2=0.180); these results were evaluated by a quadratic curve, resulting in an increased correlation (r2=0.668). ConclusionsThere was a significant correlation between plasma dabigatran concentrations and aPTT. Additionally, in daily clinical practice in Japan, plasma dabigatran concentrations and aPTT reached a peak in the 4-h post administration range. Considering the pharmacokinetics of dabigatran, aPTT can be used as an index for risk screening for excess dabigatran concentrations in Japanese patients with NVAF.

The Measure of Plasma Concentrations Is Crucial in Managing Dabigatran Treatment in Non-Valvular Atrial Fibrillation

One of the most popular features utilized to promote direct oral anticoagulants diffusion in stroke prevention in non-valvular atrial fibrillation is that these drugs do not require monitoring of their anticoagulant activity or plasma level measurements. We believe that there are clinical scenarios in which monitoring can help guide clinical management. Here we describe a clinical case in which close monitoring of thrombin inhibitor dabigatran peak plasma levels helped in maintaining the patient drug administration schedule, despite the interference of mirabegron, a beta-3 adrenergic agonist, utilized for the symptomatic treatment overactive bladder (OAB) syndrome, and of amiodarone, an effective and commonly prescribed antiarrhythmic drug. These P-glycoprotein (P-gp) inhibitors drugs may increase the blood levels and effects of dabigatran. Combining these medications may increase the risk of bleeding complications and anemia, especially when associated to mild to moderate renal insufficiency.Dabigatran etexilate is a pro-drug that has a low (3%-7%) bioavailability. Once dabigatran etexilate is absorbed, the pro-drug is hydrolyzed to the active drug, dabigatran, by carboxylesterases in the bloodstream. Dabigatran etexilate is a substrate of the efflux transporter P-gp, but dabigatran, the active drug, is not. P-gp is present on the luminal side of absorptive cells in the small intestine and takes drug molecules from the cell cytoplasm and transports them back into the intestinal lumen for excretion. An inhibitor of P-gp, as mirabegron or amiodarone, will increase bioavailability of a P-gp substrate by reducing drug efflux.Our patients suffers from persistent non-valvular atrial fibrillation, complicated by ischemic stroke. He has been assigned a dabigatran 150 mg b.i.d. treatment in may 2014. His creatinine clearance was 75 mL/min. One month later, due to OAB syndrome, he started mirabegron, a drug which might potentially increase the risk of bleeding and which generally causes to stop dabigatran administration. We decided instead to set up a monitoring plan, utilizing plasma-diluted thrombin time to measure dabigatran concentrations. Calibration material consisted of pooled normal plasma with known quantities of dabigatran. By using this calibration material, we constructed a dose-response curve ranging from 0 to 500 ng/mL of dabigatran. An examination of the published literature of the pharmacology of dabigatran indicated that the expected peak steady-state concentration of dabigatran in patients with atrial fibrillation (150 mg, 2 times daily) was approximately 180 ng/mL with a trough of approximately 90 ng/mL (12 hours after last dose).After 4 weeks of mirabegron administration, dabigatran plasma trough level of our patient was 126.4 ng/mL. We therefore decided not to modify the 150 mg b.i.d. treatment.Three weeks later, patient suffered from acute cardiac failure associated to a creatinine clearance reduction to 41 mL/min. As a consequence, mirabegron treatment was stopped. At this point, dabigatran plasma trough level was 148.6 ng/mL. Again, we decided not to modify the 150 mg b.i.d. treatment.Six weeks later, due to an elevated cardiac rate (140/min) a treatment with 300 mg o.d. of amiodarone was started. A week later dabigatran plasma trough level was 219.6 ng/mL, with a creatinine clearance of 58mL/min. We decided then to reduce the administration of dabigatran to 110 mg twice daily.After two weeks, dabigatran plasma trough level lowered to 120.2 ng/mL.Our patient is still on dabigatran treatment which has so far never been stopped.At this time, studies addressing the correlation of dabigatran concentration with risk of bleeding (overdose) or breakthrough thrombosis (underdosing) are unavailable. The lack of such studies currently limits the predictive power of the plasma-diluted thrombin time to determine hemorrhage/thrombosis risk. However, we believe that in situations like our patient’s the adoption of this assay would be a valuable tool to aid adjusting dabigatran treatment. With access to appropriate pharmacodynamic and pharmacokinetic data and relevant calibration material, the plasma–diluted thrombin time assay can easily be applied for use in the monitoring dabigatran etexilate. DisclosuresNo relevant conflicts of interest to declare.

Estimation of Dabigatran Plasma Concentrations in the Perioperative Setting. an Ex-Vivo Study Using Dedicated Coagulation Assays

▪BACKGROUNDDabigatran etexilate has received its market authorization for various indications worldwide. It was developed to be used in fixed dose regimens without the need of regular monitoring. However, the perioperative management of dabigatran could require an assessment of the drug plasma levels to ensure a safe use of the product, especially in absence of specific antidotes. The EMA stated that dabigatran concentrations under 48 ng/mL should be reached before invasive intervention. The GIHP put the threshold at 30 ng/mL. Therefore, a specific laboratory assay, accurate in the low concentration range, easily available and performable 24h/7 is requested but, until now, all coagulation tests dedicated to the measurement of plasma dabigatran concentrations showed a lower limit of quantitation from 30 to 50 ng/mL. This limits their perioperative utility and the thrombin time (TT) is currently presented as an alternative due to its very high sensitivity to dabigatran.This interesting approach has limitations because TT is affected by several analytical and biological variables that could lead to misinterpretations and expose the patient at riskier hemostatic conditions. In this study, we propose to investigate the performance of two coagulation tests (the Hemoclot Thrombin Inhibitors LOW (HTI LOW) (Hyphen BioMed) and the Ecarin Chromogenic Assay II (ECA-II) (Diagnostica Stago)) specifically developed to measure low plasma dabigatran concentrations and to compare their results with a reference LC-MS/MS. We also assessed the performance of the standard procedure of HTI and TT.MATERIALS AND METHODSThirty-three plasma samples of patients treated with dabigatran etexilate for stroke prevention in non-valvular atrial fibrillation, were included in the study. Plasma samples were taken randomly and included after a first screening using the HTI to select plasma concentrations <200 ng/mL.For HTI, tested plasma was diluted 1:8 in Owren-Koller® buffer. Fifty μl of tested plasma were mixed with 100 μl of normal pooled plasma and were incubated during 240 sec. One hundred μl of highly purified human thrombin pre-incubated at 37°C was then added to start the reaction. For HTI LOW, the dilution of the sample was reduced to 1:2 and specific calibrators at lower concentrations were used.For ECA-II, tested plasma was diluted 1:5 in Owren-Koller® buffer. Fifty μl of tested plasma were mixed with 140 μl of prothrombin and then 70 µl of chromogenic substrate were added and incubated during 240 sec. Seventy μl of ecarin pre-incubated at 37°C were then added to start the reaction.Thrombin time was performed using Thrombin Time® reagent 1.5NIH (Diagnostica Stago) and the limit of measurement was extended to 300 sec.All of these procedures were performed on a STA-R Evolution® coagulometer (Diagnostica Stago).RESULTS AND DISCUSSIONThe plasma concentrations ranged from 0 to 200 ng/mL as provided by LC-MS/MS measurements. Among these samples, 17 were between 0 and 50 ng/mL. Linear correlation, Spearman correlation and Bland-Altman analyses versus LC-MS/MS are provided in Figure 1, for assays that express results in ng/mL, i.e. HTI, HTI LOW and ECA-II. For TT, the relation is described by linear correlation.Our results show that HTI LOW performs better than HTI and ECA-II on the whole concentration range providing closer correlation and a lower systematic difference compared to LC-MS/MS. For concentrations below 50 ng/mL HTI LOW and ECA-II reveal similar systematic difference with higher 95% CI for ECA-II and perform better than HTI to estimate plasma concentrations. Thrombin time is less useful than dedicated assays to provide an estimation of the pharmacodynamics of dabigatran. For concentrations above 50 ng/mL it often exceeds the limit of measurement, i.e. 120 sec as defined by the manufacturer, and is influenced by the level of fibrinogen. However, it gives an acceptable correlation for concentration below 50 ng/mL. This may help the biologist to choose which test to use to avoid unnecessary costs and ensure the use of the more accurate dedicated assay to estimate the plasma concentrations. When TT is lower than 120 sec, HTI LOW or ECA-II should be preferred to HTI.CONCLUSIONWe recommend the use of specific coagulation assays to assess the dabigatran plasma concentrations before invasive procedure. Thrombin time may guide the biologist on the dedicated coagulation test to perform if he uses the HTI platform. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Idarucizumab, a Specific Antidote for Dabigatran: Immediate, Complete and Sustained Reversal of Dabigatran Induced Anticoagulation in Elderly and Renally Impaired Subjects

IntroductionOral anticoagulation is an effective therapy to prevent and treat thromboembolic events. So far, Vitamin K antagonists have been the main drug of choice. Recently, the advent of the direct oral anticoagulants (DOAC) has changed medical practice significantly; nevertheless all anticoagulants are associated with an increased risk of bleeding. Bleeding management can be achieved through established therapies; however specific antidotes are not yet available for these agents to further facilitate patient management in cases needed. Previously the dabigatran antidote (idarucizumab) has demonstrated immediate, complete and sustained reversal of dabigatran induced anti-coagulation in healthy male volunteers. In the present study it was determined whether and to what extent doses of up to 5 g idarucizumab would reverse the anticoagulant effects of dabigatran in male and female healthy mid-aged, elderly and renally impaired volunteers. In addition, it was tested whether oral intake of dabigatran etexilate 24 hrs after idarucizumab treatment could restore dabigatran related anticoagulation. It was further tested if a second administration of idarucizumab 2 months later was safe and well tolerated.MethodsSafety, tolerability, pharmacokinetics (PK) and pharmacodynamics (PD) of idarucizumab were investigated in a randomized, double-blind, placebo controlled two-way cross-over study in 46 male and female volunteers. Dabigatran etexilate (DE), 220 mg bid in healthy subjects and 150 mg bid in subjects with mild or moderate renal impairment (CLCR60 to <90 or 30 to <60 [mL/min], respectively) was given over 4 days to achieve the steady state conditions. Idarucizumab doses of 1 g, 2.5 g, 5 g or 5 g given as 2x2.5 g one hour apart were administered as 5 min i.v. infusion 2 hrs after the last dose of DE. Concentrations of unbound dabigatran were determined as a measure of pharmacologically active dabigatran. The anticoagulant effect of dabigatran and its reversal were assessed by coagulation time measurements, including diluted Thrombin Time (dTT, Hemoclot® DTI assay), Ecarin Clotting Time (ECT) and activated Partial Thromboplastin Time (aPTT).ResultsAll administered doses of idarucizumab were safe and well tolerated. PK measurements of unbound dabigatran indicated that idarucizumab binding and thus reversal of the anticoagulant effect of dabigatran occurred immediately after end of infusion. Prolongation of clotting times induced by dabigatran was reversed to baseline at the end of the 5 minute infusion of the antidote. This was consistently demonstrated by all clotting assays. Sustained reversal over the entire observation period was observed for idarucizumab doses of 2.5 g, 5 g and 2x2.5 g. For the 1g dose, there was partial return of dabigatran induced anticoagulation around 2-4 hours after i.v. infusion. Also a second administration of idarucizumab (two months after the first) was safe and resulted in complete reversal. In addition, PD and PK measurements at selected time points and in comparison to placebo treatment confirmed that effective dabigatran anticoagulation could be re-established 24 hours after administration of idarucizumab.ConclusionsThe dabigatran antidote, idarucizumab, was well tolerated under all conditions tested. The administration of 5 g or 2x2.5 g led to sustained reversal of dabigatran induced anticoagulation in male and female subjects of different age and renal function. In addition, idarucizumab administered 2 months apart achieved the same degree of reversal. Dabigatran anticoagulation could be re-established 24 hrs after idarucizumab dosing. These results support the use of a total dose of 5 g idarucizumab as an effective dose in further clinical testing. DisclosuresGlund:Boehringer Ingelheim: Employment. Off Label Use: Idarucizumab, a specific antidote for dabigatran, is in clinical development.. Stangier:Boehringer Ingelheim: Employment. Schmohl:Boehringer Ingelheim: Employment. Moschetti:Boehringer Ingelheim: Employment. Haazen:SGS Life Science Services (contracted by Boehringer Ingelheim to conduct the study): Employment. De Smet:SCS Boehringer Ingelheim Comm. V.: Employment. Gansser:Boehringer Ingelheim: Employment. Norris:Boehringer Ingelheim: Employment. Lang:Boehringer Ingelheim: Employment. Reilly:Boehringer Ingelheim: Employment.

Development and Validation of a Chromogenic Direct Thrombin Inhibitor Assay on the ACL Top 700 for Quantifying Dabigatran Levels in Plasma

Dabigatran is a potent, competitive, and reversible direct thrombin inhibitor, that binds to the active site of thrombin, inhibiting both free and clot-bound thrombin. It is the active form of dabigatran etexilate, which is a low molecular weight prodrug metabolized to its active form after oral administration. Although patients on therapeutic doses of dabigatran do not require routine coagulation monitoring due to its dose dependent and predictable pharmacological profile, there may be situations in which it may be beneficial to be able to accurately measure the degree of anticoagulation (ie. urgent surgery, severe bleeding, thrombosis despite treatment, overdose, bridging with other anticoagulants, patients with a high risk of dabigatran accumulation or potential drug interactions). Accordingly, we developed and validated a chromogenic direct thrombin inhibitor assay on the ACL TOP 700 automated hemostasis analyzer for quantifying dabigatran levels in human plasma.Our test principle was based on the in vitro thrombin inhibition by dabigatran, in which excess thrombin was added to the plasma sample, the thrombin in the sample was neutralized in proportion to the amount of dabigatran, and the residual thrombin hydrolyzed the chromogenic substrate releasing pNA which was measured photometrically at 405 nm on the ACL TOP 700. This assay was calibrated with purchased dabigatran calibration samples (Aniara). Each calibration curve consisted of five points, used a 3rd order polynomial curve fit, and was performed each time an assay was run. For all runs, an r2 of 1.0 was observed and all calibrators demonstrated an acceptable accuracy and precision (± 10% from assigned value for samples ≥ 100 ng/mL or < 10 ng/mL of assigned values for all other samples).Validation of this dabigatran level assay for accuracy, intra- and inter-assay precision, analytical specificity, analytical sensitivity, analyte stability, and robustness was completed and reportable result ranges established. To evaluate accuracy, three plasma samples containing dabigatran in the range of expected concentrations (high, medium, low) were compared to the value determined by mass spectrometry. Intra- and inter-assay precision was determined by analyzing these 3 samples over multiple assay runs spanning multiple days. All determined values were within 15% of the assigned values for samples ≥ 100ng/mL and within 10ng/mL of the assigned values for all other samples. Analytical specificity was confirmed by running sample pairs (plasma/serum, citrate/EDTA, hemolyzed/non-hemolyzed, icteric/non-icteric, lipemic/cleared-lipemic, 1 U/mL unfractionated heparin and 2 U/mL low molecular weight heparin-containing samples). Only the serum sample resulted in >10% difference from the standard plasma, indicating assay interference. Assay results were not affected by unfractionated heparin up to 1 U/mL and low molecular weight heparin up to 2 U/mL. Analytical sensitivity was established by assaying normal plasma containing no or low levels of dabigatran. The limit of blank was determined to be 3 ng/mL, limit of detection 10 ng/mL, and limit of quantitation (LoQ) 20 ng/mL. Analyte stability studies established samples thawed and refrozen up to 2 times were acceptable. Assay robustness was determined to be acceptable. The reportable range was determined to be 20-900 ng/mL based on the calibration curve and LoQ.In summary, we have developed and validated an accurate, precise, sensitive and robust chromogenic assay on the ACL TOP 700 for the determination of dabigatran concentration in human plasma. This assay may prove useful in certain clinical circumstances (urgent surgery, severe bleeding, or thrombosis despite treatment) for the assessment of anticoagulation status. DisclosuresNo relevant conflicts of interest to declare.

Enhanced elimination of dabigatran through extracorporeal methods.

Several pharmacokinetic studies have suggested that dabigatran possesses a number of ideal properties for expedited removal via extracorporeal methods. However, this practice has not been prospectively evaluated in patients with life-threatening bleeding or requiring emergency surgery secondary to dabigatran-associated coagulopathy. The purpose of this literature review is to evaluate the published evidence surrounding extracorporeal removal of dabigatran in the setting of emergency surgery or life-threatening bleeding. A query of MEDLINE, Web of Science, International Pharmaceutical Abstracts, and Google Scholar using the terms dabigatran, dabigatran etexilate, hemodialysis, renal replacement therapy, hemorrhage, and atrial fibrillation was used to retrieve relevant literature. Furthermore, a manual search of the references of the identified literature was performed to capture additional data. Current evidence suggests that extracorporeal removal of dabigatran may play a role in the setting of life-threatening bleeding and emergent surgery. Conflicting evidence exists with regard to the potential for redistribution based on serum dabigatran concentrations. In addition, a number of practicalities must be considered before incorporating this technique in the clinical setting. Extracorporeal removal of dabigatran may be a treatment modality in selected patients who require emergency reversal.

CHA2DS2-VASc and HAS-BLED scores and activated partial thromboplastin time for prediction of high plasma concentration of dabigatran at trough

IntroductionAlthough dabigatran, an oral direct thrombin inhibitor, does not require routine monitoring, high plasma concentration of dabigatran (PDC) at trough level is shown to be a high risk for bleeding in patients with nonvalvular atrial fibrillation (NVAF). As dabigatran prolongs the activated partial thromboplastin time (APTT), we examined relationships of PDC at trough with APTT and clinical features to identify patients at high risk for major bleeding during dabigatran treatment. Materials and MethodsIn the consecutive 48 patients with NVAF taking dabigatran at a daily dose of 220mg (n=32) or 300mg (n=16), we measured PDC using HEMOCLOT Thrombin Inhibitor assay and APTT ratio to control before (trough) and 2hours after taking dabigatran. ResultsPDC was positively correlated with APTT ratio (R2=0.64, p<0.0001). Using this regression equation and values of median trough PDC 116 (46.7-269) ng/mL observed in patients with major bleeding in the RE-LY trial, we calculated the expected value of APTT ratio corresponding to the 10th percentile of trough PDC (46.7). It was 1.20. There was a significant increase in trough PDC with increasing CHA2DS2-VASc score (p=0.01) and with increasing HAS-BLED score (p=0.01), especially in CHA2DS2-VASc score ≥4 and in HAS-BLED score ≥3, respectively. The highest trough PDC was obtained in patient group with CHA2DS2-VASc score ≥4, HAS-BLED score ≥3, or creatinine clearance ≤80, each combined with trough APTT ratio ≥1.20. ConclusionsThis study provides an important clinical implication for identifying patients at high risk for major bleeding during dabigatran treatment in clinical practice.

Point-of-Care Coagulation Testing for Assessment of the Pharmacodynamic Anticoagulant Effect of Direct Oral Anticoagulant

This investigation was carried out with already available point-of-care testing (POCT) systems for coagulation parameters to evaluate the qualitative and semiquantitative determination of the time- and concentration-dependent anticoagulant effects of the direct oral anticoagulants rivaroxaban and dabigatran. The whole blood prothrombin time (PT), activated partial thromboplastin time (aPTT), and activated clotting time (ACT) were determined using the GEM PCL Plus coagulation system. Whole blood PT was also measured on the CoaguCheck XS instrument. In addition, PT and aPTT values were obtained in citrated plasma using the PT reagent Neoplastin Plus and the STA APTT reagent. Drug concentrations of rivaroxaban and dabigatran were determined with a chromogenic anti-Xa assay and the hemoclot assay, which are reported to have good agreement with liquid chromatography coupled with tandem mass spectrometry measurements. POCT was performed in 27 consecutive patients who received rivaroxaban 10, 15, or 20 mg once daily and in 15 patients receiving dabigatran 110 or 150 mg twice daily. Blood samples were collected predose and 2 hours after observed drug intake at steady state. Two hours after observed rivaroxaban administration, the whole blood PT measured on the GEM PCL Plus was prolonged by an average of 64.5% in comparison with predose levels. Less differentiation was observed for rivaroxaban when the PT was measured on the CoaguCheck XS instrument or in plasma (prolongation of 24.1% and 36.8%, respectively). After 2 hours observed dabigatran administration, the whole blood aPTT was comparable with plasma values and was prolonged by 23.5% in comparison with trough values. Significant concentration-dependent prolongations of the activated clotting time were observed to different extents for both direct anticoagulants. Direct oral anticoagulants display variable ex vivo effects on different POCT-assays. POCT for aPTT is sensitive to increased concentrations of dabigatran, whereas the PT-POCT assessed with test systems such as the GEM PCL Plus may be helpful to measure the pharmacodynamic anticoagulant effects of rivaroxaban in emergency clinical situations.

Direct oral anticoagulants – laboratory monitoring

ZusammenfassungDie zum niedermolekularen Heparin (NMH) ähnliche Pharmakokinetik der DOAKs (Direkte Orale Antikoagulanzien) ermöglicht im klinischen Alltag den Austausch dieser Substanzen unter Beibehaltung der Anwendungsfrequenz. Vor allem bei nicht sicherer oder nicht möglicher oraler Anwendung von DOAKs erfolgt die parenterale Gabe von NMH. Die benötigte Karenzzeit vor Interventionen oder Operationen ist für beide Anwendungen präzise darstellbar. Beide Substanzklassen werden, wo nötig, durch ähnliche Labortests überwacht.Ein generelles therapiebegleitendes Gerinnungsmonitoring, wie es seit vielen Jahrzehnten unter Verwendung von Vitamin-K Antagonisten üblich ist, ist unter Einnahme der neuen Xa- und Thrombin-Inhibitoren nicht erforderlich. Treten bei Patienten, die mit den DOAK behandelt werden, spezielle klinische Situationen auf (z.B. notfallmäßige Operationen oder Interventionen, akute Blutungssituation, akutes Organversagen), so können für den behandelnden Arzt Informationen über die Wirkspiegel im Plasma des Patienten die Einschätzung des Blutungsrisikos erleichtern. Da die DOAKs an zentraler Stelle in das Gerinnungssystem eingreifen, zeigen sie eine Interferenz mit den globalen Gerinnungstesten wie z.B. Thromboplastinzeit (TPZ; Quick/INR), aktivierte partielle Thromboplastinzeit (aPTT) und Thrombinzeit (TZ)(nur Thrombininhibitoren!) und speziellen gerinnungsphysiologischen Untersuchungen. Die Veränderungen in der Gerinnungsdiagnostik sind sowohl abhängig vom Wirkmechanismus des DOAK und der entsprechenden Halbwertszeit als auch vom Zeitpunkt der Tabletteneinnahme, der Dosierung und vom dem im Gerinnungslabor verwendetem Testsystem/Reagenz. Zur Interpretation der hämostaseologischen Messergebnisse muss dem behandelnden Arzt und dem Labormediziner bekannt sein, welches DOAK zu welchem Zeitpunkt eingenommen wurde. Weiters muss die Empfindlichkeit (Dosis-Wirkungskurve) des jeweiligen verwendeten Testsystems berücksichtigt werden. Mit den Globaltesten der Gerinnung können unter Behandlung mit DOAK lediglich abschätzende (semi-quantitative) Aussagen getroffen werden:Liegt unter Einnahme von Rivaroxaban die Thromboplastinzeit (Quick / INR) im Normbereich (vorausgesetzt es wurde im Labor ein auf Rivaroxaban empfindliches Reagenz, z.B. Neoplastin Plus benutzt), so kann gefolgert werden, dass eine klinisch relevante Restwirkung von Rivaroxaban im Patientenplasma unwahrscheinlich ist. Unter Einnahme von Dabigatran weist eine aPTT &gt;80 sec im Talspiegel auf ein erhöhtes Blutungsrisiko hin, eine im Normbereich liegende TZ lässt auf die Abwesenheit von Dabigatran im Plasma schließen.Für die Quantifizierung der Plasmakonzentration von anti-Xa Inhibitoren stehen speziell auf die jeweilige Substanz kalibrierte chromogene anti-Xa-Teste zur Verfügung. Für die Quantifizierung der Plasmakonzentration von Dabigatran kann die kalibrierte verdünnte Thrombinzeit (Hemoclot®) eingesetzt werden. Eine Messung der Medikamentenkonzentrationen ist im klinischen Alltag jedoch bislang routinemäßig nicht vorgesehen, da die gemessenen Plasmakonzentrationen klinisch derzeit nicht für den einzelnen Patienten interpretiert werden können. Insgesamt muss der Einsatz hämostaseologischer Labormethoden zum Nachweis von DOAKS den lokalen Gegebenheiten entsprechend geplant und immer wieder (z. B. bei Reagenzienwechsel) zwischen Labor und Klinik abgestimmt werden.

A proposal for dose‐adjustment of dabigatran etexilate in atrial fibrillation guided by thrombin time

Dabigatran is an oral anticoagulant that is increasingly used for atrial fibrillation (AF). Presently, many authorities state that routine laboratory coagulation monitoring is not required. However, data have recently been published demonstrating that higher trough plasma dabigatran concentrations are associated with lower thromboembolic and higher haemorrhagic event rates. Using these data, we simulate a range of AF patients with varying risks for these events and derive a target range of trough plasma dabigatran concentrations (30-130 μg l(-1) ). Finally, we propose that a conventional screening coagulation assay, the thrombin time (TT), can be used to discern whether or not patients are within this range of dabigatran concentrations.