Ecarin Clotting Time Research Articles

Monitoring the efficiency of reversal on anti-Xa direct oral anticoagulants using point-of-care viscoelastic testing

Bleeding events in patients receiving direct oral anticoagulation (DOAC) can be life-threatening even at therapeutic DOAC plasma concentrations, as anticoagulation impairs hemostasis and should therefore be identified immediately after hospital admission. The anticoagulatory effects of DOAC are typically not measurable in standard coagulation tests, such as PT or aPTT. Specific calibrated anti-FXa-tests allow specific drug monitoring, but they are too time-consuming for critical bleeding events and are commonly not available for 24 h/7 days in routine care. However, recent advances in point-of-care (POC) viscoelastic testing (VET) have shown a promising approach for rapid and quantitative detection of DOAC plasma concentrations using the Russell viper venom factor V activator (RVV for FXa-inhibitors) test or the ecarin clotting time (thrombin inhibitors). In acute bleeding situations, direct FXa inhibitors can be reversed by specific antidote andexanet alfa or hemostasis can be improved by prothrombin complex factor concentrates (PCCs). After reversal, confirmation of reversal efficacy is often requested, but no routine assays are currently available. Thus, the emergency management of bleeding DOAC patients is usually “blinded” with regard to reversal efficacy. POC VET laboratory assays might therefore also be helpful for measuring DOAC effects after reversal. We present a case series demonstrating the usefulness of RVV-clotting time post-DOAC reversal with andexanet alfa.

Challenges in laboratory testing of patients suspected of antiphospholipid syndrome: practical implications for clinicians.

This paper focuses on the laboratory tests necessary for the diagnosis of antiphospholipid syndrome (APS). Diagnosis starts with the selection of patients suspicious of having APS. The timing related to the clinical event is important to avoid false classification of APS patients. For the interpretation of the test results of antiphospholipid antibodies (aPL) understanding of all pitfalls and interferences is necessary. Lupus anticoagulant (LA) measurement remains a complex procedure with many pitfalls and interferences. The effect of anticoagulant therapy, the main confounder of LA measurement, can be overcome by removal agents for direct oral anticoagulants (DOAC) or by considering assays as Taipan snake venom time / Ecarin clotting time not affected by antivitamin K therapy and anti-Xa DOAC. However, both procedures have limitations. Solid-phase assays for anticardiolipin antibodies (aCL) and anti-β2-glycoprotein 1 antibodies (aβ2GPI) show inter-assay differences. Diagnosis is based on the measurement of three groups of aPL: LA, aCL and aβ2GPI, of IgG and IgM isotype. This allows to make antibody profiles that helps in identifying patients at risk. Other aPL, such as antibodies against the domain I of β2GPI and anti-phosphatidylserine-prothrombin antibodies may be useful in risk stratification of APS patients and in some specific situations of patients with incomplete antibody profile, but are not needed for diagnosis. Laboratory diagnosis of APS remains challenging. To increase the diagnostic efficacy and reliability, an integrated interpretation of all results and an interpretative comment should be provided on the laboratory report. Therefore, a close interaction between clinical pathologists and clinicians is mandatory.

First-in-human trial of SAR107375E, a novel small molecule anticoagulant with dual inhibition of factor Xa and factor IIa

ABSTRACT Background SAR107375E is a direct dual inhibitor of both Factor Xa and Factor IIa and has shown potent anticoagulation activity in vitro and animals. This study evaluated the safety, tolerability, pharmacokinetics, and pharmacodynamics of single ascending intravenous doses of SAR107375E in healthy Chinese adult subjects. Methods In this randomized, double-blind, placebo-controlled trial, 60 healthy Chinese adult subjects were administered intravenously single ascending doses (0.5, 1.5, 3.0, 5.0, 7.5, 10.0, 15.0, or 20.0 mg) of SAR107375E (N = 44) or placebo (N = 16). Plasma and urine concentrations of SAR107375E were measured and used to calculate pharmacokinetic parameters. Coagulation functions were measured and compared with baseline values. Treatment-emergent adverse events were recorded to evaluate safety. Results In plasma, from the 0.5 to 20.0 mg dose group, t1/2 is 1.51–4.00 h, Cmax is 59.05–1360 ug/L, and AUC0-t is 25.01–528.45 h*ug/L. And it shows dose proportionality in the 5.0–20.0 mg range. Activated partial thromboplastin time and Ecarin clotting time correlated linearly with drug plasma concentration. No serious adverse events were reported during the study. Conclusion SAR107375E exhibits good safety and tolerability, predictable pharmacokinetics and pharmacodynamics. Clinical trial registration www.chinadrugtrials.org.cn, identifier is CTR20211082.

Monitoring of Argatroban in Critically Ill Patients: A Prospective Study Comparing Activated Partial Thromboplastin Time, Point-of-Care Viscoelastic Testing with Ecarin Clotting Time and Diluted Thrombin Time to Mass Spectrometry.

The direct thrombin inhibitor argatroban is indicated for the treatment of heparin-induced thrombocytopenia II, but it is also used off-label to treat critically ill patients presenting with heparin resistance, severe antithrombin deficiency, or hypercoagulability. Direct drug monitoring is not routinely available, and argatroban dosing is mainly based on global coagulation assays such as activated partial thromboplastin time (PTT) or diluted thrombin time (TT), both of which have limitations in patients with hypercoagulability. Blood samples were obtained from critically ill patients treated with argatroban. Activated PTT and diluted TT were measured with a STA R Max3 analyzer (STAGO Deutschland GmbH, Germany) using an argatroban-calibrated kit. Ecarin clotting time was measured using a point-of-care viscoelastic test device. Liquid chromatography with tandem mass spectrometry was performed using a reversed-phase column, a solvent gradient, and an API4000 mass spectrometer with electrospray. Correlation was described using Pearson correlation coefficient r and Bayesian multilevel regression to estimate relationships between outcomes and covariates. From June 2021 to March 2022, 205 blood samples from 22 patients were analyzed, allowing for 195 activated PTT-liquid chromatography with tandem mass spectrometry comparisons, 153 ecarin clotting time-liquid chromatography with tandem mass spectrometry comparison, and 105 diluted TT-liquid chromatography with tandem mass spectrometry comparisons. Compared to liquid chromatography with tandem mass spectrometry, performance of argatroban quantification was best for diluted TT (r = 0.91), followed by ecarin clotting time (r = 0.58) and activated PTT (r = 0.48). Regression analysis revealed that patients with sepsis were more prone to argatroban overdosing (coefficient, 4.194; 95% credible interval, 2.220 to 6.792). Although activated PTT monitoring of argatroban is the most commonly used test, in critically ill patients, diluted TT provides more precise measurements. Alternately, point-of-care viscoelastic ecarin clotting time also provides guidance for argatroban dosing to identify overdosing if available. The data also suggested that patients with sepsis are at greater risk for argatroban overdosing.

PB1034 Monitoring of Argatroban in Critical Ill Patients: A Prospective Study Comparing aPTT, Point-of-Care Viscoelastic Testing with Ecarin Clotting Time and dTT to LC/MS/MS

PB1034 Monitoring of Argatroban in Critical Ill Patients: A Prospective Study Comparing aPTT, Point-of-Care Viscoelastic Testing with Ecarin Clotting Time and dTT to LC/MS/MS

Anticoagulant Effects of Dabigatran on Coagulation Laboratory Parameters in Pediatric Patients: Combined Data from Five Pediatric Clinical Trials.

Background Dabigatran etexilate, a direct oral thrombin inhibitor, is approved to treat venous thromboembolism (VTE) in both adults and children. Objectives This population analysis characterized relationships between dabigatran total plasma concentrations and coagulation laboratory parameters (activated partial thromboplastin time [aPTT]; diluted thrombin time [dTT]; ecarin clotting time [ECT]). Methods Data from three phase 2a and one single-arm and one randomized, comparative phase 2b/3 pediatric studies (measurements: aPTT 2,925 [ N = 358]; dTT 2,348 [ N = 324]; ECT 2,929 [ N = 357]) were compared with adult data (5,740 aPTT, 3,472 dTT, 3,817 ECT measurements; N = 1,978). Population models were fitted using nonlinear mixed-effects modeling. Covariates (e.g., sex, age) were assessed on baseline and drug-effect parameters, using a stepwise covariate model-building procedure. Results Overall, relationships between dabigatran, aPTT, dTT, and ECT were similar in children and adults. For children aged <6 months, a higher proportion of baseline samples were outside or close to the upper aPTT and ECT adult ranges. No age-related differences were detected for dTT. With increasing dabigatran concentration, aPTT rose nonlinearly (half the maximum effect at 368 ng/mL dabigatran) while dTT and ECT increased linearly (0.37 and 0.73% change per ng/mL dabigatran, respectively). Mean baseline aPTT (45 vs. 36 seconds) and ECT (40 vs. 36 seconds) were slightly increased for those aged <6 months versus older children. Conclusion The similar relationships of laboratory parameters observed across pediatric age groups suggests that developmental changes in the hemostatic system may have little effect on response to dabigatran.

Impact of Idarucizumab and Andexanet Alfa on DOAC Plasma Concentration and ClotPro® Clotting Time: An Ex Vivo Spiking Study in A Cohort of Trauma Patients.

Specific antagonists have been developed for the reversal of direct oral anticoagulants (DOAC). We investigated the impact of these reversal agents on the plasma concentration and visco-elastic test results of dabigatran and factor Xa inhibitors. After baseline measurements of dabigatran, the plasma concentration, and the visco-elastic ClotPro® ecarin clotting time (ECA-CT), we added the reversal agent Idarucizumab in vitro and these two analyses were repeated. Likewise, the baseline plasma concentration of apixaban, edoxaban, and rivaroxaban as well as ClotPro® Russell’s viper venom test clotting time (RVV-CT) were measured and reanalyzed following Andexanet alfa spiking. We analyzed fifty blood samples from 37 patients and 10 healthy volunteers. Idarucizumab decreased the measured dabigatran plasma concentration from 323.9 ± 185.4 ng/mL to 5.9 ± 2.3 ng/mL and ECA-CT from 706.2 ± 344.6 s to 70.6 ± 20.2 s, (all, p < 0.001). Andexanet alfa decreased the apixaban concentration from 165.1 ± 65.5 ng/mL to 9.8 ± 8.1 ng/mL, edoxaban from 152.4 ± 79.0 ng/mL to 36.4 ± 19.2 ng/mL, and rivaroxaban from 153.2 ± 111.8 ng/mL to 18.1 ± 9.1 ng/mL (all p < 0.001). Andexanet alfa shortened the RVV-CT of patients with apixaban from 239.2 ± 71.7 s to 151.1 ± 30.2 s, edoxaban from 288.2 ± 65.0 s to 122.7 ± 37.1 s, and rivaroxaban from 225.9 ± 49.3 s to 103.7 ± 12.1 s (all p < 0.001). In vitro spiking of dabigatran-containing blood with Idarucizumab substantially reduced the plasma concentration and ecarin-test clotting time. Andexanet alfa lowered the concentration of the investigated factor Xa-inhibitors but did not normalize the RVV-CT. In healthy volunteers’ blood, Idarucizumab spiking had no impact on ECA-CT. Andexanet alfa spiking of non-anticoagulated blood prolonged RVV-CT (p = 0.001), potentially as a consequence of a competitive antagonism with human factor Xa.

Dabigatran etexilate for the treatment of acute venous thromboembolism in children (DIVERSITY): a randomised, controlled, open-label, phase 2b/3, non-inferiority trial

Dabigatran etexilate is a direct oral anticoagulant with potential to overcome the limitations of standard of care in children with venous thromboembolism. The aims of this clinical trial were to study the appropriateness of a paediatric dabigatran dosing algorithm, and the efficacy and safety of dabigatran dosed according to that algorithm versus standard of care in treating children with venous thromboembolism. DIVERSITY is a randomised, controlled, open-label, parallel-group, phase 2b/3 non-inferiority trial done in 65 centres in 26 countries. Standard of care (low-molecular-weight heparins, unfractionated heparin, vitamin K antagonists or fondaparinux) was compared with a paediatric oral dabigatran dosing regimen (an age-adjusted and weight-adjusted nomogram) in children younger than 18 years with acute venous thromboembolism initially treated (5-21 days) with parenteral anticoagulation, requiring anticoagulation therapy for at least 3 months. Patients were randomised 1:2 (standard of care:dabigatran) and stratified by age (12 to <18 years, 2 to <12 years, and birth to <2 years) via interactive response technology. The primary composite efficacy endpoint (intention-to-treat analysis) was the proportion of children with complete thrombus resolution, and freedom from recurrent venous thromboembolism and venous thromboembolism-related death. A non-inferiority margin of absolute differences of 20% was used. Secondary endpoints included safety (determined by major bleeding events [time-to-event analysis on the treated set]), and pharmacokinetic-pharmacodynamic relationships (descriptive analyses). This trial is registered with ClinicalTrials.gov, NCT01895777 and is completed. 328 children were enrolled between Feb 18, 2014, and Nov 14, 2019. 267 were randomly assigned (90 [34%] to standard of care and 177 [66%] to dabigatran) and included in the analyses. Median exposure to standard of care was 85·0 days (IQR 80·0-90·0) and to dabigatran was 84·5 days (78·0-89·0). Similar proportions of children treated with standard of care and dabigatran met the composite efficacy endpoint (38 [42%] of 90 vs 81 [46%] of 177; Mantel-Haenszel weighted difference, -0·04; 90% CI -0·14 to 0·07; p<0·0001 for non-inferiority). On-treatment bleeding events were reported in 22 (24%) of 90 children receiving standard of care and 38 (22%) of 176 children receiving dabigatran (hazard ratio [HR] 1·15, 95% CI 0·68 to 1·94; p=0·61); major bleeding events were similar between the groups (two [2%] of 90 and four [2%] of 176; HR 0·94, 95% CI 0·17 to 5·16; p=0·95). Pharmacokinetic-pharmacodynamic curves showed a linear relationship between total dabigatran plasma concentration and diluted thrombin time and ecarin clotting time, and a non-linear relationship with activated partial thromboplastin time; curves were similar to those for adults. Serious adverse events were reported for 18 (20%) of 90 children receiving standard of care and 22 (13%) of 176 children receiving dabigatran. The most common severe adverse events were vascular disorders (standard of care three [3%] of 90, dabigatran two [1%] of 176), and gastrointestinal disorders (standard of care two [2%] of 90 and dabigatran five [3%] of 176). One on-treatment death occurred in the standard of care group (retroperitoneal bleeding, not considered treatment related by the study investigators). An age-adjusted and weight-adjusted dabigatran dosing algorithm was appropriate in children aged birth to less than 18 years with venous thromboembolism. Dabigatran was non-inferior to standard of care in terms of efficacy, with similar pharmacokinetic-pharmacodynamic relationships as those seen in adults, and might be a suitable alternative to standard of care. Boehringer Ingelheim.

Dabigatran Etexilate in Children with Venous Thromboembolism: Results of the Open-Label, Phase IIb/III, Randomized Diversity Clinical Trial

Background: In children, current standard of care (SOC) with heparins or vitamin K antagonists is limited by the need for parenteral administration and frequent monitoring. Dabigatran etexilate (DE), a direct oral anticoagulant, is an effective oral treatment for venous thromboembolism (VTE) in adults and may be an effective and safe alternative to SOC for treating acute VTE in children. Aims: The DIVERSITY trial evaluated the efficacy and safety of DE versus SOC, and the appropriateness of a DE dosing algorithm, in children with VTE aged from birth to &amp;lt; 18 years. Methods: This was an open-label, randomized, active-controlled, multicenter, phase IIb/III trial (NCT01895777; consent obtained and ethics committee approved). Children (12 to &amp;lt; 18 years, 2 to &amp;lt; 12 years, birth to &amp;lt; 2 years) with an objectively confirmed VTE diagnosis and initially treated with unfractionated heparin or low-molecular-weight heparin were randomized (2:1) to DE (capsules, and child-friendly pellets and oral suspension, dosed using an age- and body-weight-adjusted dosing algorithm) or SOC, and treated for up to 3 months. The primary composite efficacy endpoint was the proportion of children with complete thrombus resolution, and freedom from recurrent VTE or VTE-related death. Safety endpoints included bleeding events and adverse events (AEs), and pharmacokinetic/pharmacodynamic (PK/PD) relationships. Results: Of 267 children in the randomized set, 35 were aged &amp;lt; 2 years (13 SOC, 22 DE; of these, 14 were &amp;lt; 6 months [3 SOC, 11 DE]), 64 were 2 to &amp;lt; 12 years (21 SOC, 43 DE), and 168 were 12 to &amp;lt; 18 years (56 SOC, 112 DE). Overall, similar proportions of children treated with SOC and DE met the composite efficacy endpoint (38/90 [42.2%] vs 81/177 [45.8%]; Mantel Hänszel-weighted difference -0.04; 90% confidence interval [CI] -0.14 to 0.07; p &amp;lt; 0.0001 for non-inferiority), and major bleeding events were comparable (2/90 [2.2%] and 4/176 [2.3%]; hazard ratio 0.94; 95% CI 0.17-5.16; p = 0.95). Across the three age groups (12 to &amp;lt; 18 years, 2 to &amp;lt; 12 years, birth to &amp;lt; 2 years), DE was comparable with SOC for the combined efficacy endpoint, which was achieved by 33.9-57.1% of children treated with SOC and 42.0-59.1% treated with DE. Similar numbers of children experienced major bleeding events, reported by between 0.0-3.6% of children treated with SOC and 1.8-4.5% treated with DE. In the overall population, the incidence of AEs in children treated with SOC and DE was 66.7% and 76.7%, respectively, and serious AEs were reported by 20.0% and 12.5% of all children, respectively. Across the three main age groups, the frequency of serious AEs was comparable for SOC and DE (reported by 19.0-23.1% and 9.1-14.4% of children, respectively). Dabigatran PK/PD relationships for three laboratory coagulation parameters (activated partial thromboplastin time [aPTT], diluted thrombin time [dTT], and ecarin clotting time [ECT]) were comparable across the three main age groups and were also similar in infants aged &amp;lt; 6 months (Figure). There was a linear relationship between total dabigatran plasma concentration for both dTT and ECT, and a nonlinear relationship with aPTT. These PK/PD relationships were similar to those observed in adults (data not shown). Conclusions: This randomized trial confirmed that an age- and weight-adjusted dabigatran dosing algorithm was appropriate in children aged from birth to &amp;lt; 18 yrs. Dabigatran, given as either capsules or child-friendly pellets or oral suspension, was comparable with SOC in terms of efficacy for acute VTE treatment across all age groups and, notably, in vulnerable children aged &amp;lt; 2 yrs. Disclosures Halton: Boehringer Ingelheim: Other: Member of a paediatric expert working group. Brandao:Boehringer Ingelheim: Other: Member of a paediatric expert working group. Luciani:Boehringer Ingelheim: Other: Member of a paediatric expert working group. Bomgaars:Boehringer Ingelheim: Other: Member of a paediatric expert working group. Chalmers:Shire/Takeda: Honoraria; CSL Behring: Honoraria; Bristol-Myers Squibb: Honoraria; Sobi: Honoraria; Roche: Honoraria; Grifols: Honoraria; Boehringer Ingelheim: Other: Member of a paediatric expert working group. Mitchell:Boehringer Ingelheim: Other: Member of a paediatric expert working group. Sharathkumar:Takeda: Consultancy; CSL Behring: Consultancy; Bayer: Consultancy; Kedrion: Consultancy. Svirin:Takeda: Consultancy, Other: Personal fees; CSL Behring: Consultancy, Other: Personal fees. Tartakovsky:Boehringer Ingelheim: Current Employment. Simetzberger:Boehringer Ingelheim: Current Employment. Huang:Boehringer Ingelheim: Current Employment. Sun:Boehringer Ingelheim: Current Employment. Kreuzer:Boehringer Ingelheim: Current Employment. Gropper:Boehringer Ingelheim: Current Employment. Brueckmann:Boehringer Ingelheim: Current Employment. Albisetti:Daiichi Sankyo: Membership on an entity's Board of Directors or advisory committees; Boehringer Ingelheim: Other: Member of a paediatric expert working group. OffLabel Disclosure: dabigatran etexilate in paediatric VTE

A prospective observational study of the rapid detection of clinically-relevant plasma direct oral anticoagulant levels following acute traumatic injury.

In urgent clinical situations, such as trauma, urgent surgery or before thrombolysis, rapid quantification of direct oral anticoagulant plasma drug levels is warranted. Using the ClotPro® analyser, we assessed two novel viscoelastic tests for detection of clinically-relevant plasma drug levels in trauma patients. The ecarin clotting time was used to assess the plasma concentration of dabigatran and Russell´s viper venom clotting time to determine the plasma concentration of direct factor Xa inhibitors. In parallel, plasma concentrations were analysed using plasma-based chromogenic assays. A total of 203 simultaneous measurements were performed.Strong to very strong linear correlations were detected between ecarin clotting time and plasma concentration of dabigatran (r=0.9693), and between Russell´s viper venom clotting time and plasma concentrations of apixaban (r=0.7391), edoxaban (r=0.9251) and rivaroxaban (r=0.8792), all p<0.001. An ecarin clotting time≥189seconds provided 100% sensitivity and 90% specificity for detecting plasma dabigatran concentrations≥50ng.ml-1 . Corresponding Russell´s viper venom clotting time cut-off values were≥136seconds for apixaban (80% sensitivity, 88% specificity), ≥ 168seconds for edoxaban (100% sensitivity, 100% specificity) and≥177seconds for rivaroxaban (90% sensitivity, 100% specificity). Detection of drug levels≥100ng.ml-1 was also investigated: for dabigatran, an ecarin clotting time≥315seconds yielded 92% sensitivity and 100% specificity; while Russell´s viper venom clotting time cut-offs of 191, 188 and 196seconds were calculated for apixaban (67% sensitivity, 88% specificity), edoxaban (100% sensitivity, 75% specificity) and rivaroxaban (100% sensitivity, 91% specificity), respectively. We have demonstrated strong positive correlations between plasma drug levels and clotting time values in the specific ClotPro assays. Cut-off values for detecting clinically-relevant drug levels showed high levels of sensitivity and specificity.

Idarucizumab Reverses Dabigatran Anticoagulant Activity in Healthy Chinese Volunteers: A Pharmacokinetics, Pharmacodynamics, and Safety Study

Idarucizumab is a humanized monoclonal antibody fragment that specifically binds to dabigatran with high affinity and reverses its anticoagulant effect. This study investigated the pharmacokinetics (PK) and pharmacodynamics (PD) of idarucizumab in healthy Chinese subjects at steady state of dabigatran and explored the effect of idarucizumab on PK and PD of dabigatran. Twelve subjects received dabigatran etexilate treatment alone (220mg twice daily, b.i.d., oral). After a washout period, the 12 subjects again received dabigatran etexilate (220mg b.i.d., oral) and idarucizumab (2.5 + 2.5g, intravenous) 2h after the last administration of dabigatran etexilate. The geometric mean (gMean) values of area under the plasma concentration-time curve (AUC0-∞) and maximum concentration (Cmax) were 44,200nmolh/L and 30,900nmol/L, respectively. An amount of 35.3μmol of idarucizumab, corresponding to 33.8% of the total dose, was excreted by urine over 72h. The area under the effect (AUECabove,2-12) in the presence and absence of idarucizumab was close to zero for all coagulation parameters, diluted thrombin time (dTT), ecarin clotting time (ECT), activated partial thromboplastin time (aPTT), and thrombin time (TT), which indicated the reversal of dabigatran anticoagulation by idarucizumab. There were no serious adverse events reported in this study. No subject tested positive for anti-idarucizumab antibodies. Idarucizumab was well tolerated and no subject tested positive for anti-idarucizumab antibodies in this study. PK and PD of idarucizumab in healthy Chinese subjects at a steady state of dabigatran were comparable with those in Japanese and Caucasian subjects. ClinicalTrials.gov Identifier No. NCT03086356.

Pharmacological Profile of JNJ-64179375: A Novel, Long-Acting Exosite-1 Thrombin Inhibitor

JNJ-64179375 (JNJ-9375) is a recombinant human IgG4 monoclonal antibody engineered to mimic an IgA antibody that was identified in a patient who exhibited markedly prolonged clotting times but without spontaneous bleeding episodes over several years of follow-up. The crystal structure of the JNJ-9375 antigen-binding fragment/thrombin complex showed an almost identical binding mode to that of the patient IgA. In the current study, we characterized the in vitro and in vivo properties of JNJ-9375. Surface plasmon resonance studies demonstrated that JNJ-9375 binds to α-thrombin with high affinity and specificity (K D: 0.8 nM for human thrombin). JNJ-9375 produced concentration-dependent prolongation of in vitro clotting assays in human plasma, including thrombin time (TT), ecarin clotting time, prothrombin time, and activated partial thromboplastin time, with EC2X values of 4.4, 12.4, 172.6, and 202.7 µg/ml, respectively. JNJ-9375 inhibited thrombin-induced platelet aggregation in human plasma with an IC50 value of 52.6 nM (7.8 µg/ml) and produced concentration-dependent prolongation of reaction time tested by thromboelastography. JNJ-9375 pretreatment resulted in dose-dependent reduction in thrombus formation in the rat arteriovenous (AV) shunt model of thrombosis. Robust efficacy was observed at 0.3 mg/kg accompanied by 1.5× of TT. Bleeding was increased at 3 mg/kg in a rat tail transection bleeding model demonstrating a therapeutic index of 10× compared with 1× for apixaban in the same models. Our data suggest that thrombin exosite I inhibition is efficacious against thrombosis in a pretreatment prevention animal model. SIGNIFICANCE STATEMENT: JNJ-9375 is a novel, fully human monoclonal antibody that binds to the exosite I region of thrombin with high affinity and specificity. JNJ-9375 concentration dependently prolonged clotting times and inhibited thrombin-induced platelet aggregation in in vitro assays based on its mechanism of action. In an in vivo rat AV shunt model, JNJ-9375 prevented thrombus formation in a dose-dependent fashion while demonstrating reduced bleeding risk. The present study demonstrated the antithrombotic effects of inhibiting the exosite I region of thrombin when given in a prevention mode in preclinical animal models.

Pharmacokinetics of idarucizumab and its target dabigatran in patients requiring urgent reversal of the anticoagulant effect of dabigatran

BackgroundIdarucizumab is a monoclonal antibody fragment that reverses dabigatran anticoagulation. Pharmacokinetics (PK) of idarucizumab have been described in healthy, elderly, or renally impaired (RI) volunteers, but PK data in patients are lacking. ObjectivesThis analysis describes the PK of idarucizumab and its target dabigatran in bleeding/surgical patients. Patients and MethodsResults from the Reversal Effects of Idarucizumab on Active Dabigatran study, a prospective, multicenter, single‐arm study demonstrated the reversal of dabigatran anticoagulation by idarucizumab in patients with uncontrollable bleeding (group A) or who needed urgent surgery (group B). Idarucizumab and unbound dabigatran concentrations, immunogenicity, and pharmacodynamics were assessed. ResultsTotal and unbound dabigatran levels at baseline were 165 ng/mL vs 110 ng/mL and 103 ng/mL vs 69.5 ng/mL in group A and B patients, respectively. Maximum plasma concentrations and area under the curves (AUC0‐24) of idarucizumab in group A vs B, respectively, were 24 900 nmol/L vs 25 000 nmol/L and 76 600 nmol/h/L vs 68 000 nmol/h/L. Idarucizumab AUC0–24 increased by 38% in mild, 90% in moderate, and 146% in severe RI patients vs normal renal function. Hepatic impairment or geographical region had no relevant effect on idarucizumab PK. Idarucizumab immediately decreased unbound dabigatran concentration (<20 ng/mL). A linear correlation was observed between unbound dabigatran and diluted thrombin time and ecarin clotting time. Antidrug antibody titers were low (1‐64 at day 30; 0‐16 at day 90) and had no impact on idarucizumab PK and pharmacodynamics. ConclusionIdarucizumab PK in target patients was consistent with phase I data. Patient characteristics had no impact on PK, whereas RI increased the exposure of idarucizumab and dabigatran.Trial registration number: ClinicalTrials.gov NCT02104947.

Complications of Pharmacotherapy with New Oral Anticoagulants Caused by Inter-Drug Interactions: Focus on Gastrointestinal Bleeding

New oral anticoagulants are effective for the prevention of thromboembolic complications in patients with atrial fibrillation, or after orthopedic surgery. The use of these drugs may be associated with the risk of bleeding from the gastrointestinal tract — a dangerous complication, which can potentially lead to death. The aim of this research was systematization and analysis of information on the interactions of new oral anticoagulants with other drugs and food products, and the identification of potentially dangerous combinations that increase the risk of gastrointestinal bleeding. To assess the risk of a heavy bleeding in patients with atrial fibrillation taking oral anticoagulants to prevent thromboembolic complications, HAS-BLED scale is used. In some cases activated partial thromboplastin time, ecarin clotting time, anti-FXa, etc. can be used to assess the effectiveness of the oral anticoagulants. Potential combinations that increase the risk of bleeding include the simultaneous administration of new oral anticoagulants with antiplatelet agents, anticoagulants, including vitamin K antagonists, unfractionated heparin, low molecular weight heparins, nonsteroidal anti-inflammatory drugs, antidepressants along with other drugs and foods (curcumin, grapefruit juice, peppermint, eucalyptus, etc.). The administration of proton pump inhibitors, as well as the elimination of potentially dangerous combinations of drugs and food with new oral anticoagulants, can help prevent gastrointestinal bleeding.

Pharmacokinetic and Pharmacodynamic Drug Monitoring of Direct-Acting Oral Anticoagulants: Where Do We Stand?

For decades, oral anticoagulation has been based on vitamin K antagonist such as warfarin, which requires pharmacodynamic (PD) drug monitoring to guide the therapy. The drug effect is measured by the clotting test prothrombin time and expressed as international normalized ratio. New direct oral anticoagulants are increasingly used in fixed-dose regimens but are licensed without any therapy monitoring. However, extensive clinical experiences have demonstrated that interindividual variations in the response to the therapy with direct oral anticoagulants do exist. In situations such as bleeding or thrombosis, therapeutic drug monitoring could be useful. Unfortunately, global coagulation assays such as the prothrombin time or the activated partial thrombin time are not suitable for this purpose. To measure drug concentrations, more specific coagulation test can be used if they are externally calibrated with the respective drugs. For the direct thrombin inhibitor dabigatran etexilate, a calibrated diluted thrombin time or ecarin clotting time can be used, whereas for anti-factor Xa drugs such as rivaroxaban, apixaban, edoxaban, and betrixaban, calibrated anti-factor Xa assays are appropriate. However, the gold standard to measure drug concentrations is LC-MS/MS. The variation in bleeding and thrombotic events noted with both drug classes under fixed-dose conditions suggests additional interindividual PD differences. Therefore, PD monitoring to individualize the therapy may be an option. For dabigatran, this is the inhibition of thrombin formation and for anti-factor Xa drugs, the inhibition of factor Xa activity, which can be followed using the functional assays mentioned above but without calibration. Alternatively, thrombin generation assays have been proposed for both drug classes. So far, not many clinical data have been published about the potentially beneficial effects of PD monitoring for dose individualization. The assay platforms for PD monitoring are present in many clinical laboratories, but efforts are needed to validate and standardize available assays to perform appropriate clinical trials.

Microfluidic Point-of-Care Ecarin-Based Clotting and Chromogenic Assays for Monitoring Direct Thrombin Inhibitors

Direct thrombin inhibitors (DTIs), such as bivalirudin and dabigatran, have maintained steady inpatient and outpatient use as substitutes for heparin and warfarin, respectively, because of their high bioavailability and relatively safe “on-therapy” range. Current clinical methods lack the capacity to directly quantify plasma DTI concentrations across wide ranges. At present, the gold standard is the ecarin clotting time (ECT), where ecarin maximizes thrombin activity and clotting time is evaluated to assess DTIs’ anticoagulation capability. This work focused on the development of a microfluidic paper analytic device (µPAD) that can quantify the extent of anticoagulation as well as DTI concentration within a patient’s whole blood sample. Capillary action propels a small blood sample to flow through the nitrocellulose paper channels. Digital images of whole blood migration are then captured by our self-coded Raspberry Pi and/or the Samsung Galaxy S8 smartphone camera. Both the flow length and the blue absorbance from the plasma front on the μPAD were measured, allowing simultaneous, dual assays: ecarin clotting test (ECT) and ecarin chromogenic assay (ECA). Statistically significant (p &lt; .05) changes in flow and absorbance were observed within our translational research study. Currently, there are no quantitative, commercially available point-of-care tests for the ECT and ECA within the United States. Both the ECT and ECA assays could be instrumental to differentiate between supratherapeutic and subtherapeutic incidents during bridging anticoagulant therapy and limit the unwarranted use of reversal agents.

Contemporary Reversal of Oral Anticoagulation in Intracerebral Hemorrhage.

Author(s): Bower, Matthew M; Sweidan, Alexander J; Shafie, Mohammad; Atallah, Steven; Groysman, Leonid I; Yu, Wengui

Bivalirudin and ECLS

Bivalirudin and ECLS

Limitations of Specific Coagulation Tests for Direct Oral Anticoagulants: A Critical Analysis.

BackgroundDuring treatment with direct oral anticoagulants (DOAC), coagulation assessment is required before thrombolysis, surgery, and if anticoagulation reversal is evaluated. Limited data support the accuracy of DOAC‐specific coagulation assays around the current safe‐for‐treatment threshold of 30 ng/mL.Methods and ResultsIn 481 samples obtained from 96 patients enrolled at a single center, DOAC concentrations were measured using Hemoclot direct thrombin inhibitor assay, Biophen direct thrombin inhibitor assay or ecarin clotting time for dabigatran, chromogenic anti‐Xa assay (AXA) for factor Xa inhibitors (rivaroxaban, apixaban) and ultraperformance liquid chromatography–tandem mass spectrometry as reference. All dabigatran‐specific assays had high sensitivity to concentrations >30 ng/mL, but specificity was lower for Hemoclot direct thrombin inhibitor assay (78.2%) than for Biophen direct thrombin inhibitor assay (98.9%) and ecarin clotting time (94.6%). AXA provided high sensitivity and specificity for rivaroxaban, but low sensitivity for apixaban (73.8%; concentrations up to 82 ng/mL were misclassified as <30 ng/mL). If no DOAC‐specific calibration for AXA is available, results 2‐fold above the upper limit of normal indicate relevant rivaroxaban concentrations. For apixaban, all elevated results should raise suspicion of relevant anticoagulation.Conclusions DOAC‐specific tests differ considerably in diagnostic performance for concentrations close to the currently accepted safe‐for‐treatment threshold. Compared with Biophen direct thrombin inhibitor assay and ecarin clotting time, limited specificity of Hemoclot direct thrombin inhibitor assay poses a high risk of unnecessary anticoagulation reversal or treatment delays in patients on dabigatran. While AXA accurately detected rivaroxaban, the impact of low apixaban levels on the assay was weak. Hence, AXA results need to be interpreted with extreme caution when used to assess hemostatic function in patients on apixaban.Clinical Trial Registration URL: https://www.clinicaltrials.gov. Unique identifiers: NCT02371044, NCT02371070.

Abstract 521: Pharmacological Profile of JNJ-64179375: a Novel, Long Acting Exosite-1 Thrombin Inhibitor

Background: Thrombin is a key serine protease involved in hemostasis and thrombosis by mediating the conversion of soluble fibrinogen to insoluble fibrin that forms the meshwork of a clot. In addition, it is the most potent activator of platelets. Hence thrombin inhibition has been a target for the development of anticoagulant drugs. Recently, an IgA that binds to the exosite 1 region of thrombin was identified in a patient that was profoundly anticoagulated but without any abnormal bleeding episodes over a prolonged follow-up. JNJ-64179375 (JNJ-9375) is a monoclonal antibody derivative of the IgA which by selectively inhibiting exosite 1 may allow for some of thrombin functions mediated by exosite 2 and the active site which might be important for hemostasis. JNJ-9375 is being developed as a new long-acting anti-coagulant with a potential for better efficacy with a reduced bleeding liability. Objectives: To assess the in vitro effects of spiked JNJ-9375 on various clotting assays in human and animal plasma and to evaluate the antithrombotic activity in a rat A-V shunt model of thrombosis. Methods and results: JNJ-9375 produced a concentration-dependent prolongation of thrombin time (TT), ecarin clotting time (ECT), prothrombin time (PT), and activated partial thromboplastin time (aPTT) when added to normal human plasma. The concentrations required to prolong clotting time by twofold (EC2Xs) were 4.4, 12.4, 202.7, and 172.6 μg/ml for TT, ECT, aPTT, and PT respectively. Therefore, TT was the most sensitive assay to show pharmacodynamic activity of JNJ-9375. Similar effects were observed in other species studied including monkey, rat, and mouse. In the rat A-V shunt model of thrombosis, JNJ-9375 dose dependently inhibited thrombus weight with an efficacious dose of 0.3 mg/kg, which was accompanied by prolongation of TT, ECT, PT, and aPTT from ex vivo plasma samples. Conclusion: Results from these studies demonstrate that selective inhibition of exosite 1 on thrombin leads to robust anticoagulation and antithrombotic efficacy. JNJ-9375 is currently in early clinical development.