Andexanet alfa effectively reverses edoxaban anticoagulation effects and associated bleeding in a rabbit acute hemorrhage model

<div><p>Introduction</p><p>Increasing use of factor Xa (FXa) inhibitors necessitates effective reversal agents to manage bleeding. Andexanet alfa, a novel modified recombinant human FXa, rapidly reverses the anticoagulation effects of direct and indirect FXa inhibitors.</p><p>Objective</p><p>To evaluate the ability of andexanet to reverse anticoagulation <i>in vitro</i> and reduce bleeding in rabbits administered edoxaban.</p><p>Materials and methods</p><p><i>In vitro</i> studies characterized the interaction of andexanet with edoxaban and its ability to reverse edoxaban-mediated anti-FXa activity. In a rabbit model of surgically induced, acute hemorrhage, animals received edoxaban vehicle+andexanet vehicle (control), edoxaban (1 mg/kg)+andexanet vehicle, edoxaban+andexanet (75 mg, 5-minute infusion, 20 minutes after edoxaban), or edoxaban vehicle+andexanet prior to injury.</p><p>Results</p><p>Andexanet bound edoxaban with high affinity similar to FXa. Andexanet rapidly and dose-dependently reversed the effects of edoxaban on FXa activity and coagulation pharmacodynamic parameters <i>in vitro</i>. In edoxaban-anticoagulated rabbits, andexanet reduced anti-FXa activity by 82% (from 548±87 to 100±41 ng/ml; <i>P</i><0.0001), mean unbound edoxaban plasma concentration by ~80% (from 100±10 to 21±6 ng/ml; <i>P</i><0.0001), and blood loss by 80% vs. vehicle (adjusted for control, 2.6 vs. 12.9 g; <i>P</i> = 0.003). The reduction in blood loss correlated with the decrease in anti-FXa activity (r = 0.6993, <i>P</i><0.0001) and unbound edoxaban (r = 0.5951, <i>P</i> = 0.0035).</p><p>Conclusion</p><p>These data demonstrate that andexanet rapidly reversed the anticoagulant effects of edoxaban, suggesting it could be clinically valuable for the management of acute and surgery-related bleeding. Correlation of blood loss with anti-FXa activity supports the use of anti-FXa activity as a biomarker for assessing anticoagulation reversal in clinical trials.</p></div

Correlation between blood loss and pharmacodynamic markers in edoxaban-anticoagulated rabbits.

<p>Blood loss correlated with (A) anti-FXa activity and (B) unbound edoxaban plasma concentrations. Each data point represents the measurements from an individual rabbit at the end of andexanet infusion (Time 25 minutes) for anti-FXa and unbound edoxaban, and 15 minutes after liver injury for blood loss (Time 40 minutes). △ vehicle alone (both edoxaban + andexanet vehicle); ○ edoxaban + andexanet; ◇ edoxaban + andexanet vehicle.</p

Total blood loss at end of study.

<p>The procedure for the rabbit liver injury prophylactic blood loss model was a modification of a previously published hepatosplenic liver injury model as described in Materials and Methods. Rabbits were administered edoxaban (1 mg/mL) or edoxaban vehicle at Time 0. Andexanet or andexanet vehicle was administered IV over 5 minutes (from Time 20 to Time 25 minutes). A standardized liver injury was made into 2 liver lobes with 5 incisions in each lobe and allowed to bleed for 15 minutes (from Time 25 to Time 40 minutes). The blood loss was measured in grams by weighing pre-weighed dry gauze. Each data point represents the measurement from an individual rabbit at Time 40 minutes (15 minutes after liver injury). Horizontal bars represent mean values.</p

Edoxaban anti-FXa activity and andexanet effects <i>in vitro</i>.

<p>(A) Inhibition of FXa chromogenic activity by edoxaban in a buffer system with purified enzyme. Human FXa at 0.5 nM (□) and 1.0 nM (○) were pre-incubated with increasing concentrations of edoxaban (0, 0.5, 0.8, 1, 1.5, 2, 3, 4, 6, 8, 10, 12 nM) at RT for 2 hrs. Residual FXa activity was determined by measuring the initial rates of peptidyl substrate hydrolysis (mOD₄₀₅/min) at RT over 5 min. The initial velocity was fitted with Dynafit to obtain the kinetic parameters Ki and kcat using the pre-determined Km (82.2 μM) as described in Materials and Methods. Fig 2A shows a representative result from one of the three experiments shown in Fig 2D (Test a for Ki). The symbols represent the measured mean initial rate from quadruplicate wells at each edoxaban concentration. The solid lines were drawn using the best fitted values with Ki = 0.101 nM, kcat = 156 1/s. (B) Reversal of edoxaban-induced inhibition of FXa chromogenic activity by andexanet in a buffer system with purified enzyme. Human FXa (3.0 nM); different concentrations of edoxaban at 0 (□), 2.5 (△), 5.0 (◇),and 7.5 nM (○); and increasing concentrations of andexanet (0, 12.5, 25, 37.5, 50, 75, 100, 125, 188, 250, 500 nM) were pre-incubated at RT for 2 hrs. Residual FXa activity was determined by measuring the initial rates of peptidyl substrate hydrolysis (mOD₄₀₅/min) at RT over 5 min. The initial velocity was fitted with Dynafit to obtain the kinetic parameters Kd and kcat using the pre-determined Km (82.2 μM) and Ki (0.122 nM) as described in Materials and Methods. Fig 2B shows a representative result from one of the three experiments shown in Fig 2D (Test b for Kd). The symbols represent the measured mean initial rate from duplicate wells at each andexanet concentration. The solid lines were drawn using the best fitted values with Kd = 0.98 nM, kcat = 176 1/s. (C) Reversal of edoxaban-induced anti-FXa activity by andexanet in human plasma. Edoxaban (76 ng/mL, 0.136 μM) and increasing concentrations of andexanet (0, 0.05, 0.1, 0.15, 0.2, 0.3, 0.5, 0.8, 1, 1.5, 2.5 μM) were prepared in human plasma and pre-incubated at RT for 30 min. Residual anti-FXa activity for edoxaban was measured as described in Materials and Methods. Fig 2C shows edoxaban anti-FXa activity (%) after normalization of the results to the mean anti-FXa value at 0 nM andexanet. Data were plotted as the mean ± standard deviation from three separate experiments. (D) Constants for edoxaban interaction with FXa (Ki) and andexanet (Kd) determined by the kinetic measurements as described in panel (A) and panel (B), respectively.</p

<i>In vitro</i> reversal of edoxaban-induced inhibition of thrombin generation in human plasma.

<p>Plasma samples contained edoxaban at 0 (◇), 250 (△), 500 (□), and 1000 (○) ng/mL (0, 0.46, 0.91, 1.82 μM) and increasing concentrations of andexanet (0, 0.05, 0.1, 0.5, 1.0, 2.0, 2.5, 3.0 μM). Data (ETP) were plotted as mean ± standard deviation from two separate experiments. Additional thrombin generation parameters are provided in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0195122#pone.0195122.s003" target="_blank">S2 Fig</a>.</p

Change in pharmacokinetic parameters following administration of andexanet in edoxaban-anticoagulated rabbits.

<p>(A) Total edoxaban plasma concentration. (B) Unbound edoxaban plasma concentration. Total and unbound (pharmacologically active) edoxaban plasma levels were determined by LC/MS/MS with a turbo-ion spray source using an internal standard (edoxaban-D6) as described in Materials and Methods. Pharmacologically active unbound edoxaban was separated using an HTD96b apparatus at 37°C for approximately 4 hours with gentle vortexing. (C) Andexanet plasma concentration was determined by ELISA with paired antibodies recognizing human FX/FXa. Andexanet standard was freshly prepared in blocking buffer with the same lot material used in this study for 8-point curve (0–200 ng/mL). Data are plotted as mean ± standard deviation.</p

Design of rabbit liver laceration model of acute hemorrhage study.

<p>Design of rabbit liver laceration model of acute hemorrhage study.</p

Next directions in measurement of the home mathematics environment: an international and interdisciplinary perspective

This article synthesizes findings from an international virtual conference, funded by the National Science Foundation (NSF), focused on the home mathematics environment (HME). In light of inconsistencies and gaps in research investigating relations between the HME and children’s outcomes, the purpose of the conference was to discuss actionable steps and considerations for future work. The conference was composed of international researchers with a wide range of expertise and backgrounds. Presentations and discussions during the conference centered broadly on the need to better operationalize and measure the HME as a construct – focusing on issues related to child, family, and community factors, country and cultural factors, and the cognitive and affective characteristics of caregivers and children. Results of the conference and a subsequent writing workshop include a synthesis of core questions and key considerations for the field of research on the HME. Findings highlight the need for the field at large to use multi-method measurement approaches to capture nuances in the HME, and to do so with increased international and interdisciplinary collaboration, open science practices, and communication among scholars