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

Fig 1. Design of rabbit liver laceration model of acute hemorrhage study.

Fig 2. Edoxaban anti-FXa activity and andexanet effects in vitro. (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,…

Fig 3. In vitro reversal of edoxaban-induced prolongation of PT and aPTT in human plasma. Plasma samples contained edoxaban at 0 (○), 1.0 (□), and 2.0 μM (◇) and increasing concentrations of andexanet (0, 0.38, 0.76, 1.14, 1.52, 1.9, 2.5, and 3.0 μM). PT and aPTT were measured on Stago Compact as described in Materials and Methods. Data were plotted as the mean ± standard deviation from two separate experiments.

Fig 4. In vitro reversal of edoxaban-induced inhibition of thrombin generation in human plasma. 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 S2 Fig.

Fig 5. Total blood loss at end of study. 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.

Fig 6. Change in pharmacodynamic parameters following administration of andexanet in edoxaban-anticoagulated rabbits. (A) Edoxaban anti-FXa activity was measured by a modified anti-FXa activity assay. The anti-FXa activity in unknown samples was quantified using the known concentrations of edoxaban in rabbit plasma as standards and expressed as ng/mL. Data are plotted as mean ± standard deviation. (B) PT measurements in seconds. (C) aPTT measurements in seconds. Determination of PT and aPTT was performed on an Instrumentation Laboratory ACL Elite. HemosIL PT-Fibrinogen kit was used for PT measurements, and HemosIL SynthASil kit was used for aPTT measurements.

Fig 7. Change in pharmacokinetic parameters following administration of andexanet in edoxaban-anticoagulated rabbits. (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.

Fig 8. Correlation between blood loss and pharmacodynamic markers in edoxaban-anticoagulated rabbits. 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.