Supplementary Appendix

Transcription

1 Supplementary Appendix This appendix has been provided by the authors to give readers additional information about their work. Supplement to: Siegal DM, Curnutte JT, Connolly SJ, et al. Andexanet alfa for the reversal of factor Xa inhibitor activity. N Engl J Med 2015;373: DOI: /NEJMoa

2 SUPPLEMENTARY APPENDIX Table of contents: 1- Trial Organization and Committees Page 2 2- Supplementary Methods Page 3 3- Figure S1: Study design diagram Page 8 4- Figure S2: Time course of activated clotting time with andexanet administration Page 9 5- Figure S3: Study consort diagram Page Table S1: Baseline subject characteristics Page Table S2: Descriptive endpoint statistics Page References Page 13 1

3 ANNEXA Trial Organization and Committees: Sponsor: Portola Pharmaceuticals. Study Investigators: Dr. Dennis Swearingen (ANNEXA-A), Dr. Anson Lam (ANNEXA-R) Independent Safety Committee: Dr. Harry Buller, Dr. Richard Becker, Dr. Jeffrey Weitz Acknowledgment: We would like to thank Gallia Levy and Shelly Goodman for their critical contribution to the design, conduct, and analysis of the study. 2

4 METHODS Subjects: The use of a volunteer population was deemed appropriate for demonstration of reversal of anticoagulation because blocking anticoagulation in the target population would expose these patients underlying thrombotic risk by reversing their medically necessary anticoagulation. Instead, it is possible to achieve therapeutic anticoagulation in a volunteer population and to directly measure the reversal of anticoagulation using assays for anti-fxa activity and other coagulation markers. In order to approximate the ultimate target population for andexanet, older (ages years) subjects, including those with existing but stable chronic medical conditions, were included. Study treatment: The doses in this study were chosen based on the pre-clinical animal model data, data from Phase 2 dose-ranging studies, and a pharmacokinetic/pharmacodynamic model used to predict the andexanet dose required to reverse anticoagulation activity of each direct FXa inhibitor at the highest approved dose. Apixaban and rivaroxaban were tested at the C max of the highest approved dose for stroke prevention in atrial fibrillation. Effective reversal of inhibition of coagulation requires a molar excess of andexanet relative to the FXa inhibitor (>1:1 molar ratio). The pharmacokinetic/pharmacodynamic model identified the andexanet doses for each inhibitor by accounting for both the initial intravascular FXa inhibitor concentration, as well as the amount that redistributes into the vasculature upon andexanet administration and decreases in unbound plasma FXa levels. 3

5 Endpoints: At various time points, blood samples for pharmacodynamic and pharmacokinetic endpoints were collected into 3.2% trisodium citrate tubes. Plasma or platelet-poor plasma were prepared and frozen as small aliquots and then stored at -70 o C for pharmacokinetic and pharmacodynamic measurements, respectively, in centralized laboratories. Anti-FXa activity, thrombin generation, F1+2, D-dimer, and neutralizing antibody activity were performed by Quest Diagnostics; free anticoagulant concentrations were performed by Alturas Analytics, Inc; immunoassays for anti-drug antibodies and anti-human FX and FXa antibodies were performed by BioAgilytix Laboratories. Anti-FXa activity was measured at a central laboratory using a validated anti-fxa calibrated chromogenic assay described previously. 1 The anti-fxa activity assay is an enzymatic assay directly measuring fxa activity by cleavage of a fxa chromogenic peptide substrate in plasma using reagents from a commercial kit (Coamatic Heparin, DiaPharma). The protocol provided in the package insert was modified to minimize the sample dilution effect. The inhibitor-specific calibrators and controls were freshly prepared in pooled human plasma using the same anticoagulant present in the unknown samples (e.g., apixaban, rivaroxaban). The calibrators for each inhibitor were used to construct the standard curve with the inhibitor concentrations ranging from 0.0 to ng/ml. Samples with anti-fxa activity higher than ng/ml were reanalyzed following minimal sample dilution. The assay reportable ranges are ng/ml for apixaban and ng/ml for rivaroxaban, respectively. 4

6 The free anticoagulant fraction, which was not bound to either plasma protein or andexanet, was separated by a HTD96b high throughput rapid equilibrium micro-dialysis device (HTDialysis, Gales Ferry, CT) and quantified by a validated HPLC/MS/MS method at a central laboratory. Total anticoagulant concentration in plasma was quantified by HPLC/MS/MS following solid-liquid extraction using the same validated method. Due to the lack of clinical data to determine with certainty the therapeutic levels of rivaroxaban and apixaban, we calculated the estimated no-effect levels of the unbound FXa inhibitor concentrations. We multiplied the approximate no-effect level of the total FXa inhibitor concentration (30 ng/ml) 2 by 10% (the average measured unbound fraction) and corrected for the estimated dilution caused by the equilibrium dialysis method. Based on these calculations, the no-effect level is 3.5 ng/ml for unbound apixaban and 4 ng/ml for unbound rivaroxaban. Therefore, the unbound FXa inhibitor plasma concentrations associated with a therapeutic effect are higher than 4 ng/ml. Thrombin generation was measured using Calibrated Automated Thrombogram (CAT) according to the manufacturer s instruction (Diagnostica Stago). The reaction was initiated by addition of the PPP-reagent containing 5pM tissue factor (TF). CAT parameters analyzed include ETP, peak thrombin, lag time, time-to-peak, and velocity index. ETP was prospectively chosen as the endpoint because it reflects the overall thrombin generation potential. The ETP baselinederived range was calculated for each part of each study using all individuals who had at least a baseline value (pre-anticoagulant) for ETP. 5

7 Activated clotting time (ACT) was tested locally using Hemochrom Jr. Signature Elite coagulation device, according to the manufacturer s instruction. ACT+ cuvette and native whole blood were used. Safety: Samples for anti-drug antibodies (ADA) were assessed in a central laboratory using a validated bridging immunoassay based on electrochemical luminescence (ECL). Biotinylated and ruthenium conjugated (SULFO-TAG-labeled) andexanet were used as the capture and detecting reagents, respectively, in a MSD Streptavidin Gold plate. Three different assays with similar formats have been validated for screening, confirmation, and antibody titer analysis. For plasma samples with a confirmed presence of ADA, potential neutralizing activity of the ADA was tested using an anti-fxa assay similar to that described previously, after adding exogenous FXa inhibitor and andexanet to the plasma samples. Neutralizing antibodies were defined as those that impaired the ability of andexanet to block anti-fxa activity in this assay. Antibody neutralization was determined by the effect of the antibody s potential interference with the ability of andexanet to reverse anti-fxa activity of FXa inhibitors ex-vivo. D-dimer was analyzed with a validated immunoturbidity method using STA Liatest D-Di kit (Diagonostica Stago), per manufacturer s instructions, with an analytical range of µg/ml FEU. Following sample dilutions, the reportable range was µg/ml FEU with an upper limit of normal of 0.5 µg/ml FEU. 6

8 F1+2 was analyzed with a validated enzyme immunoassay (EIA) method using the Enzygnost F1+2 micro kit (Siemens), per manufacturer s instructions, with an analytical range 20 1,200 pmol/l. Following sample dilutions, the reportable range was 20 12,000 pmol/l, with a reference interval of pmol/l. 7

9 Figure S1: ANNEXA-A and ANNEXA-R study designs Subjects were dosed to steady state with apixaban or rivaroxaban. On Day 4, andexanet or placebo IV bolus was administered for 13 or 27 minutes in ANNEXA-A and ANNEXA-R, respectively. In Part 2 of the studies, a continuous 120 minute infusion of andexanet or placebo was administered after the IV bolus. The timing of the primary and secondary endpoints with respect to the end of the bolus or the infusion is shown. 8

10 Figure S2: Time course of activated clotting time before and after andexanet administration Activated clotting time (ACT) was measured prior to and after andexanet or placebo administration on study Day 4. (A) Apixaban with andexanet 400 mg IV bolus; (B) Rivaroxaban with andexanet 800 mg IV bolus; (C) Apixaban with andexanet 400 mg IV bolus + 4 mg/min infusion for 120 minutes; (D) Rivaroxaban with andexanet 800 mg IV bolus + 8 mg/min infusion for 120 minutes. The points on the graph represent the mean ACT and error bars illustrate standard error. The upper and lower limits of the baseline range are defined as one standard deviation above or below the mean ACT, respectively. 9

11 CONSORT 2010 Flow Diagram 1 One subject did not receive study drug due to inadequate IV access 2 Drug withdrawn due to adverse event of mild hives Randomized ANNEXA-A = 66 ANNEXA-R = 80 Received allocated andexanet ANNEXA-A = 48/49 1 ANNEXA-R = 53/53 Allocation Received allocated placebo ANNEXA-A = 17/17 ANNEXA-R = 27/27 Lost to follow-up ANNEXA-A = 0 ANNEXA-R = 1 Discontinued intervention ANNEXA-A = 1 2 ANNEXA-R = 0 Follow-Up Lost to follow-up ANNEXA-A = 0 ANNEXA-R = 0 Discontinued intervention ANNEXA-A = 0 ANNEXA-R = 0 Analysis Analysed efficacy; safety ANNEXA-A = 47/49; 48/49 ANNEXA-R = 53/53; 53/53 Analysed efficacy; safety ANNEXA-A = 17/17; 17/17 ANNEXA-R = 27/27; 27/27 Figure S3: Study consort diagram. A total of 66 subjects were randomized in ANNEXA-A: 49 to the andexanet arm (N = 25 in Part 1 and N = 24 in Part 2) and 17 to the placebo arm (N = 9 in Part 1 and N = 8 in Part 2). One subject (ANNEXA-A, Part 1) received apixaban but was not treated with andexanet due to inadequate IV access and was therefore excluded from both the efficacy and safety analyses. One subject (ANNEXA-A, Part 2) was withdrawn partway through the study drug infusion due to mild hives; this subject was excluded from the efficacy analysis due to not having anti-fxa values recorded after the infusion but was included in the safety population. A total of 80 subjects were randomized in ANNEXA-R: 53 to the andexanet arm (N = 27 in Part 1 and N = 26 in Part 2) and 27 to the placebo arm (N = 14 in Part 1 and N = 13 in Part 2). In ANNEXA-R Part 2, one subject was lost to follow-up and one withdrew from the study. Both subjects completed study drug administration and were included in the efficacy and safety analyses. 10

12 Apixaban Rivaroxaban Age - Yr Part 1 bolus only Part 2 bolus + infusion Part 1 bolus only Part 2 bolus + infusion Andexanet Placebo Andexanet Placebo Andexanet Placebo Andexanet Placebo N Median Female sex, N (%) 11 (45.8) 3 (33.3) 7 (29.2) 3 (37.5) 9 (33.3) 6 (42.9) 11 (42.3) 6 (46.2) BMI, Mean (SD) 26.7 (2.5) 27.4 (2.5) 27.5 (2.1) 27.8 (2.4) 27.0 (3.4) 25.9 (3.4) 27.8 (3.0) 27.6 (2.6) Creatinine, Mean (SD) (mg/dl) 0.8 (0.2) 0.8 (0.1) 0.9 (0.2) 0.9 (0.2) 0.9 (0.2) 0.8 (0.2) 0.9 (0.2) 0.9 (0.2) Race, N (%) White 24 (100) 9 (100) 21 (87.5) 8 (100) 22 (81.5) 10 (71.4) 20 (76.9) 8 (61.5) Ethnicity, N (%) Hispanic or Latino 10 (41.7) 4 (44.4) 11 (45.8) 2 (25) 9 (33.3) 4 (28.6) 4 (30.8) 10 (38.5) Table S1: Clinical characteristics of the subjects 11

13 Primary Endpoint Mean change (SD) in anti-fxa activity from baseline to nadir post-bolus (Part 1) or post-infusion (Part 2) % change (SD) in anti-fxa activity p-value % change (vs. placebo) Secondary Endpoints Mean change (SD) in anti-fxa activity from baseline to nadir post-bolus (Part 2) % change (SD) in anti-fxa activity from baseline to postbolus nadir (Part 2) p-value % change (vs. placebo) Number of patients (%) with 80% reduction in anti-fxa activity Apixaban Rivaroxaban Part 1 Part 2 Part 1 Part 2 bolus only bolus+infusion bolus only bolus+infusion Andexanet Placebo Andexanet Placebo Andexanet Placebo Andexanet Placebo N (60.8) (1.7) (24.5) (8.6) (49.3) (2.8) (18.1) (5.6) (75.9) (10.7) (42.2) (14.7) (89.2) (1.8) <0.001 <0.001 <0.001 < (48.8) (1.5) (7.4) (4.1) (84.8) (1.6) <0.001 < (58.8) (11.7) (36.7) (10.3) 24 (100) 0 (0) 23 (100) 0 (0) 26 (96.3) 0 (0) 26 (100) 0 (0) p-value (vs. placebo) <0.001 <0.001 <0.001 <0.001 Mean (SD) change in FXa inhibitor free fraction to nadir (ng/ml) -9.3 (3.2) -1.9 (1.6) -6.5 (2.8) -3.0 (1.2) (6.2) -4.2 (2.9) (8.1) (5.3) p-value (vs. placebo) <0.001 <0.001 <0.001 <0.001 Mean (SD) change in thrombin generation to peak (nm.min) (335.4) 88.2 (125.8) (263.3) (184.8) (331.2) (104.2) (344.8) p-value (vs. placebo) <0.001 <0.001 <0.001 <0.001 Number of patients (%) with ETP above lower limit of derived normal range (140.7) 24 (100) 1 (11.1) 23 (100) 2 (25) 26 (96.3) 1 (7.1) 26 (100) 0 (0) p-value (vs. placebo) <0.001 <0.001 <0.001 <0.001 Table S2: Descriptive endpoint statistics

14 References: 1. Lu G, DeGuzman FR, Hollenbach SJ, et al. A specific antidote for reversal of anticoagulation by direct and indirect inhibitors of coagulation factor Xa. Nat Med 2013;19: Pernod G, Albaladejo P, Godier A, et al. Management of major bleeding complications and emergency surgery in patients on long-term treatment with direct oral anticoagulants, thrombin or factor-xa inhibitors: Proposals of the Working Group on Perioperative Haemostasis (GIHP) March Archives of Cardiovascular Diseases 2013;106: