Study Design and Oversight

The ANNEXA-A and ANNEXA-R trials were randomized, double-blind, placebo-controlled studies that were designed to evaluate the ability of andexanet to reverse anticoagulation with apixaban (Eliquis; Pfizer and Bristol-Myers Squibb) or rivaroxaban (Xarelto; Bayer and Johnson & Johnson) and to evaluate the safety of andexanet in healthy older volunteers. The study was conducted at two clinical sites (Celerion in Tempe, Arizona [ANNEXA-A], and West Coast Clinical Trials in Cypress, California [ANNEXA-R]). The study protocols were approved by the institutional review board at each study site, and all participants provided written informed consent before enrollment. Healthy volunteers 50 to 75 years of age were randomly assigned, with the use of an interactive Web-response system, in a 3:1 ratio (ANNEXA-A) or a 2:1 ratio (ANNEXA-R), to receive andexanet or matching placebo. Each study was performed in two consecutive parts: in part 1, we examined the intravenous andexanet bolus alone, and in part 2 we studied an intravenous bolus followed by a continuous 120-minute infusion. Study participants were housed at the study site for 8 days, and safety outcomes were assessed on days 15, 36, and 43 after administration of the study drug. An independent safety committee whose members were aware of the study-group assignments reviewed safety data on an ongoing basis.

The studies were conducted in accordance with International Conference on Harmonisation Good Clinical Practice guidelines and the principles of the Declaration of Helsinki. They were designed by the academic investigators in collaboration with Portola Pharmaceuticals. All the authors contributed to the writing of the manuscript, made the decision to submit the manuscript for publication, and vouch for the completeness of the data and the accuracy of the results and for the fidelity of this report to the study protocol, which is available with the full text of this article at NEJM.org. Nondisclosure agreements were in place between the sponsor and the academic authors, but the authors had unrestricted access to the data. The study was conducted under the sponsorship of Portola Pharmaceuticals, with additional financial and scientific support from Bayer, Bristol-Myers Squibb, Johnson & Johnson, and Pfizer.

Study Treatment

In the ANNEXA-A study, participants received 5 mg of apixaban orally twice daily for 3.5 days to achieve steady-state plasma levels at the highest approved dose. Three hours after the last dose of apixaban on day 4 (at or near the time of the highest plasma concentration), andexanet was administered as a 400-mg intravenous bolus (30 mg per minute) (part 1) or as a 400-mg intravenous bolus followed by a continuous infusion of 4 mg per minute for 120 minutes (480 mg in total) (part 2). In the ANNEXA-R study, participants received 20 mg of rivaroxaban orally once daily (the highest approved dose) for 4 days. On day 4, at 4 hours after the last dose of rivaroxaban (at or near the maximum plasma concentration), andexanet was administered as an 800-mg intravenous bolus (30 mg per minute) (part 1) or as an 800-mg intravenous bolus followed by a continuous infusion of 8 mg per minute for 120 minutes (960 mg in total) (part 2). The doses were selected on the basis of the phase 2 development program that established the stoichiometric ratio needed for reversal of the effects of each anticoagulant with the use of andexanet.8,10,11 The dose of andexanet required to reverse the effects of 20 mg of rivaroxaban once daily is higher than that required to reverse the effects of 5 mg of apixaban twice daily because of both the higher initial maximum plasma concentration of rivaroxaban and the larger volume of distribution of rivaroxaban.

Study End Points

The primary end point for both studies was the percent change in anti–factor Xa activity, measured with the use of a validated chromogenic assay of factor Xa enzymatic activity,12 from baseline (before administration of andexanet or placebo) to nadir (after administration of andexanet or placebo). For part 1, the nadir was defined as the value of anti–factor Xa activity at 2 minutes or 5 minutes (whichever value was smaller) after the end of the bolus; for part 2, it was defined as the smallest value between 10 minutes before and 5 minutes after the end of the continuous infusion (Fig. S1 in the Supplementary Appendix, available at NEJM.org).

The secondary efficacy end points were the proportion of participants with an 80% or greater reduction in anti–factor Xa activity from baseline to the nadir after administration of andexanet or placebo; the change in unbound inhibitor plasma concentration from baseline to the nadir after administration of andexanet or placebo; the change in thrombin generation, measured as the change in endogenous thrombin potential, from baseline to peak after administration of andexanet or placebo; and the occurrence of an endogenous thrombin potential above the lower limit of the baseline-derived range at its peak after administration of andexanet or placebo (between 2 and 10 minutes after the end of the bolus) or after the infusion. Because of the absence of a clinically validated reference range for endogenous thrombin potential, the baseline-derived range (hereafter referred to as the normal range) was prospectively defined as the mean endogenous thrombin potential at baseline on day 1 (before anticoagulant administration) plus or minus 1 standard deviation. For part 2, an additional secondary end point was the percent change in anti–factor Xa activity from baseline to the post-bolus nadir. Patients were followed for evaluation of clinical outcomes, including symptomatic thrombosis and bleeding. Details regarding the methods for measurement of the end points are provided in the Supplementary Appendix.

Statistical Analysis

The primary efficacy analysis was performed with the modified intention-to-treat population, which included all participants who underwent randomization, who received any amount of andexanet or placebo, and for whom a baseline measurement of anti–factor Xa activity (before administration of andexanet or placebo) and at least one measurement of anti–factor Xa activity after administration of andexanet or placebo were available for analysis. All efficacy analyses were performed on data from the modified intention-to-treat population, and end points were compared with the use of an exact Wilcoxon rank-sum test.13 A total of 145 participants treated with apixaban or rivaroxaban were randomly assigned to andexanet or placebo in a 3:1 ratio (ANNEXA-A) or a 2:1 ratio (ANNEXA-R). The samples were sufficient to provide greater than 99% power to detect a difference between andexanet and placebo in the percent change in anti–factor Xa activity from baseline to the primary time point (nadir), at a two-sided alpha level of 5%, within each part of each study. The study was powered under the assumption that the differences relative to placebo that were observed in the previous studies of andexanet represent the true differences (additional information on power calculations are provided in the protocol and statistical analysis plan). The sample size of 145 participants was sufficient to provide safety data on at least 100 participants treated with andexanet and to retain power if the observed difference in previous studies overestimated the true difference. To control the type I error rate, the primary end point was tested first within each part of each study.14 For the primary end point of each part of each study, a two-sided P value of less than or equal to 0.05 for the percentage change in anti–factor Xa activity was considered to indicate statistical significance. The secondary end points were evaluated in sequence, and a difference between andexanet and placebo was considered to be significant if the two-sided P value was less than or equal to 0.05 and the differences with regard to all previous end points in the sequence were also significant.