This was a phase II/III, multicenter, prospective, open-label, non-controlled study to evaluate the efficacy and safety of rpFVIII for the treatment of severe bleeding episodes in Japanese patients with AHA (ClinicalTrials.gov ID: NCT04580407). The study protocol, informed consent form, and all amendments were reviewed and approved by the local institutional review boards of each investigator site before study initiation. The study was conducted in accordance with the Declaration of Helsinki and the principles and guidelines described in the study protocol. Informed consent was received from all patients in this study.

Eligible patients included men and women aged at least 18 years who were suspected of having, or had previously received, an AHA diagnosis (based on clinical evaluation and laboratory testing) and who presented with a severe bleeding episode (e.g., threatening vital organ function, requiring a blood transfusion, compromising muscle viability or neurovascular integrity, or affecting a major joint). Once a diagnosis of AHA had been confirmed, patients were able to start treatment with rpFVIII. Prior treatment with rFVIIa, aPCC, and pd-FVIIa/FX was not an exclusion criterion, provided that a washout period was allowed (3 h, 6 h, or 8 h, respectively) before the initial rpFVIII infusion.

rpFVIII was administered at an initial dose of 200 U/kg. The need for additional doses was determined by the investigator based on clinical bleeding status (reviewed every 6–12 h as per expert consensus recommendations) [26] and clinical laboratory evaluation (FVIII:C [measured with a one-stage clotting assay using the World Health Organization hFVIII plasma standard], activated partial thromboplastin clotting time [aPTT], hemoglobin [Hgb] and hematocrit [Hct] at 30 min, 8 h, 16 h, and 24 h after the first infusion, then every 12 h until 72 h, and then every 24 h until the end of infusion/study). If additional doses of rpFVIII were administered, FVIII:C, aPTT, Hgb, and Hct were also measured at each subsequent dose until 24 h after the dose was administered. Additional doses of rpFVIII were administered as frequently as every 4–12 h with dose and frequency determined based on the post-infusion FVIII:C result and the target FVIII:C. For bleeds of particular clinical concern (e.g., severe mucosal, intracranial, retro- or intra-abdominal, genitourinary, neck, traumatic, or postoperative bleeds), the target trough FVIII:C was at least 80% for the first 24 h. For all other severe bleeding episodes (e.g., joint, muscle, soft tissue) in the first 24 h and all bleeding episodes after the first 24 h, the target trough FVIII:C was at least 50%. The dose of rpFVIII could not exceed 800 U/kg every 4 h. Treatment with rpFVIII was continued until bleeding was successfully controlled, until the investigator concluded a lack of efficacy, or until the patient withdrew from the study.

The primary efficacy endpoint was the proportion of severe bleeding episodes with a demonstrated positive response to rpFVIII therapy at 24 h after the initiation of treatment using a well-defined four-point ordinal scale (Supplementary Table S1). A positive response was defined as effective or partially effective assessment of efficacy. Control of bleeding was evaluated based on obvious blood loss (external blood loss and bodily fluids), hematology results, blood transfusion and blood component requirements, physical or technological examination of the bleeding site, neurological examination, and imaging studies. Patients who experienced a therapeutic response to rpFVIII were eligible for treatment with rpFVIII for any subsequent major bleeding episode but outcomes for these bleeding episodes were not part of the primary efficacy analysis. Re-bleeding was considered to have occurred if there was bleeding from a previously successfully controlled site within 2 weeks of the last dose of rpFVIII. In this case, the event was recorded as an adverse event (AE) and treated appropriately. Key secondary efficacy endpoints included: the overall proportion of severe bleeding episodes successfully controlled with rpFVIII therapy; the proportion of bleeding episodes responsive to rpFVIII therapy at 30 min, 8 h, and 16 h after the initiation of therapy, and every 14 days until the end of the study (90 days after the final dose of rpFVIII for initial bleeding, to cover the average time to remission of AHA [3, 27] including resolution of other bleeding besides initial bleeding); and the frequency, total dose, and total number of infusions of rpFVIII required to successfully control qualified bleeding episodes.

Safety endpoints included AEs, serious adverse events (SAEs), clinical laboratory measurements, vital signs, and the presence of anti-BHK cell antibodies. AEs of special interest included hypersensitivity reactions, the development of de novo inhibitors to pFVIII, anamnestic reactions with an increase of inhibitor titer to pFVIII and/or hFVIII, and thromboembolic events.

The planned total sample size for this study was five patients, which was based on feasibility considerations given the low incidence of AHA in Japan. The primary efficacy endpoint was calculated as the proportion of patients with a positive response to rpFVIII therapy at 24 h post-treatment and the corresponding exact two-sided Clopper–Pearson 95% confidence intervals (CIs). Patients who had hemostatic response and stopped treatment because bleeding was controlled were assumed to be responders at the 24-h assessment time point.