The present analysis is based on the results from the testing of serum samples from the predefined efficacy set of patients with a baseline imputed PaO2/FiO2 (P/F) >75 and ≤200 who enrolled in CARDEA (ClinicalTrials.gov identifier, NCT04345614). All patients were adults with ≥ 1 symptom consistent with COVID-19 infection, had a diagnosis of COVID-19 confirmed by laboratory testing using polymerase chain reaction or other assay, and pneumonia documented by chest imaging. At the time of enrollment, patients were receiving oxygen therapy via either a high flow (HFNC) or low flow nasal cannula but not non-invasive or invasive mechanical ventilation. All patients received dexamethasone or equivalent dose of another corticosteroid and 99% received pharmacological prophylaxis against development of venous thromboembolic disease. Remdesivir use was recommended for all patients, and convalescent plasma administration was allowed according to local standard of care. Other immunomodulators for the treatment of COVID-19 pneumonia, including tocilizumab and JAK inhibitors, were prohibited due to regulatory guidance. Auxora was administered by a 4-h IV infusion at 2.0 mg/kg (1.25 mL/kg) at 0-hour and 1.6 mg/kg (1 mL/kg) at 24 and 48 hours. Placebo was a matching formulation without the active pharmaceutical ingredient and was also dosed as a 4-h IV infusion at equivalent volumes of 1.25 mL/kg at 0-hour and 1 mL/kg at 24 and 48 hours.
After obtaining informed consent and before randomization, the clinical status of each patient was assessed using an 8-point ordinal scale (Table S1) in a standardized manner as described in the electronic case report form. The patient’s medical record was also reviewed to document the lowest SpO2/FiO2 (S/F) recorded during the previous 24 hours. The SpO2 was obtained using pulse oximetry. The FiO2 was read from the controlled oxygen source in patients requiring HFNC. For patients on an uncontrolled oxygen source, a conversion table was provided to all sites to estimate the FiO2 based on the method of oxygen delivery and oxygen flow rate. After randomization and the infusion of the first dose of study drug, an assessment of the clinical status of the patient using the ordinal scale and review of the lowest S/F were again performed before each subsequent infusion, then every 24 h until 240 h, and then continued every 48 h until Day 30 or discharge. A blood sample to check a D-dimer level was obtained prior to randomization and then every 72 hours while the patient remained hospitalized. The blood sample for D-dimer was processed, and results obtained, at each individual institution using their standard operating procedures and laboratory equipment.
Patients were also asked to consent separately for additional blood draws to obtain samples for Angiopoietin-1, Angiopoietin-2, renin and sCD25. These samples were collected prior to randomization and again at 96 hours in those patients who provided the additional consent. Blood for biomarker testing was placed in a red top serum separator tube, gently mixed by inverting the tube five times, and then allowed to clot undisturbed for 30 minutes with the tube standing upright. This was followed by centrifugation at 1800g until the clot and serum separated. The serum was then transferred by pipette to a collection kit and stored at -80o C. The samples were batched at each site and shipped frozen to Cincinnati Children’s Hospital Medical Center for testing. The levels of Angiopoietin-1, Angiopoietin-2, renin and soluble CD25 were determined by the enzyme-linked immunosorbent method utilizing kits from R&D Systems (Minneapolis, MN, USA) on the Dynex Elisa Processor.
Objectives
The objective of the analyses was to confirm the decrease in D-dimer levels that had been noted after treatment with Auxora in the initial open-label trial and to correlate a decrease, if observed, to changes in the daily imputed P/F and clinical outcomes as categorized by the 8-point ordinal scale. In addition, a secondary objective was to determine if Auxora decreased levels of Angiopoietin-2, soluble CD25, and renin, as well as increased Angiopoietin-1 levels, and whether those changes correlated with the changes in D-dimer levels, oxygenation, and clinical outcomes.
Statistical Analysis
We used descriptive statistics with 95% confidence intervals (CIs) to summarize data according to treatment group. We analyzed differences between treatment groups using MMRM modeling for the daily imputed P/F, ANCOVA modeling for other continuous variables, proportional odds testing for ordinal variables, and Cochran-Mantel-Haenszel testing for discrete variables. The ANCOVA model included the baseline value of the endpoint as a covariate and the treatment as fixed effect. The Cochran-Mantel-Haenszel test was stratified by the baseline imputed PaO2/FiO2 of <100 vs. >100. The PaO2 was imputed from the SpO2 using a published table based on Ellis’s inversion of the Severinghaus equation [15]. A two-sided alpha amount of 0.05 was used to test for differences in treatment outcomes without adjustments for multiplicity. The pairwise Pearson correlations between change from baseline of the clinical endpoints and the change from baseline of lab parameters were conducted by treatment groups.