Introduction

Vascular leakage is a major feature of pathogen-induced acute respiratory distress syndrome (ARDS) [1]. Triggered by inflammation following endothelial and epithelial lesions, it is thought to play an important role in altering gas exchanges. Consequently, the extravascular lung-water index (EVLWi), a marker of pulmonary vascular leakage measured by transpulmonary thermodilution, is independently associated with ARDS patients’ outcomes [2, 3]. Because EVLWi is highly elevated during SARS-CoV-2-induced ARDS [4, 5], which causes high mortality [4, 6], controlling vascular leakage might be of major interest in managing this disease.

FX06, an innovative drug containing fibrin-derived peptide Bβ15-42, stabilizes vascular endothelial (VE)-cadherin–dependent interendothelial cell junctions [7,8,9]. It reduced capillary leakage in several animal models of lipopolysaccharide- or HCl-induced acute lung injury [9, 10] and prolonged survival in a murine model of dengue-virus infection [9]. In a phase II trial conducted on 234 patients suffering from ischemia–reperfusion injuries during acute coronary syndrome, FX06-treated patients had 58% smaller early necrotic core zones [11]. Importantly, adverse events were comparable between groups, indicating the drug’s high safety profile. FX06 was then used as salvage therapy for a patient with severe ARDS following Ebola-virus infection, with a temporal link between its injection and sharply decreased EVLWi [12]. More recently, FX06 (400 mg/d for 4–7 days) was given as compassionate therapy to 6 patients receiving extracorporeal membrane oxygenation (ECMO) for coronavirus disease 2019 (COVID-19) [13]; 4 experienced improvement and 2 died. No clear treatment-related adverse event occurred.

Taken together, those findings indicate that FX06 is well-tolerated by patients and is a potent regulator of vascular leakage during ARDS. We hypothesized that FX06 might limit pulmonary vascular hyperpermeability during ARDS induced by SARS-CoV-2 infection, thereby improving gas exchanges and patients’ outcomes.

Methods

Trial design

We conducted a multicenter, double-blinded, randomized trial. The independent ethics review board CPP Ouest VI, Brest, France, and the ANSM (Agence Nationale de Sécurité du Médicament et des Produits de Santé) approved the trial protocol (available in  Additional file 1). F4-Pharma Ges.m.b.H. (Vienna, Austria) provided FX06. An independent Data- and Safety-Monitoring Committee periodically reviewed safety outcomes, with recruitment interruptions planned after inclusions of 10 and 30 patients. Neither F4-Pharma nor trial sponsors participated in the trial design, data collection, analysis or interpretation, or the writing or submission of the manuscript. The study protocol was registered at ClinicalTrials.gov (NCT04618042).

Participants

To be eligible for inclusion, patients had to be ≥ 18 year old and receiving invasive mechanical ventilation for < 5 days for polymerase-chain reaction-confirmed SARS-CoV-2-induced ARDS, according to the Berlin definition [14]. Exclusion criteria were mechanical ventilation for > 4 days; participation in another interventional clinical trial; severe renal, hepatic or cardiac insufficiency, or in a moribund state at randomization (see Additional file 1); contraindication for vascular access implantation for transpulmonary thermodilution monitoring; chemotherapy, radiotherapy or immunotherapy for malignancy; pregnancy or lactation; any history of severe allergic drug reaction. Patients taking drugs interfering with inflammation were also excluded, unless the drug’s use during COVID-19 was stated in the hospital center’s written policy.

According to the specifications of emergency consent, randomization was possible without a close relative’s or surrogate’s consent, but informed consent by the patient or patient’s relatives was obtained for all patients.

Treatment allocation

Patients were randomly assigned to receive either FX06 or its vehicle (phosphate-buffered saline) as the placebo. The randomization list was computer-generated with a 1:1 ratio and undisclosed block sizes, stratified by center. Concealment of the study-group assignments used a centralized, secure, interactive, web-based response system (CleanWeb, Telemedicine Technologies S.A.S., Boulogne-Billancourt, France) accessible from each study center. All investigators, statisticians, and data analysts were blinded to arm assignments until the study and analysis were completed.

Interventions

Patients were randomized to receive intravenous FX06, 400 mg/d or the placebo for 5 days. Each dose was administered in two boluses separated by a 10-min interval. The dose regimen chosen was based on the results of previous studies, in animals and humans, that suggested safety and mechanistic engagement with this dosing (additional Methods in Additional file 2). The manufacturer provided each treatment in unrecognizable ready-to-use form (numbered and sealed therapeutic units containing 10 vials of active treatment or placebo solution), stocked in each intensive care unit (ICU) under the supervision of each facility’s pharmacy department.

Patients were monitored using transpulmonary thermodilution systems (EV1000/Volume View, Edwards Lifesciences, Irvine, CA, USA, or PiCCO2, Pulsion Medical Systems, Feldkirchen, Germany), with thermistor-tipped catheters introduced in a femoral artery and an internal jugular vein [2,3,4]. Extravascular lung water and other thermodilution-derived parameters were averaged from three injections of cold physiological saline solution, in supine position, and indexed to the patient’s predicted body weight. Thermodilution measurements were taken before treatment administration during the first 5 days post-inclusion, with a measurement repeated 3 h post-administration on day 2, to detect a possible short-time effect of the drug. For patients receiving venovenous (VV)-ECMO, measurements were taken during a transient diminution of ECMO blood flow to < 2 L/min. Preliminary study results showed that thermodilution parameters were not affected by ECMO blood flow under that level (see Additional file 2: Table S1 and Fig. S1).

For ARDS management, investigators were asked to follow the most recent recommendations from the French Society of Intensive Care Medicine (https://www.srlf.org/rfe-srlf-prise-en-charge-du-syndrome-de-detresse-respiratoire-aigue-sdra-de-ladulte-a-la-phase-initiale/). Specific treatments targeting COVID-19 were discouraged, unless the drug’s use during COVID-19 was stated in the center’s written policy.

Serum interleukin (IL)-6, IL-10, and soluble (s)VE-cadherin were quantified with DuoSet Elisa kits (R&D systems, Minneapolis, MN, USA).

Outcomes

The primary endpoint was the EVLWi change, assessed by transpulmonary thermodilution, between day 1 and day 7. Secondary endpoints included the evolution of daily EVLWi, cardiac index, global end-diastolic volume index, and pulmonary vascular permeability index measured by transpulmonary thermodilution for 7 days; daily fluid balance; serum albumin; systolic, diastolic, and mean blood pressures; and heart rate for 7 days; partial oxygen pressure/fraction of inspired oxygen (PaO2/FiO2) ratio and Sequential Organ-Failure Assessment (SOFA) score over 15 days; rate of rescue with VV-ECMO; durations of invasive mechanical ventilation, vasopressor support, and renal replacement therapy over 30 days; Weinberg Radiological Severity score over 30 days [15]; survival at 30 and 60 days; nature and frequency of adverse events. Kinetics of serum d-dimers and C-reactive protein over 7 days were extracted from medical charts afterward. Serum IL-6, IL-10, and sVE-cadherin measurements on days 1 and 7 in available biological samples were added as post hoc measurements.

Statistical analyses

Assuming a baseline (inclusion) mean EVLWi of 13 mL/kg and standard deviation (SD) of 5 mL/kg [16], and a 30% EVLWi decrease in FX06-treated patients compared to controls on day 7 [9, 10, 12], for 80% power and an overall 5% two-sided α-risk, the required sample size was 25 patients/group.

Baseline characteristics are reported as number (%) for categorical variables and median [interquartile range, IQR] for continuous variables. Efficacy endpoints were analyzed according to intention-to-treat principles. Safety endpoints were analyzed for all patients who received at least one assigned-treatment dose.

Missing primary endpoints were replaced by imputation values for patients who died or whose conditions no longer warranted the transpulmonary thermodilution system before 7 days; the last thermodilution value was retained for the primary analyses. Primary endpoints were compared between groups using an adjusted analysis of covariance (ANCOVA) of EVLWi at randomization. Results are expressed in terms of adjusted mean change with 95% confidence interval (CI).

Three sensitivity analyses were computed: complete case analysis, worst-case analysis or using a different statistical method (Mann–Whitney U test). Prespecified subgroup analyses were conducted according to VV-ECMO or EVLWi > 10 mL/kg at inclusion.

Qualitative and quantitative secondary outcome measures were compared between groups using, respectively, Pearson’s Chi-square tests and t tests, or Mann–Whitney U tests. Overall survival was estimated with the Kaplan–Meier method. Longitudinal quantitative endpoints were compared using linear-mixed models with a random effect for subjects. This model was fitted to fixed effect by an interaction between treatment arm and time (since the date of randomization), with the slope parameter estimating the difference between groups. A restricted likelihood maximization-estimation method was used. The p-values associated with the fixed effects were calculated using the analysis of variance (ANOVA) function with Kenward–Roger approximation for calculating the number of degrees of freedom.

Analyses were computed with a 2-sided α risk of 5%. All analyses were performed using R software (R Foundation for Statistical Computing, Vienna, Austria), version 4.0.3.

Results

Forty-nine patients were randomized from November 2020 to April 2021 and retained for analysis (Fig. 1). Their main characteristics are reported in Table 1. They were very severely ill at baseline, with median PaO2/FiO2 ratio at 104, static pulmonary compliance of < 30 mL/cmH2O, and more than one-third of them were on VV-ECMO. One-third were receiving vasopressors. Specific therapies targeting COVID-19 were marginal, except corticosteroids, given to all participants.

Fig. 1
figure 1

Flow chart of inclusion, randomization, and follow-up of patients included in this clinical trial

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Table 1 Characteristics of patients with COVID-19-associated ARDs at trial inclusion, according to assigned treatment arm
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Study drug

All but 5 patients received the complete treatment. One patient allocated to receive the placebo was accidentally included in another interventional study; the assigned treatment was interrupted after 3 days. One patient allocated to the FX06 arm stopped treatment after 3 days because of fungal co-infection. Two patients allocated to the placebo arm died on day 3 or day 4. Lastly, one patient’s treatment was accidentally withheld on day 4.

Primary outcome

The primary outcome of EVLWi change between days 1 and 7 did not differ between FX06-treated patients and controls (Fig. 2 and Table 2). EVLWi kinetics and their individual variations were also comparable between groups (Additional file 2: Figs. S2 and S3). Patients’s EVLWis were high at inclusion, comparable for the 2 groups, and remained elevated during the first 7 days. Eight patients—1 FX06 recipient and 7 controls—did not undergo transpulmonary thermodilution on day 7; their last available values were retained for the primary analysis: 3 had died (1 FX06 recipient and 2 controls), 3 had recovered sufficiently to allow removal of their thermodilution catheters, 1 had a catheter infection necessitating its removal, and 1 withdrew consent to participate. Excluding those patients did not affect the primary-analysis results.

Fig. 2
figure 2

Primary outcome: extravascular lung-water index (EVLWi) variations from day 1 to day 7 for FX06 recipients and placebo controls. The panels show Tukey’s boxplot visualization

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Table 2 Primary and secondary outcomes according to assigned treatment arm
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Several sensitivity analyses were computed: analyzing the primary outcome for patients with ECMO vs without; most severely ill (EVLWi > 16 mL/kg) vs less severely ill; or indexing the dose received above vs below 4.2 mg/kg/d (median dose received). All failed to detect any significant FX06 effect on EVLWi (not shown). Interestingly, EVLWi measured before and 3 h after FX06 bolus injections was very similar (median variation − 0.02 [IQR − 0.57; 0.55], n = 22).

Secondary outcomes

The Pulmonary Vascular Permeability Index (PVPI) was also very high during the first 7 days and did not differ between groups (Additional file 2: Fig. S4). Cardiac index and global end-diastolic volume index (GEDVI) were also comparable (not shown). Daily fluid balance remained positive during the first week, with comparable levels for the 2 groups (Additional file 2: Fig. S5). Serum albumin, another marker of vascular leakage, was very low at inclusion (median 23 [IQR 19; 26] g/L; n = 48); it remained stable with no between-group differences during the first week.

PaO2/FiO2 remained very low during the first 15 days (Additional file 2: Fig. S6), and very few patients survived to be extubated on 30 days (Table 2). Interestingly, the Weinberg Radiological Severity score decreased less for FX06 recipients (estimated effect 0.13 [95% CI 0.07–0.18]; p < 0.001). However, for a post hoc analysis taking into account missing values and mortality as a competing risk by imputing the last score available for survivors and a score of 12 after the patient died, score kinetics did not differ between groups (Additional file 2: Fig. S7). Three patients (2 FX06 recipients and 1 control) received VV-ECMO rescue therapy after inclusion.

Although catecholamine-free days were comparable for the 2 groups (Table 2), FX06 recipients had significantly lower systolic and diastolic blood pressure values after day 4 (Additional file 2: Fig. S8). FX06 did not affect heart rate (not shown).

The baseline SOFA score, reflecting the extent of multiple organ failure, was high and remained stable for both groups for 15 days (Additional file 2: Fig. S9). Finally, survival was comparable for both groups, with 34/49 (69%) day-60 survivors (Table 2 and Additional file 2: Fig. S10). C-reactive protein and serum d-dimers, elevated at baseline, remained stable and comparable. Serum cytokine measurements were available for 24 patients. For both groups at inclusion, IL-6 was elevated and continued to rise during the first week, while IL-10 levels were also high and declined slightly during the first week. FX06 recipients had slightly higher baseline sVE-cadherin values that remained comparable to those of the placebo group thereafter.

Safety

Adverse event rates were comparable for the 2 groups (Table 3). Although overall secondary infections were not more common in FX06 recipients, they did develop more episodes of microbiologically confirmed ventilator-associated pneumonia [17].

Table 3 Serious adverse events (SAEs) reported according to assigned treatment arm
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Discussion

In this multicenter, double-blinded, randomized trial, FX06 did not alter the thermodilution-measured EVLWi evolution during SARS-CoV-2-induced ARDS. Other markers of pulmonary vascular leakage, e.g., patients’ functional outcomes reflecting pulmonary function and 60-day survival, were also not affected. Although elevated at baseline, circulating markers of inflammation and endothelial lesions were comparable for the 2 groups. Despite their similar rates of serious adverse events, FX06 was associated with higher rates of ventilator-associated pneumonia.

Inflammation-induced pulmonary vascular leakage is widely diffused during severe SARS-CoV-2 infection. Autopsies of COVID-19 patients revealed markedly elevated lung weights [18, 19] and disruption of interendothelial VE-cadherin-dependent junctions [7] and to dampen neutrophil recruitment in the lung in two models of lipopolysaccharide- or HCl-induced acute lung injury, indicating a risk of impaired bacterial clearance [10]. Importantly, incubation of FX06 with monocytes or alveolar macrophages did not impact their in vitro activation and capacity to release pro-inflammatory cytokines. Moreover, on the contrary, FX06 was shown to enhance bacterial clearance and ultimately survival in a model of secondary Pseudomonas aeruginosa infection [10]. Although this potential effect of FX06 needs to be better clarified, these findings indicate the need for close monitoring of secondary infections in future evaluations of it.

Conclusions

FX06 did not lower thermodilution-derived EVLWi during severe ARDS induced by SARS-CoV-2 infection. Whether other time-lines for its administration or other dosing regimens might be more efficient remains to be determined.