Nitric Oxide Gas Inhalation Therapy for Mechanically Ventilated Patients With Severe Acute Respiratory Syndrome Caused by SARS-CoV2: a Randomized Clinical Trial.
Phase: PHASE2
Conditions: SARS (Severe Acute Respiratory Syndrome), Coronavirus
Sponsor: Massachusetts General Hospital

 
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High-Dose Inhaled Nitric Oxide in Acute Hypoxemic Respiratory Failure due to COVID-19: A 
Multicenter Phase 2 Trial  
Raffaele Di Fenza, Naman S Shetty, Stefano Gianni, Vibhu Parcha, Valentina Giammatteo, Bijan Safaee 
Fakhr, Daniel Tornberg, Olof Wall, Piotr Harbut, Peggy S Lai, Jonathan Z Li, Sabrina Paganoni, Stefano 
Cenci, Ariel L Mueller, Timothy T Houle, Oluwaseun Akeju, Edward A Bittner, Somnath Bose, Louie K 
Scott, Ryan W Carroll, Fumito Ichinose, Magnus Hedenstierna, Pankaj Arora and Lorenzo Berra, on 
behalf of the Nitric Oxide Investigators.  
Additional Nitric Oxide Investigators include: Caio C Araujo Morais, Lauren E Gibson, Takamitsu Ikeda, 
Eizo Marutani, Yusuke Miyazaki, Anna Fischbach, Lisa Traeger, Martin I Capriles, Eduardo Diaz 
Delgado, Grant M Larson, Roberta Ribeiro De Santis Santiago, Carolyn La Vita, Binglan Yu, Maurizio F. 
Cereda, Nattaly Greene, Paula Restrepo, James P Flynn, James Regan, Riccardo Pinciroli, Elizabeth I 
Caskey, Kimberley Hutchinson, N Stuart Harris, Josanna Rodriguez-Lopez, Marvin G Chang, Jacob 
Wideaus, Matilda Widaeus Kambiz Shahgaldi, Karl Hagman, Garima Arora, Robert Johnson.  
Clinicaltrials.gov Protocol Number: NCT04306393  
Publication Date: December 15, 2023

 
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STUDY METHODS 
Study Design 
This was an investigator-initiated multicenter, single-blinded, randomized (1:1), controlled, parallel-arm 
clinical trial conducted at five sites in the United States (Massachusetts General Hospital, University of 
Alabama at Birmingham, Louisiana State University, and Beth Israel Deaconess Medical Center) and 
Sweden (Danderyd Hospital). All participants or their medically authorized representatives provided 
written informed consent, and the study was approved by the Institutional Review Boards at each of the 
respective study sites. The detailed study protocol and the statistical analysis plan are included in this 
Online Data Supplement document. This study was registered on ClinicalTrials.gov as NCT04306393 
(Registered on March 12th, 2020).  
Participants  
The study enrolled adult patients with SARS CoV-2 infection (confirmed diagnosis using RT-PCR) 
admitted to the intensive care units and were intubated and mechanically ventilated. Individuals were 
excluded if they had been intubated for >72 hours or the physician of record opposed the enrollment due 
to safety concerns. In July 2020, exclusion criteria were expanded to additionally exclude those who: 1) 
were enrolled in another intervention study, 2) had a prior medical history of lung malignancy, 
pneumonectomy, or lung transplant, 3) were receiving tidal volume support <3 ml/kg of ideal body 
weight at the screening to assure NO delivery, 4) had severe burns (>40% of total body surface area), 5) 
had experienced cardiac arrest with cardiopulmonary resuscitation for longer than 30 minutes, 6) had a 
presumed severe deficit in cerebral function with fixed dilated pupil, 7) were receiving renal replacement 
therapy at the time of screening, 8) had a history of malignancy or other irreversible disease/conditions 
with an estimated six- month mortality > 50%, 9) had received inhaled nitric oxide gas before screening, 
or 10) were admitted to the hospital for reasons unrelated to COVID-19. Expansion of the exclusion 
criteria occurred during the first months of the trial to ensure a uniform patient population among centers 

 
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during the evolving pandemic. Figure 1 describes patient enrolment and follow-up as per CONSORT 
recommendations (Appendix, p 42).  
Randomization and Masking  
After informed consent was obtained, eligible participants were randomized on the same day using a 
centralized, secure computer platform stratified by site, age (≤ or >60 years-old), and sex. Because of the 
uncertainty of site enrolment due to the variable nature of the pandemic, a small, fixed block size of two 
was used for the first 20 allocations at each site. Future assignments were conducted using randomized 
permuted blocks of size 2 and 4 to respective study arms. All assignments were generated using a 1:1 
ratio using a randomization scheme prepared by the study statistician. Allocation concealment was 
accomplished using a centralized computer system. The study interventions included either the 
institutional usual care alone (control arm) or the institutional usual care with the addition of high-dose 
inhaled nitric oxide (treatment arm). Usual care was delivered according to each institution’s protocols 
(including ventilation strategies and use and dosage of antivirals and antimicrobials, anti-inflammatory 
agents including steroids, inhaled nitric oxide as a rescue therapy at 5-20 ppm when PaO2/FiO2<100 
mmHg, inotropic-vasopressor agents and initiation of extracorporeal membrane oxygenator). Enrolment 
of participants occurred at the onset of the pandemic and before vaccines were available. In order to 
minimize aerosolization procedures and associated possible contamination and infection of healthcare 
professionals from the break of the ventilator circuit per recommendations of the World Health 
Organization, no placebo was used.16 Masking was only possible for participants and/or their legally 
authorized representative, as patients were unconscious and no one other than medical personnel was 
allowed in the participant’s room. Healthcare professionals and study team members assessing outcomes 
were not blinded to the treatment.  
Procedures  

 
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Participants in the treatment arm received inhaled nitric oxide at 80 ppm for the first 48 hours after 
enrolment. The gas was started immediately after randomization within the first 72 hours of mechanical 
ventilation. After the first 48 hours of treatment, the gas was reduced to 40 ppm and maintained at this 
concentration until severe hypoxemia resolved (PaO2/FiO2>300 mmHg). The gas administration was 
synchronized with the ventilator and delivered through an injector module at constant concentration 
throughout the respiratory cycle into the inspiratory limb of the patient's breathing circuit. Weaning from 
inhaled nitric oxide was initiated when PaO2/FiO2>300 mmHg was recorded for a duration of greater than 
24 hours. Gradual weaning by 50% every four hours was employed with cautious evaluation for rebound 
hypoxemia and acute hypotension. The previous dose was used when rebound hypoxemia or acute 
hypotension occurred. The detailed inhaled nitric oxide weaning protocol is described in the Appendix 
(pp 13-14).  
Participants from all centers were evaluated by the research staff up to 90 days after study completion. In 
participating U.S. centers, alive participants were contacted on day 90 for a follow-up phone call. In 
addition, data were retrieved from physicians’ notes to determine outcomes after hospital discharge (e.g., 
hospital readmission, admission to a rehabilitative facility, death) and to investigate functional status. The 
phone interviews were conducted by research staff through a structured questionnaire provided in the 
Appendix (pp 22-31). During the follow-up phone calls, participants were asked if they were experiencing 
any sensory symptoms or motor deficits. No phone calls were performed in Sweden, and data were 
retrieved only from the medical records (following the same format as the U.S. questionnaire). 
Electromyography and imaging (magnetic resonance imaging or computed tomography) reports were 
collected from medical records for participants reporting sensory or motor symptoms. For the final 
analysis, participants from any center were considered positive for neurological findings if sensory 
symptoms and/or motor deficits were confirmed and reported by a physician caring for the patient, such 
as a neurologist or physiatrist, in the medical charts.  

 
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Outcomes  
The primary outcome of this study was the change in arterial oxygenation (PaO2/FiO2) at 48 hours. The 
secondary outcomes were all-cause mortality at 28 and 90 days, time to reach normoxaemia (defined by a 
PaO2/FiO2 >300 mmHg for at least 24 hours among survivors), and the proportion of normoxaemic 
participants in the two groups at 28 days. The safety outcomes for this clinical trial included 
methaemoglobinaemia defined as methaemoglobin (MetHb) exceeding 5%, inhaled nitrogen dioxide >3 
ppm, haemodynamic instability (rebound hypotension) during weaning, the occurrence of acute kidney 
injury by 28-days, or the initiation of renal replacement therapy by 90 days. Exploratory study outcomes 
included change in viral load (Log10 copies of SARS-CoV-2 per mL) in plasma and sputum, duration of 
mechanical ventilation, use of venous-venous extracorporeal membrane oxygenator (VV-ECMO), and 
neurological signs and symptoms (motor and sensory) at 90 days.  
Plasma and Sputum Preparation  
Starting June 24th, 2020, at Massachusetts General Hospital, plasma and sputum (endotracheal aspirate in 
intubated participants, spontaneously expectorated in extubated participants) were collected 
prospectively. Biological samples of those participants who consented were stored at -80 oC for 
quantification of the SARS-CoV-2 viral load. There was no post-hoc selection of participants for this 
analysis. Quantification of viral load in blood and sputum was obtained as previously described.17 
Briefly, samples were centrifuged at 21,000 g for 2 hours at 4 oC. The supernatant was removed, and 
Trizol-LS was added to the samples and vortexed briefly. The samples were incubated at 4 oC for 15 
minutes and subsequently treated with chloroform. Samples were vortexed briefly and then centrifuged at 
21,000 g for 15 minutes at 4 oC. The resulting supernatant containing RNA was then concentrated using 
isopropanol precipitation, and SARS- CoV-2 RNA was measured by RT-qPCR.  
Statistical Analysis  

 
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Participants randomized to inhaled nitric oxide were hypothesized to have at least 20% greater 
improvement in PaO2/FiO2 at 48 hours after gas initiation compared with the usual care alone. Assuming 
a two-tailed alpha of 0.05, the enrolment of 182 participants would provide 90% power to detect an effect 
size of 38 mmHg PaO2/FiO2 change based on the effect estimates in a previous investigation in 
hypoxemic intubated and mechanically ventilated patients.18 Presuming a 10% dropout, the target sample 
size was 100 in each group (n=200 total). The original sample size estimates were derived using a 
frequentist approach to the analysis. However, due to reporting requirements posed by the FDA and IRB 
and the need for rapid learning in the context of the COVID-19 crisis, the analysis plan was revised (April 
2020) to employ Bayesian estimation, which allowed for sequential group analyses. Prior probability 
distributions were specified for the intercept, the individual predictors, and the error term(s) (i.e., sigma 
for linear regression). Traditionally, weakly informative priors specified as ~ usual (0, 2.5 x SD of the 
outcome) were used. For the appropriateness of the choice of priors, a prior predictive check was 
conducted by simulating the distributions. Generally, the predicted values from the priors appeared 
reasonable, as the predicted values resided in a diverse range of plausible values for the outcome.  
The baseline characteristics were summarized as the median and interquartile range for continuous data 
and counts and percentages for categorical data. Standardized mean difference (SMD) is reported to 
quantify the differences between the two study arms, with values greater than 0.20 suggesting a potential 
imbalance between groups. All study outcomes were analyzed using Bayesian estimation of generalized 
linear models. Bayesian estimation utilized in this trial allowed interpretation of the posterior probability 
distributions for respective effects. The primary and secondary outcomes analysis was conducted using a 
Bayesian framework that estimates the treatment effect conditional on several pre-specified additional 
variables included in the model (defined a priori: age, age2, sex, BMI, and APACHE II score and 
variables with SMD>0.20). The PaO2/FiO2PaO2/FiO2 ratio was modeled for the primary outcome using a 
normal distribution and identity link function. The PaO2/FiO2 ratio at 48 hours was regressed on baseline 
(i.e., enrollment) PaO2/FiO2 ratio, randomized group assignment, and additional covariates, as specified 

 
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above. Secondary endpoints were conducted using outcome distributions and link functions appropriate to 
the outcome (e.g., normal, gamma, negative binomial) using the same covariates (i.e., age, age2, sex, 
BMI, and APACHE II score and variables with SMD>0.20) and baseline PaO2/FiO2 ratio. The primary 
and secondary study outcomes are reported as adjusted effect estimates and 95% credible interval (CrI).  
To assess the change in viral load in 149 plasma samples from 33 patients and 86 sputum samples from 
22 patients, a Bayesian linear mixed-effect model was used to determine the effect of high dose inhaled 
nitric oxide on viral load (log10 ΔCT [threshold cycle]) over time. This model included fixed effects of 
the treatment arm, time from randomization, and an interaction term (arm × time), using a repeated 
measures covariance structure (first-order autoregressive structure). A random intercept at the level of the 
participant was used in these models.  
All study outcomes were analyzed in the modified intention-to-treat population, excluding participants for 
whom respiratory treatment was unavailable at the study site or who did not meet the secondary review of 
the inclusion/exclusion criteria within 24 hours of enrolment. A total of seven patients were excluded 
after randomization for the modified intention-to-treat analysis (mITT, Figure 1). Due to the ongoing 
pandemic, relatives and family members were not allowed to come to the hospital. This limited the 
information on the medical history of critically ill patients entering the intensive care unit, rendering a 
mandatory secondary review of inclusion/exclusion criteria after randomization. Additionally, the 
pandemic impacted the supply of nitric oxide gas tanks at the respective study sites, which was sometimes 
intermittent.  
All statistical analyses were performed using R 4.0.2 (R Core Team) with Bayesian estimation conducted 
in RStan. Bayesian posterior distributions are summarized using posterior means and two-sided 0.95 CrI. 
No formal adjustments were made to the inferences that would be appropriate for statistical significance 
thresholds using frequentist adaptive designs. Full details of the statistical analysis plan – including prior 
probability definitions, full model details, and stopping rules – are outlined in Appendix (p 32-41). The 

 
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trial was overseen by an independent data safety monitoring board (DSMB) which evaluated unmasked 
interim data for futility, efficacy, and safety. The DSMB met to review interim data analysis after every 
25 patients were enrolled. The study continued until the target recruitment was reached. The only 
stopping rule for the trial was defined as a significant increase of mortality with nitric oxide gas.