Abstract
Background
Inhaled nitric oxide (iNO) in high concentration inhibits SARS-CoV-2 replication in epithelial cells and may prevent severe disease in hospitalised patients. The aim of this study was to evaluate safety and efficacy of iNO 160 ppm on supplemental oxygen in patients hospitalised with COVID-19.
Methods
We conducted an open-label, randomised clinical trial in hospitalised patients with COVID-19 receiving supplemental oxygen. Patients were randomly assigned to receive iNO for 6 h in addition to standard of care. The primary safety end-point was assessed by incidence of adverse events. The secondary efficacy end-point was the number of days free of supplemental oxygen within 15 days after randomisation.
Results
55 patients were enrolled, 27 in the iNO group and 27 in the control group, and one patient was excluded. No adverse events occurred with the inhalation of nitric oxide. Median (IQR) number of days free of supplemental oxygen was 11 (8.0–13.0) in the iNO group and 8 (2.5–10.5) in the control group (p=0.044). The iNO group had a shorter length of hospital stay (4.5 days (3.0–6.3) compared to 7.0 days (6.0–10.0) in the control group; p=0.004). Clinical score was lower in the iNO group on days 3 and 5 (p=0.010 and p=0.033). The number of patients weaned from ventilatory support on day 3 was higher in the iNO group (n=9 (33%)) when compared to controls (n=1 (4%)); p=0.005. Respiratory failure and mortality did not differ between the groups.
Conclusion
The iNO treatment was well tolerated and safe. Among adults hospitalised for COVID-19, iNO 160 ppm for 6 h increased the number of days free from supplemental oxygen, shortened the days of ventilatory support and the length of hospital stay, and improved the clinical score.
Shareable abstract
In summary, we report a successful novel strategy to treat SARS-CoV-2 respiratory infection with inhaled nitric oxide in high concentration (>160 ppm) for 6 h, and this approach could potentially be applied to other pulmonary infections https://bit.ly/40ztJE9
Introduction
COVID-19 is a respiratory and vascular disease that presents as an acute upper and lower respiratory tract infection of varying severity, and its complications include endothelial cell dysfunction, hyperinflammation, intravascular coagulation and thrombosis [1]. Antiviral and anti-inflammatory therapies are the main alternatives to prevent disease progression [2].
Inhaled nitric oxide (iNO) is a promising adjunct treatment for patients with COVID-19 [3, 4] and other respiratory infections [5]. In 2003, during the SARS epidemic in China, a small observational study of patients with SARS pneumonia receiving noninvasive support was treated with low-dose iNO. This therapy improved oxygenation, accelerated the resolution of chest radiographic infiltrates, reduced the need for intubation, and led to a faster and sustained resolution of acute respiratory disease syndrome [6]. These respiratory improvements with low-dose iNO (<40 ppm) were also shown in more recent studies in patients with severe pneumonia due to COVID-19 [7–9].
iNO in high concentrations (>160 ppm) has a well-described antimicrobial effect [10], expanding its potential usefulness in the treatment of COVID-19 beyond pulmonary vasodilation. Additionally, available data show that treatments using nitric oxide can specifically inhibit SARS-CoV-2 viral replication [11–13].
During the current coronavirus pandemic, high-concentration iNO has been used in patients with moderate and severe pulmonary disease with favourable and promising results [14–16]. High-concentration iNO treatments have been limited to a maximum of 30–60 min to avoid producing methaemoglobin (MetHb) and compromising oxygen transport in patients experiencing hypoxaemia. Based on emerging evidence, this study tested the hypothesis that early administration of high-dose iNO in hospitalised patients requiring supplemental oxygen could prevent the progression of coronavirus disease to more severe forms. We opted for a continuous 6-h period of high-dose iNO to enhance practicality and efficiency during the pandemic. Continuous treatment allows multiple patients to be treated with a single system, reducing the need for repeated equipment sterilisation. In contrast, intermittent treatment (e.g., 30-min sessions four times per day) would require multiple visits over several days, with 1- to 2-h intervals for sterilisation, making it highly inefficient.
To assess the safety and potential benefits of high-dose iNO therapy, we designed a phase I clinical trial in hospitalised patients with COVID-19 pneumonia. In this randomised controlled trial, we investigated whether an intervention with sustained high concentration iNO with a total time of 6 h in the first day of hospitalisation is safe and would improve the clinical course of the disease.
Methods
Study design
The iNO-COVID-19 study was an open-label, parallel, phase 1, randomised controlled trial designed to assess the safety and secondary efficacy of inhaling high-concentration nitric oxide by evaluating the incidence of adverse events and increase the number of days free of supplemental oxygen in hospitalised patients infected with SARS-CoV-2 and receiving low-flow supplemental oxygen (maximum of 4 L·min−1). The trial was conducted according to the resolution no. 466/12 of the Brazilian National Health Council at a single centre, Hospital São Lucas in Porto Alegre, Brazil. The study protocol was approved by the Brazilian National Commission for Research Ethics (approval number 4.755.149, CAEE: 34578820.6.1001.5336). Written informed consent was obtained from all study participants before randomisation.
This study is registered with the Brazilian Registry of Clinical Trials (ReBEC, number RBR-8nfx26; https://ensaiosclinicos.gov.br/rg/RBR-8nfx26/1) [17].
Study subjects
To be eligible for participation, patients had to be aged 10 years or older, admitted to the hospital, recruited within the first 48 h of admission prior to randomisation, and with moderate COVID-19 (WHO Clinical Progression Scale score 4, hospitalised with supplemental oxygen). Details of patient selection are shown in the supplementary methods.
Randomisation and stratification
Participants were randomly assigned (1:1) to receive a single high dose of iNO (>160 ppm) for 6 h and standard of care (experimental group) or standard of care (control group) stratified by age and presence of comorbidities as registered in the medical records. Randomisation was performed through an online web-based system using computer-generated random numbers and blocks of two and four individuals in each risk stratum using the Research Electronic Data Capture (REDCap).
Procedures
In the experimental group, iNO was administered for 6 h at a sustained concentration of 160 ppm using a medical grade pressurised cylinder of 500 ppm with a custom digital gas regulator device (supplementary figure S1). Further details of participant monitoring, systems and devices used for iNO administration are shown in the supplementary methods.
Primary and secondary outcomes
The primary safety end-point was assessed by the incidence of adverse events and by monitoring safety parameters during treatment. The secondary efficacy end-point was the number of days free of supplemental oxygen within 15 days from enrolment. The exploratory end-points were: 1) clinical score at days 3, 5 and 7 [18]; 2) proportion of patients receiving supplemental oxygen at days 3, 5 and 7; 3) proportion of patients that progress to severe disease within 15 days from enrolment, 4) length of hospital stay; 5) all-cause mortality; and 6) proportion of patients with positive qPCR test for SARS-CoV-2 at day 5. All end-points were assessed in all randomised patients.
Statistical analysis
Details of sample size estimation and statistical analysis are shown in the supplementary methods. Descriptive statistics were obtained for numerical variables, including measurement of central tendency, median and interquartile range (IQR). Descriptive statistics for categorical variables were made in the tabulation of frequencies with counts and percentages. Safety and secondary efficacy analyses were performed in the intention-to-treat population.
Statistical analysis was performed using the R computing environment (version 4.3.1, R Foundation for Statistical Computing, Vienna, Austria). No adjustments for multiplicity were performed. Thus, the results of secondary outcomes and subgroup analyses (supplementary results) should be interpreted as exploratory. All analyses were with a significance level of 0.05.
Results
Baseline demographics and disease characteristics
From November 2020 to March 2022, 55 patients were enrolled, 28 allocated to the iNO group and 27 allocated to the control group. The study participant recruitment flowchart is shown in figure 1. The baseline characteristics were similar between the intervention and control groups (table 1).
FIGURE 1.
Trial profile. iNO: inhaled nitric oxide; SoC: standard of care.
TABLE 1.
Baseline characteristics of patients
| Characteristics | Control | iNO |
|---|---|---|
| Patients, n | 27 | 27 |
| Sex, male, n (%) | 18 (66.7) | 19 (70.4) |
| Age, years, median (IQR) | 53 (39–65) | 57 (40–63) |
| Weight, kg, median (IQR) | 85 (80–100) | 88 (81–97) |
| Height, cm, median (IQR) | 167 (159–176) | 174 (166–181) |
| BMI, kg·m−2, median (IQR) | 32.17 (29.02–33.97) | 30.07 (27.07–32.50) |
| Unvaccinated, n (%) | 17 (70.8) | 16 (66.7) |
| Obesity, n (%) | ||
| Healthy weight | 0 (0) | 2 (7.4) |
| Overweight | 9 (33.3) | 11 (40.7) |
| Obese | 18 (66.7) | 14 (51.9) |
| Race/skin colour, n (%) | ||
| White | 20 (74.1) | 21 (77.8) |
| Black | 2 (7.4) | 5 (18.5) |
| Brown | 5 (18.5) | 1 (3.7) |
| Tobacco use, n (%) | 1 (3.7) | 1 (3.7) |
| Alcohol use, n (%) | 8 (29.6) | 13 (48.1) |
| Symptoms, days#, median (IQR) | 8 (7.00–10.00) | 9 (8.00–11.00) |
| Respiratory symptoms, days ¶ , median (IQR) | 3 (2.00–5.00) | 4 (2.00–5.00) |
| Comorbidities, n (%) | 13 (48.1) | 9 (33.3) |
| Hypertension | 13 (48.1) | 9 (33.3) |
| CHF | 1 (3.7) | 0 (0) |
| Diabetes | 8 (29.6) | 1 (3.7) |
| Medication use, n (%) | ||
| Non-steroidal anti-inflammatory drugs (NSAIDs) | 15 (55.6) | 15 (55.6) |
| Azithromycin | 15 (55.6) | 17 (63.0) |
| Antibiotics | 6 (22.2) | 5 (18.5) |
| Hydroxychloroquine | 5 (18.5) | 3 (11.1) |
| Corticosteroid | 15 (55.6) | 14 (51.9) |
| Pulmonary involvement %+, median (IQR) | 50 (30–60) | 50 (40–50) |
| Oxygen saturation (emergency room) %, median (IQR) | 90 (89–91) | 91 (90–92) |
| Inflammatory markers | ||
| C-reactive protein mg·dL−1, median (IQR) | 7.70 (4.75–21.45) | 7.70 (4.30–22.10) |
| D-dimer µg·mL−1, median (IQR) | 602 (360–791) | 783 (549–1065) |
iNO: inhaled nitric oxide; BMI: body mass index; CHF: congestive heart failure. #: the number of days from symptom onset to hospital admission; ¶: the number of days from respiratory symptom onset to hospital admission; +: by chest tomography.
Adverse events
Patients in the iNO group were continuously monitored (MetHb%, haemaglobin saturation (SatHb%), respiratory rate, heart rate) during and up to 30 min after the inhalation of nitric oxide 160 ppm (figure 2). One subject reported discomfort after 3 h of inhalation and the intervention was suspended. This was considered a non-serious adverse event, not related to the iNO and not associated with changes in SatHb% or MetHb%. No other adverse events were observed during or immediately after treatment with iNO in the remaining subjects. All patients were monitored daily for 15 days for adverse events. No significant differences in adverse events were observed between the iNO and control groups (table 2). In the iNO group, hypotension and acute kidney injury were not reported during or within 48 h following the intervention. Creatinine levels (mg·dL−1) remained stable, measuring 1.00 (0.81–1.26) before the intervention and 1.05 (0.80–1.26) afterward, while D-dimer levels (µg·mL−1) were 783 (549–1065) prior to the intervention and 799 (729–2953) post-intervention. Vital signs, including oxygen saturation measured by pulse oximetry (%), heart rate, respiratory rate, blood pressure and supplemental oxygen flow (L·min−1), were derived from routine daily assessments recorded in electronic medical records not linked to the study (table 3).
FIGURE 2.
Monitoring of safety parameters during nitric oxide inhalation.
TABLE 2.
Safety end-points (overall summary of all adverse events by treatment)
| iNO group subjects | Control group subjects | |
|---|---|---|
| Patients, n | 27 | 27 |
| Any AE | 4 (14.8) | 2 (7.7) |
| Any SAE | 3 (11.1) | 2 (7.7) |
| AE drug-related | 0 (0) | 0 (0) |
| AE device-related | 0 (0) | 0 (0) |
| AE procedure-related | 1 (0.03) | 0 (0) |
| AE leading to early treatment termination | 0 (0) | 0 (0) |
| SAE related to drug | 0 (0) | 0 (0) |
| SAE related to device | 0 (0) | 0 (0) |
| SAE procedure related | 0 (0) | 0 (0) |
Data are presented as n (%) of subjects across treatment arms. iNO: inhaled nitric oxide; AE: adverse event; SAE: serious adverse event.
TABLE 3.
Vital sign measurements taken from the daily assessments
| Parameter/time point | Median (IQR) |
|---|---|
| SpO2, % | |
| Pre-inhalation | 95 (93–97) |
| Post-inhalation (day 1) | 95 (94–97) |
| Post-inhalation (day 2) | 95 (93–97) |
| Post-inhalation (day 3) | 94 (94–97) |
| Heart rate, beats·min−1 | |
| Pre-inhalation | 82 (76–86) |
| Post-inhalation (day 1) | 79 (67–85) |
| Post-inhalation (day 2) | 74 (64–81) |
| Post-inhalation (day 3) | 70 (62–84) |
| Respiratory rate, breaths·min−1 | |
| Pre-inhalation | 19 (18–20) |
| Post-inhalation (day 1) | 20 (18–20) |
| Post-inhalation (day 2) | 20 (19–22) |
| Post-inhalation (day 3) | 20 (19–20) |
| Systolic blood, pressure mmHg | |
| Pre-inhalation | 123 (120–130) |
| Post-inhalation (day 1) | 120 (120–130) |
| Post-inhalation (day 2) | 120 (111–130) |
| Post-inhalation (day 3) | 120 (119–133) |
| Supplemental O2, flow L·min−1 | |
| Pre-inhalation | 3 (2–4) |
| Post-inhalation (day 1) | 2 (1–5) |
| Post-inhalation (day 2) | 1 (0–5) |
| Post-inhalation (day 3) | 0.5 (0–3.7) |
Values used in analysis are based on vital sign measurements taken from the daily assessments. Data are presented as median (IQR). SpO2: oxygen saturation measured by pulse oximetry.
Treatment effects on outcomes parameters
For the secondary efficacy end-point, patients in the iNO group had a higher median number of days free of supplemental oxygen compared to the control group (11 (IQR 8–13) versus 8 (IQR 2.5–10.5); p=0.044; table 4 and supplementary figure S2A). The intention-to-treat analysis, including 54 enrolled patients, demonstrated a significant benefit in favour of the iNO group for the secondary efficacy end-point. The cumulative probability of remaining on supplemental oxygen at 15 days was significantly higher in the control group (p=0.047, figure 3). The adjusted common hazard ratio (HR) for more days free of supplemental oxygen was 1.79 (95% confidence interval (CI) 1.03–3.13; p=0.040), favouring the iNO group (figure 4).
TABLE 4.
Study secondary end-points
| Control | iNO | p-value | |
|---|---|---|---|
| Patients, n | 27 | 27 | |
| Primary outcome | |||
| Days free of supplemental oxygen, median (IQR) | 8.0 (2.5–10.5) | 11.0 (8.0–13.0) | 0.044 |
| Secondary outcomes | |||
| Eight-level ordinal outcome, median (IQR) | |||
| At 3 days | 4.0 (4.0–5.0) | 4.0 (3.0–4.0) | 0.010 |
| At 5 days | 4.0 (3.5–5.0) | 3.0 (2.0–4.0) | 0.033 |
| At 7 days | 4.0 (2.0–5.0) | 2.0 (1.5–3.5) | 0.083 |
| Patients without supplemental oxygen, n (%) | |||
| At 3 days | 1 (4) | 9 (33) | 0.005 |
| At 5 days | 7 (26) | 14 (52) | 0.051 |
| At 7 days | 13 (48) | 20 (74) | 0.051 |
| Positive qPCR for COVID at 5 days, n (%) | 16 (59) | 16 (59) | 1.000 |
| Progression to severe disease, n (%) | 5 (18.5) | 5 (18.5) | 1.000 |
| Length of hospital stay days, median (IQR) | 7.0 (6.0–10.0) | 4.5 (3.0–6.3) | 0.004 |
| Mortality, n (%) | 2 (7.7) | 3 (11.1) | 1.000 |
Progression to severe disease: development of respiratory failure, septic shock or multiple organ failure. p-values from Mann–Whitney test and Chi-square test. iNO: inhaled nitric oxide; qPCR: quantitative PCR.
FIGURE 3.
Cumulative probability of remaining on supplemental oxygen at 15 days after randomisation. iNO: inhaled nitric oxide.
FIGURE 4.
Forest plot showing the effect size in the secondary outcome variable (common hazard ratio (HR) for a greater number of days free of supplemental oxygen at 15 days, analysed according to Cox proportional hazards regression and adjusted for the prespecified subgroups). iNO: inhaled nitric oxide.
For the exploratory end-points, patients in the iNO group exhibited a lower median clinical score on day 3 (48 h after intervention) and day 5 (96 h after intervention) compared to the control group; p=0.010 and p=0.033, respectively. However, there were no significant differences in the clinical scores on day 7 and subsequent days (up to day 60 after enrolment). The median length of hospital stay was shorter in the iNO group (4.5 days (IQR 3.0–6.3)) compared to the control group (7.00 days (IQR 6.0–10.0)); p=0.004 (table 4 and supplementary figure S2B).
There were no significant differences between the groups in terms of respiratory failure and mortality rates (table 4). Three deaths were recorded in the iNO group. According to the electronic medical records of the attending physician not linked to the study, the cause of death in all three cases was septic shock, occurring 12, 22 and 35 days after inclusion in the study, respectively. These deaths were not associated with nitric oxide inhalation.
The proportion of patients without ventilatory support at 3 days was higher in the iNO group: 9 (33%) versus 1 (4%) in the control group; p=0.005 (table 4 and supplementary figure S3). On average, 3.4 patients would have to receive iNO (instead of control treatment) for one additional patient to be free of ventilatory support on day 3. The same analysis at 5 and 7 days also shows a higher proportion of patients without ventilatory support in the iNO group but did not reach statistical significance; p=0.051 for both (table 4 and supplementary figure S3). The proportion of patients that remained PCR positive for COVID-19 at day 5 was not different in both groups.
Discussion
This is the first study to evaluate the safety and secondary efficacy of continuous administration of high-dose iNO for 6 h in a single application for treating hospitalised patients with moderate COVID-19. We found that compared to the control group, high-dose iNO increases the number of days free of supplemental oxygen and reduces the length of hospital stay. Respiratory failure and mortality did not differ between the groups. Additionally, the intervention was not associated with adverse effects, such as high levels of MetHb, hypotension or worsening of the hypoxia during the prolonged inhalation.
Our data confirm recent results observed in a pre-clinical animal study, where continued high-dose iNO was feasible and safe, without any detectable functional or inflammatory side-effects after 6 or 12 h of inhalation [5, 19]. In our study, MetHb and NO2 levels remained within safe limits. Other studies used iNO by prolonged inhalation [20, 21] and intermittent [22, 23] inhalation with which we can estimate the safety of iNO [4]. In studies with intermittent application, there is no report of toxicity since inhalation is brief (30 to 50 min) or interrupted when MetHb reaches 5–7% [20–23].
Our clinical findings are in agreement with previous results from a pilot study with patients hospitalised with severe SARS-CoV infection and suggested reduced duration of ventilatory support and hospitalisation in patients receiving low concentration nitric oxide [6]. In this study, inhalation of nitric oxide was beneficial, reducing the need for supplemental oxygen in 7 days. Data from a study that evaluated the effects of nitric oxide administration at a concentration of 160 ppm in children with acute viral bronchiolitis [24] showed a significant reduction in length of hospitalisation, suggesting that the clinical benefit is related to the nonspecific antiviral activity of iNO. Antiviral effects have been demonstrated in studies with HIV [25], herpes virus [26] and hantavirus [27] in addition to the effective in vitro effect on SARS-CoV [12, 13, 28]. In our study, no differences were observed between the intervention and control groups regarding PCR positivity rates on day 5. This observation challenges the proposed antiviral mechanism of action of iNO. However, these data should be interpreted with caution, as the presence of viral RNA does not imply active virus replication. Additionally, PCR testing was not conducted daily in these patients, and the time interval used for the analyses may have been short.
Another recent case series [15] showed the safety and efficacy of iNO for COVID-19 in hospitalised patients receiving supplemental oxygen. The administration of iNO at 160 ppm for 30 min twice daily improved the respiratory rate and oxygenation of hypoxaemic patients. High-dose iNO was safe, well tolerated and showed clinical benefit in adolescents with cystic fibrosis and pulmonary colonisation with multidrug-resistant pathogens [29]. Another study carried out during the COVID-19 pandemic recruited six pregnant women with severe COVID-19 pneumonia. The patients received iNO at high dose (>160 ppm) for 30 min, twice daily, resulting in improved oxygenation and a reduction in breathing frequency and dyspnoea [30]. A retrospective study of pregnant women with severe COVID-19 pneumonia compared the outcomes of standard of care versus iNO at high dose showing an increase in oxygen supplementation-free days and a reduction in hospital length of stay [31].
A recent study has shown that the use of high-dose iNO resulted in an improvement in arterial oxygen tension/inspiratory oxygen fraction within 48 h compared to standard care in adults with acute hypoxaemic respiratory failure due to COVID-19 [32]. This study in a more severe population corroborates with the clinical benefit observed in our findings in subjects with early non-severe disease. The results underscore the importance of planning future dose–response investigations on the antimicrobial and clinical properties of high-dose inhaled nitric oxide therapy in different populations. Another study [8] suggests that iNO may be beneficial in patients with more severe hypoxaemia with elevated levels of brain natriuretic peptide and hs-troponin, likely indicative of right ventricular strain. This specific phenotypic response to iNO supports the interaction between pulmonary vascular blood flow and right heart function in COVID-19 pneumonia, suggesting that these biomarkers may be useful for phenotypic enrichment in future clinical trials.
An important novelty of this clinical trial was the use of a sustained high dose of iNO for 6 h. We believe that the extended treatment with iNO was the key factor in shortening the hypoxaemic period of COVID-19, as several studies show that the antimicrobial effect is strongly dependent on the time of exposure to nitric oxide or its concentration [33–35]. Previous studies have used cumulative doses even greater than this but spread over several days over periods of up to 30 min.
One of the main concerns of treatment involving high concentration iNO are the increasing MetHb levels and nitrogen dioxide formation. In our study, MetHb levels above 6% were uncommon, even after hours of exposure [36]. In only one patient inhalation was stopped because MetHb was close to 7%. In our study the amount of NO2 inspired was kept below 5 ppm during the whole study, never exceeding the pre-established safety threshold levels.
The lack of drug-related adverse events observed in our study is in line with previous human studies with intermittent high-dose iNO in adults and infants with viral infections [22–24]. In addition, animal data from pigs exposed to high-dose iNO (160 ppm) for 6 h and receiving methylene blue to limit MetHb to 6% did not show any clinical or physiological adverse effect during inhalation and up to 3 days after the intervention [19]. An ex vivo experiment with 12 h of iNO 200 ppm in pigs also showed no effects on oxygenation, lung compliance and inflammatory markers [37].
The mechanism of iNO activity responsible for oxygenation improvement requires further investigation. Nitric oxide exhibits an array of potential beneficial effects that may explain the efficacy observed in the present study. iNO enhances oxygenation through selective pulmonary vasodilation [38], induces bronchodilation and exerts anti-inflammatory effects [39]. Leveraging its specific antimicrobial properties, several clinical studies have been conducted with high iNO doses [5, 15, 29, 32, 34, 35, 40]. Consistent with our findings, these studies demonstrated that high-dose iNO treatment (160 ppm) was safe, reduced tachypnea and improved oxygenation in hypoxaemic, spontaneously breathing high-risk patients with COVID-19 pneumonia.
Several constraints inherent in our clinical trial must be acknowledged. A significant drawback is the limited sample size, which impairs our ability to detect differences between groups effectively. This reduced sample size likely limited our ability to detect significant differences in respiratory failure and mortality outcomes between groups. A larger sample size could have provided the statistical power needed to uncover such effects. Furthermore, the findings of this study served as a data-generating trial to inform larger double-blind and placebo-controlled clinical trials with a larger sample size. At the time of study suspension, a notable proportion exceeding 80% of the adult population in Brazil had achieved full vaccination, leading to a noticeable reduction in hospitalisations within Porto Alegre. Another limitation worth noting is that we did not assess PCR quantitatively. This measure could have allowed for greater precision in demonstrating the antiviral mechanism of iNO, as proposed in previous studies. Additionally, the open nature of the study introduces the potential for bias. However, we have mitigated these biases through the implementation of stringent randomisation protocols and the collection of primary and secondary outcome data via medical records, thus minimising interactions with assisting physicians. Another important limitation is that we were unable to directly measure physiological response factors, such as pulmonary vasodilation, improved V/Q matching and bronchodilation. These measurements could have helped us better understand our findings.
Full immunisation constituted one of the exclusionary criteria within our trial. Nonetheless, given the emergence of immune-evading mutants and the potential decline in immunity, iNO may prove beneficial for COVID-19 patients who need supplemental oxygen therapy, irrespective of their vaccination status. While partial vaccination may have influenced clinical outcomes, the proportion of subjects with such conditions remained comparable in both groups.
In conclusion, our findings suggest that in hospitalised patients with COVID-19, high-concentration iNO administration reduced the duration of supplemental oxygen therapy and hospital stay. The intervention was demonstrated to be safe but required continuous monitoring of MetHb% and NO2% using specialised devices that are not widely available. Our results bolster the call for larger trials examining the safety and efficacy of high-dose iNO in respiratory infections. Furthermore, future investigations should explore dose–response studies, repeated dosing and the combination of iNO with other therapies (e.g., antivirals or corticosteroids), particularly to understand the specific mechanisms of action of nitric oxide better.
Acknowledgements
The authors thank CAPES, CNPq, Ministry of Health and Ministry of Science, Technology and Innovation (Brazil), AirLiquide do Brasil, University Health Network (Toronto, Canada), patients, nurses, physical therapists, and physicians of Hospital Sao Lucas, and students of PUCRS.
Footnotes
Provenance: Submitted article, peer reviewed.
This clinical trial is prospectively registered with ensaiosclinicos.gov.br as RBR-8nfx26.
Ethics statement: The trial was conducted according to the resolution number 466/12 of the Brazilian National Health Council at a single centre, Hospital São Lucas in Porto Alegre, Brazil. The study protocol was approved by the Brazilian National Commission for Research Ethics (approval number 4.755.149, CAEE: 34578820.6.1001.5336). Written informed consent was obtained from all study participants before randomisation.
Author contributions: F. Friedrich, M. Cypel, V.S. Michaelsen and M.H. Jones participated in the conception of the study. M.H. Jones, M. Cypel, F. Friedrich, V.S. Michaelsen and R.G. Rosa participated in design and definition of the outcomes of the study. F. Friedrich, M.O.A. Brum, F. Ramos, G. Chatkin, R. Marco, L.K. Grun, L.S.M. dos Santos, A.P. Santos, M.T.C. Fernandes, L.M. Paungartner, M.S. Maranghello, C. Lantmann, G. Eggers, G.D. Sanches, F.M. Barbé-Tuana and M.H. Jones participated in data acquisition, quality control, clinical assessment and review of medical records. The first draft of the manuscript was written by F. Friedrich, M. Cypel, V.S. Michaelsen, F.M. Barbé-Tuana and M.H. Jones, and then complemented with suggestions from all the other authors. All authors contributed to interpretation of results and revision of the paper. All authors shared the decision to submit the manuscript for publication, and all authors attest accuracy and completeness of the data, and compliance with the study protocol.
Conflict of interest: All authors declare no conflicts of interest in the study.
Support statement: This study was supported by MCTIC/CNPq/FNDCT/MS/SCITIE/Decit - Chamada No 07/2020, University Health Network and The University of Toronto. M.O.A. Brum, L.K. Grun and F.O. Friedrich received scholarships from CAPES. This study was financed in part by the Coordenação de Aperfeiçoamento de Pessoal de Nivel Superior – Brasil (CAPES) – Finance Code 001. L.S.M. dos Santos was supported by the PRO-Stricto Scholarship Program, PUCRS. M.H. Jones and F.M. Barbé-Tuana are investigators from CNPq. Funding information for this article has been deposited with the Open Funder Registry.
Data availability
We are committed to sharing with qualified external researchers access to patient-level data and supporting clinical documents. These requests are reviewed and approved by the authors based on scientific merit. All data provided are anonymised to respect the privacy of patients who have participated in the trial, in line with applicable laws and regulations. This trial data availability is according to the criteria and process described at http://www.clinicalstudydatarequest.com.
References
- 1.Bonaventura A, Vecchié A, Dagna L, et al. Endothelial dysfunction and immunothrombosis as key pathogenic mechanisms in COVID-19. Nat Rev Immunol 2021; 21: 319–329. doi: 10.1038/s41577-021-00536-9 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 2.Chen JT, Ostermann M. Review of anti-inflammatory and antiviral therapeutics for hospitalized patients infected with severe acute respiratory syndrome coronavirus 2. Crit Care Clin 2022; 38: 587–600. doi: 10.1016/j.ccc.2022.02.002. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 3.Pieretti JC, Rubilar O, Weller RB, et al. Nitric oxide (NO) and nanoparticles: potential small tools for the war against COVID-19 and other human coronavirus infections. Virus Res 2021; 291: 198202. doi: 10.1016/j.virusres.2020.198202 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 4.Kamenshchikov NO, Berra L, Carroll RW. Therapeutic effects of inhaled nitric oxide therapy in COVID-19 patients. Biomedicines 2022; 10: 369. doi: 10.3390/biomedicines10020369 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 5.Sorbo LD, Michaelsen VS, Ali A, et al. High doses of inhaled nitric oxide as an innovative antimicrobial strategy for lung infections. Biomed 2022; 10: 1525. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 6.Chen L, Liu P, Gao H, et al. Inhalation of nitric oxide in the treatment of severe acute respiratory syndrome: a rescue trial in Beijing. Clin Infect Dis 2004; 39: 1531–1535. doi: 10.1086/425357 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 7.Abou AO, Huette P, Debouvries F, et al. Inhaled nitric oxide for critically ill Covid-19 patients: a prospective study. Crit Care 2020; 24: 645. doi: 10.1186/s13054-020-03371-x [DOI] [PMC free article] [PubMed] [Google Scholar]
- 8.Garfield B, Fadyen C, Briar C, et al. Potential for personalised application of inhaled nitric oxide in COVID-19 pneumonia. Br J Anaesth 2021; 126: e72–e75. doi: 10.1016/j.bja.2020.11.006 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 9.Longobardo A, Montanari C, Shulman R, et al. Inhaled nitric oxide minimally improves oxygenation in COVID-19 related acute respiratory distress syndrome. Br J Anaesth 2021; 126: e44–e46. doi: 10.1016/j.bja.2020.10.011 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 10.Bath PM, Coleman CM, Gordon AL, et al. Nitric oxide for the prevention and treatment of viral, bacterial, protozoal and fungal infections. F1000Res 2021; 10: 536. doi: 10.12688/f1000research.51270.2 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 11.Garren MR, Ashcraft M, Qian Y, et al. Nitric oxide and viral infection: recent developments in antiviral therapies and platforms. Appl Mater Today 2021; 22: 100887. doi: 10.1016/j.apmt.2020.100887 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 12.Keyaerts E, Vijgen L, Chen L, et al. Inhibition of SARS-coronavirus infection in vitro by S-nitroso-N-acetylpenicillamine, a nitric oxide donor compound. Int J Infect Dis 2004; 8: 223–226. doi: 10.1016/j.ijid.2004.04.012 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 13.Akaberi D, Krambrich J, Ling J, et al. Mitigation of the replication of SARS-CoV-2 by nitric oxide in vitro. Redox Biol 2020; 37: 101734. doi: 10.1016/j.redox.2020.101734 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 14.Wiegand SB, Safaee FB, Carroll RW, et al. Rescue treatment with high-dose gaseous nitric oxide in spontaneously breathing patients with severe coronavirus disease 2019. Crit Care Explor 2020; 2: e0277. doi: 10.1097/CCE.0000000000000277 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 15.Safaee Fakhr B, Fenza R, Gianni S, et al. Inhaled high dose nitric oxide is a safe and effective respiratory treatment in spontaneous breathing hospitalized patients with COVID-19 pneumonia. Nitric Oxide 2021; 116: 7–13. doi: 10.1016/j.niox.2021.08.003 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 16.Ferrari M, Santini A, Protti A, et al. Inhaled nitric oxide in mechanically ventilated patients with COVID-19. J Crit Care 2020; 60: 159–160. doi: 10.1016/j.jcrc.2020.08.007 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 17.Brazilian Registry of Clinical Trials – REBEC. Date last accessed: 10 October 2024. https://ensaiosclinicos.gov.br/
- 18.Marshall JC, Murthy S, Diaz J, et al. A minimal common outcome measure set for COVID-19 clinical research. Lancet Infect Dis 2020; 20: e192–e197. doi: 10.1016/S1473-3099(20)30483-7 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 19.Michaelsen VS, Ribeiro RVP, Brambate E, et al. A novel pre-clinical strategy to deliver antimicrobial doses of inhaled nitric oxide. PLoS ONE 2021; 16: e0258368. doi: 10.1371/journal.pone.0258368 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 20.Young JD, Dyar O, Xiong L, et al. Methaemoglobin production in normal adults inhaling low concentrations of nitric oxide. Intensive Care Med 1994; 20: 581–584. doi: 10.1007/BF01705726 [DOI] [PubMed] [Google Scholar]
- 21.Young JD, Sear JW, Valvini EM. Kinetics of methaemoglobin and serum nitrogen oxide production during inhalation of nitric oxide in volunteers. Br J Anaesth 1996; 76: 652–656. doi: 10.1093/bja/76.5.652 [DOI] [PubMed] [Google Scholar]
- 22.Bentur L, Gur M, Ashkenazi M, et al. Pilot study to test inhaled nitric oxide in cystic fibrosis patients with refractory Mycobacterium abscessus lung infection. J Cyst Fibros 2020; 19: 225–231. doi: 10.1016/j.jcf.2019.05.002 [DOI] [PubMed] [Google Scholar]
- 23.Tal A, Greenberg D, Gay Y, et al. Nitric oxide inhalations in bronchiolitis: a pilot, randomized, double-blinded, controlled trial. Pediatr Pulmonol 2018; 53: 95–102. doi: 10.1002/ppul.23905 [DOI] [PubMed] [Google Scholar]
- 24.Goldbart A, Golan TI, Pillar G, et al. Inhaled nitric oxide therapy in acute bronchiolitis: a multicenter randomized clinical trial. Sci Rep 2020; 10: 9605. doi: 10.1038/s41598-020-66433-8 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 25.Hermann E, Idziorek T, Kusnierz JP, et al. Role of nitric oxide in the regulation of lymphocyte apoptosis and HIV-1 replication. Int J Immunopharmacol 1997; 19: 387–397. doi: 10.1016/S0192-0561(97)00060-X [DOI] [PubMed] [Google Scholar]
- 26.Croen KD. Evidence for antiviral effect of nitric oxide. Inhibition of herpes simplex virus type 1 replication. J Clin Invest 1993; 91: 2446–2452. doi: 10.1172/JCI116479 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 27.Klingström J, Åkerström S, Hardestam J, et al. Nitric oxide and peroxynitrite have different antiviral effects against hantavirus replication and free mature virions. Eur J Immunol 2006; 36: 2649–2657. doi: 10.1002/eji.200535587 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 28.Akerström S, Gunalan V, Keng CT, et al. Dual effect of nitric oxide on SARS-CoV replication: viral RNA production and palmitoylation of the S protein are affected. Virology 2009; 395: 1–9. doi: 10.1016/j.virol.2009.09.007 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 29.Bartley BL, Gardner KJ, Spina S, et al. High-dose inhaled nitric oxide as adjunct therapy in cystic fibrosis targeting Burkholderia multivorans. Case Rep Pediatr 2020; 2020: 1536714. doi: 10.1155/2020/1536714 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 30.Safaee FB, Wiegand SB, Pinciroli R, et al. High concentrations of nitric oxide inhalation therapy in pregnant patients with severe coronavirus disease 2019 (COVID-19). Obstet Gynecol 2020; 136: 1109–1113. doi: 10.1097/AOG.0000000000004128 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 31.Valsecchi C, Winterton D, Safaee FB, et al. High-dose inhaled nitric oxide for the treatment of spontaneously breathing pregnant patients with severe coronavirus disease 2019 (COVID-19) pneumonia. Obstet Gynecol 2022; 140: 195–203. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 32.Fenza R, Shetty NS, Gianni S, et al. High-dose inhaled nitric oxide in acute hypoxemic respiratory failure due to COVID-19: a multicenter phase II trial. Am J Respir Crit Care Med 2023; 208: 1293–1304. doi: 10.1164/rccm.202304-0637OC [DOI] [PMC free article] [PubMed] [Google Scholar]
- 33.Schairer DO, Chouake JS, Nosanchuk JD, et al. The potential of nitric oxide releasing therapies as antimicrobial agents. Virulence 2012; 3: 271–279. doi: 10.4161/viru.20328 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 34.Miller C, Mullin B, Ghaffari A, et al. Gaseous nitric oxide bactericidal activity retained during intermittent high-dose short duration exposure. Nitric Oxide 2009; 20: 16–23. doi: 10.1016/j.niox.2008.08.002 [DOI] [PubMed] [Google Scholar]
- 35.Bogdanovski K, Chau T, Robinson CJ, et al. Antibacterial activity of high-dose nitric oxide against pulmonary Mycobacterium abscessus disease. Access Microbiol 2020; 2: acmi000154. doi: 10.1099/acmi.0.000154 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 36.Dhont S, Derom E, Van BE, et al. The pathophysiology of ‘happy’ hypoxemia in COVID-19. Respir Res 2020; 21: 198. doi: 10.1186/s12931-020-01462-5 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 37.Michaelsen VS, Ribeiro RVP, Ali A, et al. Safety of continuous 12-hour delivery of antimicrobial doses of inhaled nitric oxide during ex vivo lung perfusion. J Thorac Cardiovasc Surg 2022; 163: 841–849.e1. doi: 10.1016/j.jtcvs.2020.11.150 [DOI] [PubMed] [Google Scholar]
- 38.Ichinose F, Roberts JD, Zapol WM. Inhaled nitric oxide: a selective pulmonary vasodilator: current uses and therapeutic potential. Circulation 2004; 109: 3106–3111. doi: 10.1161/01.CIR.0000134595.80170.62 [DOI] [PubMed] [Google Scholar]
- 39.Wolak T, Dicker D, Shifer Y, et al. A safety evaluation of intermittent high-dose inhaled nitric oxide in viral pneumonia due to COVID-19: a randomised clinical study. Sci Rep 2024; 14: 17201. doi: 10.1038/s41598-024-68055-w [DOI] [PMC free article] [PubMed] [Google Scholar]
- 40.Kamenshchikov NO, Safaee FB, Kravchenko IV, et al. Assessment of continuous low-dose and high-dose burst of inhaled nitric oxide in spontaneously breathing COVID-19 patients: a randomized controlled trial. Nitric Oxide Biol Chem 2024; 149: 41–48. doi: 10.1016/j.niox.2024.06.003 [DOI] [PubMed] [Google Scholar]
Associated Data
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Data Availability Statement
We are committed to sharing with qualified external researchers access to patient-level data and supporting clinical documents. These requests are reviewed and approved by the authors based on scientific merit. All data provided are anonymised to respect the privacy of patients who have participated in the trial, in line with applicable laws and regulations. This trial data availability is according to the criteria and process described at http://www.clinicalstudydatarequest.com.