Nitric Oxide Gas Inhalation for Prevention of COVID-19 in Healthcare Providers
Phase: PHASE2
Conditions: Coronavirus Infections, Healthcare Associated Infection
Sponsor: Massachusetts General Hospital

1 
Nitric oxide gas inhalation for healthcare providers  
to prevent COVID-2019 disease. Version 7.0 
 
May 22, 2020 
Nitric oxide gas inhalation for healthcare providers  
to prevent COVID-2019  
 
1Stefano Gianni, MD 
1Bijan Safaee Fakhr, MD 
1Raffaele Di Fenza, MD  
1Riccardo Pinciroli, MD   
1Caio Cesar Araujo Morais, PhD  
3Ryan Carroll, MD, PhD 
4Stuart N. Harris, MD  
5Kathryn Hibbert, MD 
1,2Robert Kacmarek, PhD 
1Warren Zapol, MD PhD 
1Edward Bittner, MD PhD 
1Fumito Ichinose, MD PhD 
1Oluwaseun Johnson-Akeju, MD 
1.2Lorenzo Berra, MD  
 
1Surgical Intensive Care Unit, Department of Anesthesia, Critical Care and Pain Medicine 
(DACCPM), Massachusetts General Hospital, Boston, Massachusetts, USA. 
2Respiratory Care Service, Patient Care Services, Massachusetts General Hospital, Boston, 
Massachusetts, USA. 
3Department of Pediatrics, Massachusetts General Hospital, Boston, Massachusetts, USA. 
4Department of Emergency Medicine, Massachusetts General Hospital, Boston, Massachusetts, 
USA. 
5Pulmonary and Critical Care Medicine, Department of Medicine, Massachusetts General 
Hospital, Boston, Massachusetts, USA. 
 
 
ClinicalTrials.gov Protocol Number: NCT04312243 
Detailed Protocol Version 7 
 
This document has been written following the PHRC instructions about Detailed Protocol 
writing.  
 
 
Version 7.0, May 22, 2020 
 

2 
Nitric oxide gas inhalation for healthcare providers  
to prevent COVID-2019 disease. Version 7.0 
 
May 22, 2020 
Synopsis and Study Schema 
 
Title: Nitric oxide gas inhalation for healthcare providers to prevent COVID-2019 disease 
 
Principal Investigator: Lorenzo Berra, MD 
 
Study Objective(s): To assess if intermittent inhaled NO might prevent the risk of COVID-19 
among healthcare workers. 
 
Study Design: Randomized clinical trial 
 
Inclusion Criteria. 
1. Age ≥18 years  
2. Scheduled to work with SARS-CoV-2 RT-PCR patients for at least 3 days in a week. 
3. Healthcare providers or other professional (doctors, nurses, respiratory therapists, 
technician, administrative staff, others) working at a participating hospital 
 
Exclusion Criteria.  
1. Proven previous SARS-CoV2 infection and subsequent negative rt-PCR 
2. Pregnancy 
3. Known hemoglobinopathies 
4. Known anemia 
 
Primary endpoint. 
Incidence of subjects with COVID-19 disease at 14 days 
 
Secondary endpoints. 
1. Incidence of subjects with positive SARS-CoV-2 rt-PCR test at 14 days 
2. Proportion of healthcare providers requiring quarantine 
3. Total number of quarantine days 
 
Total expected number of subjects: 470 
 
Coordinating center: Massachusetts General Hospital, Boston, MA, USA 
 
Study Drug or Intervention: The treatment group will inhale 160 parts per million (ppm) of 
nitric oxide for 15 minutes before and after each work shift.  
 
Evaluation Criteria: Laboratory confirmation of real time reverse transcriptase–polymerase 
chain reaction (RT-PCR) positive for SARS-CoV-2. 
 
Safety Assessment: Continuous Monitoring of MetHb with a non-invasive co-oximetry monitor 
in order to prevent methemoglobinemia (defined as MetHb > 5%). Continuous monitoring of 
Nitrogen Dioxide (NO2) in inhaled breath (threshold level < 5 ppm) in order to prevent reaching 
dangerously high levels. 
 

3 
Nitric oxide gas inhalation for healthcare providers  
to prevent COVID-2019 disease. Version 7.0 
 
May 22, 2020 
Statistical Considerations: 
Reported rates of intra-hospital SARS-CoV-2 infection rate are extremely variable and depend on time 
elapsed since the epidemic outbreak. We hypothesized an infection rate of 15% in control subjects, 
provided that adequate protective measures are followed, and 5% in those treated with iNO. Assuming 
an alpha of 0.05 and a beta (power) of 0.9, we calculated with a two-sided test a need for N1=207 and 
N2=207 subjects that we increased to a total of 470 by considering a dropout of 12.5%. Estimated 
sample sizes where calculated are based on a test of equality of two proportions performed on a specific 
software. 
 
Duration of Study Period: Study participants will be followed-up for 14 days year after-
randomization 
 
Data Safety Monitoring Board 
 
 
Richard Pino MD, PhD 
Associate Professor of Anesthesia at Harvard Medical School 
Department of Anesthesia Massachusetts General Hospital 
Email: RPINO@mgh.harvard.edu 
 
John Thomas MD, PhD 
Professor Emeritus in Microbiology at West Virginia University 
Email: jgthomas@hsc.wvu.edu 
 
Carl Nathan, MD 
R.A. Rees Pritchett Professor of Microbiology 
Weill Cornell Medicine 
Email: cnathan@med.cornell.edu 
 
 

4 
Nitric oxide gas inhalation for healthcare providers  
to prevent COVID-2019 disease. Version 7.0 
 
May 22, 2020 
 
 
Figure 1. Study schema 
 
 
 

5 
Nitric oxide gas inhalation for healthcare providers  
to prevent COVID-2019 disease. Version 7.0 
 
May 22, 2020 
I. BACKGROUND AND SIGNIFICANCE 
 
Historical background 
The current COVID-19 pandemia sparked in December 2019 from Wuhan (China). It is believed 
that the virus was transmitted at first from a bat (zoonosis) followed by human-to-human 
transmission [3]. The disease spread throughout the Asian continent, to Europe and more 
recently to the United States [1].The first case in the US has been reported on January 19, 2020, 
in a patient who had travelled from Wuhan. By March 2020, the disease is threatening cities 
from East to the West Coast [2]. 
The infection, known as COVID-19, is caused by a novel coronavirus called SARS-CoV-2 due 
to several genetic and clinical similarities with the coronavirus SARS-CoV which caused SARS 
epidemic in 2002-2003 [2]. The microbiological diagnosis is performed with Real Time Reverse 
Transcription - Polymerase Chain Reaction (RT-PCR) from oro/naso pharyngeal swabs or stool 
samples, or broncho-alveolar lavage in intubated patients.  
COVID-19 is predominantly a respiratory infection that spans from a mild involvement of the 
upper respiratory tract to a severe pneumonia leading to respiratory distress, shock and death [4]. 
In the early case series, fever, cough and dyspnea/tachypnea, together with myalgia and fatigue, 
have been identified as the most common presentation symptoms [6]. The incubation period 
ranges from 1 to 14 days with an estimated median of 5 to 6 days [3]. Most of the patients are 
affected by an upper respiratory tract disease for a relatively long period of time (a median of 8-
10 days), after that as much as 25% of affected patients may develop severe symptoms that 
require ICU admission [4]. Incidence of overall mortality is about 3 up to 4% [4]. Among the 
severe cases, 61% of patients might die [5].  
The disease is commonly transmitted in a hospital settings and healthcare providers are at highest 
risk. The hospital transmission of the infection is not unusual during an outbreak, as shown 
during SARS outbreak in 2003. In February 2020, about one thousand and seven hundreds 
Chinese healthcare workers were confirmed to be infected by SARS-CoV-2 with at least 6 
reported casualties including Dr Li Wenliang, the first physician to identify this new disease [6]. 
More recently in the United States, two ER physicians are in critical conditions in the Intensive 
Care Unit, after being in contact with COVID-19 patients. Unfortunately, at present time, there is 
no vaccine or pharmacological prophylaxis to decrease the risk in healthcare providers to 
contract the infection. Remdesivir may speed up a patient’s recovery and discharge from the 
hospital, however it has not been shown to prevent transmission of the virus.  
Thus, a safe and prophylactic therapy to reduce COVID-19 disease in healthcare workers would 
be of a great benefit the individuals and the society.  
 
 
Previous clinical and pre-clinical studies leading up and sustaining the proposed research  
Nitric oxide (NO) is an anti-infective agent at high concentrations. In-vitro studies have 
identified a significant activity against pathogens broadly resistant to antibiotic therapy, such as 
bacteria responsible for hospital acquired pneumonia [7]. In a small size clinical trial, nitric oxide 
gas showed to be safe and effective against bacterial colonization/superinfections in the context 
of chronic respiratory diseases [8]. 
NO gas showed also anti-viral activities against SARS-CoV [9] [10]. In an in-vitro study, NO-
donor compound S-nitroso-N-acteylpenicillamine added to a cell culture preparation increased 

6 
Nitric oxide gas inhalation for healthcare providers  
to prevent COVID-2019 disease. Version 7.0 
 
May 22, 2020 
the survival rate of mammalian cells infected with SARS-CoV [11]. In patients affected by 
SARS, NO inhalation decreased the need of oxygen therapy, ventilatory support, and a faster 
resolution of chest x-ray abnormalities [12].  
Due to the genetic similarities, both Coronaviridae SARS-CoV and SARS-CoV-2 might behave 
similarly to NO gas exposure. We hypothesized that high dose of exogenous inhaled NO is 
viricidal agent in COVID-19 disease. A dose of 160 ppm of nitric oxide has been found to be 
bactericidal, fungicidal, viricidal [13, 14]and safe for the patient when adequately managed. In 
fact, high dose of NO administered continuously for long time can generate an excess of 
methemoglobinemia. Miller et al found that intermittent administration, 160 ppm for 4 cycles of 
30 min, have the same antibacterial effect and can be safely administered [15]. 
The administration of high doses of nitric oxide, with potent viricidal effect, could prevent 
development of COVID-19 disease in healthcare providers exposed to SARS-CoV-2 positive 
patients. 
 
Rationale and hypothesis  
Preliminary data support the anti-viral effects of high concentration, intermittent inhaled NO gas. 
We hypothesized that high concentration inhaled NO can decrease the risk for COVID-19 
infection in healthcare providers.  
Sparing healthcare workers is highly desirable, especially during a pandemic infection. 
To test this hypothesis, we propose a randomized clinical trial.  
 
 
II. SPECIFIC AIMS  
 
The proposed randomized clinical trial will test the efficacy of inhaled NO at 160-180 ppm for 2 
daily cycles of 15 minutes each in: 
 
Aim 1  
To assess whether intermittent delivery of inhaled NO gas in air at a high dose may protect 
healthcare workers (physician, nurse, respiratory therapist, administrative staff, technician or 
other healthcare professional) from COVID-19. 
 
Methods to achieve Aim 1  
Primary outcome of the study is the proportion of healthcare providers who present COVID-19 
at 14 days. COVID is defined as SARS-CoV-2 infection (positive real-time polymerase chain 
reaction (RT-PCR) plus one of the following: 
• Fever: defined as temperature: 
o > 36.6 °C, axillary site 
o > 37.2°C, oral site 
o >37.7°C, tympanic or rectal site 
• Cough 
• Shortness of breath 
 

7 
Nitric oxide gas inhalation for healthcare providers  
to prevent COVID-2019 disease. Version 7.0 
 
May 22, 2020 
Aim 2  
Secondary outcome is the proportion of healthcare providers who present positivity to real time 
RT-PCR at 14 days.  
 
Methods to achieve Aim 2  
The secondary outcome (RT-PCR for SARS-CoV-2 positivity at 14 days) will be evaluated as 
the proportion of subjects who have a positive swab for SARS-CoV-2 at 14 days.  
 
Aim 3  
Proportion of healthcare providers requiring quarantine  
 
Methods to achieve Aim 3 
The aim number 3 will be evaluate as the proportion of the subjects who require quarantine 
during the study periods.  
 
Aim 4 
Total number of quarantine days in the two groups 
 
Methods to achieve Aim 4   
The aim number 3 will be evaluate as the difference in the mean of the quarantine days between 
the 2 groups 
 
 
III. SUBJECT SELECTION  
 
Inclusion /Exclusion criteria  
Inclusion criteria  
1. Age ≥18 years  
2. Scheduled to work with SARS-CoV-2 infected patients for at least 3 times/week (at least 
6 shifts in 14 days). 
3. Healthcare providers or other professional (doctors, nurses, respiratory therapists, 
technician, administrative staff, others) working at a participating hospital  
 
Exclusion criteria  
1. Proven previous SARS-CoV2 infection and subsequent negative rt-PCR 
2. Pregnancy. All eligible women in childbearing age must undergo urinary pregnancy test 
(will be excluded from testing women over 50 years of age or with more than 12 months 
from the last menstrual cycle. Will be excluded, as well, women with well documented 
surgical sterilization).  
3. Known hemoglobinopathies 
4. Known anemia 
 
Source of subject and recruitment methods 
This study is targeted for a population of healthcare provider (physician, nurse, respiratory 
therapist, administrative staff, technician or other healthcare professional) working in a hospital 
with COVID-19 patients. At the admission of the first SARS-CoV-2 positive patient in a hospital 

8 
Nitric oxide gas inhalation for healthcare providers  
to prevent COVID-2019 disease. Version 7.0 
 
May 22, 2020 
unit a recruitment email will be sent to the department chief to inform the staff about the 
research. Individual hospital staff members may be contacted via email and asked to participate 
using the recruitment email.  Staff distribution lists may also be utilized to identify potential 
participants.  Participants who are interested in learning more about the study will be able to 
contact the study team to learn more.  
 
All the healthcare professionals that met all the inclusion and not the exclusion criteria become 
eligible for the study. The clinical staff of each unit will be adequately informed about the 
protocol details. No clinical care procedure will be interrupted or delayed due to the study 
procedure.  
 
IV. SUBJECT ENROLLMENT  
 
Methods of enrollment, including procedures for patient registration  
The study was reviewed and approved by the MGH Anesthesia-in-Chief (Oluwaseun Johnson-
Akeju, investigator on this study), by the leadership of the Respiratory Care Services (Dr. Robert 
Kacmarek, investigator on this study) and by the Medical Director of Respiratory Care (Dr. 
Lorenzo Berra, PI of this study). 
 
An email containing the details of the study, the link to ClinicalTrial.gov and the study staff 
contacts will be sent to all the medical directors of the hospital responsible for the COVID-19 
patients in order to inform all their staff about the possibility to join this study. The study staff 
will approach the subject to evaluate the inclusion/exclusion criteria.  
 
In case eligibility criteria are met, investigator(s) will proceed with the enrollment procedures. 
The member of the research staff will explain to the subject all the general details of the research 
protocol. Then, if the subject is willing to, an MD investigator will go through further details and 
answer potential questions regarding the research study. Written informed consent will be 
obtained from the subject. To avoid any form of coercion the department chiefs involved in the 
study (Dr.Robert Kacmarek and Dr. Oluwaseun Johnson-Akeju) are not allowed to know the 
names of the study participants. Furthermore, they cannot participate to study gas administration. 
Since all the subjects will be healthy with intact decision-making capacity, no surrogate consent 
will be accepted. A de-identified code will be assigned to the subject and registered on a 
dedicated enrollment log. 
 
Procedures for obtaining informed consent  
If the subject will autonomously decide to take part of the study, contacting the study staff, will 
be approached by an investigator who will go through the details of the study. Signing of an 
Informed Consent Form will be requested for participation in the study. Consent can be 
withdrawn at any time. The investigators will access Personal Medical Information (PMI) for 
study purposes.  
We expect all the subjects involved in this study to be able to personally consent to participation.  
 
Treatment assignment and randomization  
Randomization will occur through random allocation sequence generated by a computerized 
random generation program (REDCap). Parallel allocation to treatment or control group will 

9 
Nitric oxide gas inhalation for healthcare providers  
to prevent COVID-2019 disease. Version 7.0 
 
May 22, 2020 
occur with a 1:1 ratio. Randomization will not be stratified for pre-specified demographic 
conditions (e.g., sex, age). Results will be adjusted for such conditions in analysis phase as 
specified in the statistical analysis section. 
Subjects will be randomly allocated to the treatment group with NO administration or to the 
control group in which no nitric oxide gas will be delivered.  
 
Blinding  
No masking is going to be used in this study.  
 
 
V. STUDY PROCEDURES  
 
Study subjects will be the healthcare providers (physician, nurse, respiratory therapist, 
administrative staff, technician or other healthcare professional) part of the units assigned to the 
COVID-19 outbreak management. Fixed time of treatment administration (14 days) and 
evaluation (14 days) has been established. All the outcomes will be evaluated at the end of this 
period. Subjects will stop receiving NO before the 14 days period under the conditions specified 
in the section “Study time frame”. In any case the subject will be monitored for 14 days. The 
workflow of the study has been summarized in Appendix 7.  
Since pregnancy is an exclusion criterion every woman in childbearing age will undergo a 
urinary pregnancy test before study gas administration. Menopausal women, defined as 12 
months without menstrual period AND over 50 years of age, as well as women with well 
documented surgical sterilization do not need to undergo a pregnancy test. During the study 
period the women in childbearing age must declare that they are not seeking pregnancy and that 
they are using acceptable contraception or are not sexually active with men.  
 
Drug to be used: NO administration  
NO will be delivered in 2 daily sessions (before and after the work shift) during 14 consecutive 
days. To consider the study gas administration effective the subject must receive at least 12 
sessions in 14 days (weekend days included). 
Each session will last 15 minutes, for a total of 30 minutes/day for each subject. The gas will be 
delivered as previously described in IRB #2016P001629 and 2020P000786. We have enrolled to 
date 220 patients in trial IRB #2016P001629. The system is easy to use and safe. No adverse 
events have been reported. Methemoglobin remained always below 5% and NO2 never higher 
than 2 ppm. The non-invasive ventilation is set at a range between 2 and 5 cmH2O of Positive 
End Expiratory Pressure (PEEP) (as illustrated in Appendix 5). A tank will be connected to the 
inspiratory limb of the circuit, and the output will be adjusted to deliver a target concentration 
between 140 and 180 ppm. Commercially available tanks or a nitric oxide generator (Appendix 
A) will be used to deliver the gas. The choice to use a tank or the nitric oxide generator will 
depend on the number of available nitric oxide tanks that day, and whether they are already in 
use. The nitric oxide generator will only be used if the tanks are otherwise unavailable. The NO 
generator is a box that creates nitric oxide from air by electrical spark. The air (nitrogen and 
oxygen) is flown in the box and the production of nitric oxide is proportional to the voltage 
difference and the rate of sparks per minute. This nonsignificant risk device and its use will 
comply with the abbreviated IDE requirements found in 21 CFR 812.2(b).  Thus, this device will 
appropriately be labeled as an investigational device, and requirements for device accountability, 

10 
Nitric oxide gas inhalation for healthcare providers  
to prevent COVID-2019 disease. Version 7.0 
 
May 22, 2020 
informed consent, monitoring, records, reports, and prohibition against promotion and IRB 
approval will be followed.  Further the PHRC will be notified immediately if any problems 
associated with the use of the device are encountered. 
 
Connection to the non-invasive ventilation circuit will be through a blender mounted on the 
ventilatory machine. Desired mixture of air, O2 and NO in the blender will be titrated with the 
blender knob, so to reach a concentration between 140 and 180 ppm at the inspiratory limb. An 
HEPA filter will be positioned on the expiratory line. 
Alternatively, a non-rebreathing mask will be used to deliver the gas (as illustrated in Appendix 
6). The choice of the system to delivery NO will depend on the availability of respiratory 
equipment and on study subject preference.     
The gas delivery process will follow these sequences: an investigator or a respiratory therapist 
will describe to the subject the equipment and the delivery of gas, like we generally do to 
patients that require home-CPAP. Once learned the CPAP settings, the subject will wear the 
mask. The respiratory therapist and the investigator will step out of the room and the CPAP 
system in stand-by until then will start delivery nitric oxide. The investigator with the respiratory 
therapist will monitor the subject's oxygenation and EKG during the gas therapy and will keep 
the contact with the subject on an audio/video monitoring. The subject at the end of the time will 
remove the mask and the ventilator will go in a stand-by mode. The subject will remain in the 
room for the next 10 minutes of observation. 
Inspiratory concentration of NO and NO2 (never higher than 3 ppm) will be continuously 
monitored. Methemoglobin also will be continuously monitored non-invasively with a dedicated 
pulse oximeter system and should remain always below 5%. NO deliver will be titrated in order 
to keep methemoglobin under 5%. Values will be recorded in a dedicated paper data sheet, 
specifically: (I) before starting NO delivery, (II) at the end of the delivery and (III) at 5 to 10 
minutes after completion of the NO delivery to demonstrate a decrease of methemoglobin after 
cessation of gas delivery. If MetHb doesn’t decrease, we will continue monitor until there is a 
decrement. Subjects assigned to the control group will not receive any gas therapy.   
 
During the session continuous SpO2 and heart rate will be monitored by study staff, non-invasive 
arterial pressure will be cycled every 5 minutes. Values will be recorded in a dedicated paper 
data sheet, specifically: (I) before starting NO delivery, (II) at the end of the delivery and (III) at 
5 to 10 minutes after completion of the NO delivery to demonstrate a decrease of methemoglobin 
after cessation of gas delivery.  
The study gas will be administered in the respiratory care room in Ellison 4 (next to the surgical 
ICU). 
Subjects assigned to the control group will not receive any gas therapy.  
 
Throat Swab 
We will be collecting a nasal/oropharyngeal swab at baseline and at 14 days. Sample collection 
for participants will depend on the availability of the specimen collection kits.  Usual precautions 
will be utilized for collection of these samples. Following collection samples will be labelled and 
immediately frozen in a clean double bag in a -20°C or colder freezer for storage. These samples 
will be stored in the BSL2 lab under the direction of Dr. Hohmann.  These samples will be 
analyzed to determine if the amount of detectable virus is different between groups.  These 

11 
Nitric oxide gas inhalation for healthcare providers  
to prevent COVID-2019 disease. Version 7.0 
 
May 22, 2020 
samples will be analyzed in batches, therefore we will not return individual results to 
participants. These samples will only be collected at MGH.   
 
Study time frame  
Study enrollment will last for 14 days. The NO gas administration will be performed every day 
twice a day for 14 days (before and after the work shift). Nitric oxide administration will be 
stopped if one of the following events happens:  
1) Development of COVID-19 disease (see “Methods to assess Aim 1”).  
2) Positive RT-PCR for SARS-CoV-2 (see “Methods to assess Aim 2”) 
3) Subject quarantined (see “Methods to assess Aim 3 and 4”) 
4) Change of the assigned ward 
  
For subjects who will exit the study before the completion of the defined time period the 
outcomes will be evaluated the same way as who will not exit at 14 days.  
 
Data To be collected and when the data is to be collected  
 
Collection from subject past medical history:  
• Subject demographics  
• Past and current medical history of heart, lung and neoplastic disease 
• Cigarette smoke 
 
Prospective collection of subject data will include:  
• Hour of exposition to SARS-CoV-2 positive patients in 14 days 
• COVID-19 disease development. 
• Time to COVID-19 development (in days). 
• Results of SARS-CoV-2 RT-PCR tests carried out by the study subject (according to 
Hospital policy) 
 
VI. BIOSTATISTICAL ANALYSIS 
 
Study Endpoint 
Primary endpoint: incidence of subjects with COVID-19 disease at 14 days 
 
Secondary endpoints: 
1. Incidence of subjects with positive SARS-CoV-2 rt-PCR test at 14 days 
2. Proportion of healthcare providers requiring quarantine 
3. Total number of quarantine days 
 
Statistical methods 
Data will be analyzed following the intention to treat analysis principle. Demographic and 
clinical data will be presented as proportions for categorical outcomes and mean plus standard 
deviation or median plus interquartile range for continuous outcomes. Comparison between 
groups for the primary outcome will be made with the X2 test or the Fisher exact test for 
categorical variables. Secondary outcomes will be analyzed with the X2 test or the Fisher exact 

12 
Nitric oxide gas inhalation for healthcare providers  
to prevent COVID-2019 disease. Version 7.0 
 
May 22, 2020 
test for categorical variables and with the t-test or Wilcoxon rank-sum test for continuous 
variables, after assessing the total number of quarantine days for normal distribution. 
A subgroup analysis will be performed with multiple linear regression, as well as logistic and 
cox regression models to adjust for age, pulmonary comorbidities, history of neoplastic disease 
and hour of exposition to SARS-CoV-2 positive patients in 14 days. 
 
Power analysis 
Based on available data from Italy and China, we predict an incidence of 15% of SARS-CoV-2 
infection in healthcare providers, and we assume that the incidence will be reduced to 5% with 
iNO cycles. Consider an alpha level of 0.05 and a power of 0.9, we determined a sample size of 
n1=207, n2=207, by a two-sided test run on a dedicated software [16]. In order to account for 
possible dropouts, we increased the sample size of about 10%, thus, from 414 to a total of 470 
subjects (235 each group). 
 
VII. RISKS AND DISCOMFORTS 
 
Procedure and drug-related risks for this study: Non-Invasive Ventilation and nitric oxide 
administration. 
 
Non-invasive ventilation risk: 
• Due to the tightness of the facial mask the risk of pressure sores is described between 2 
and 50%. This kind of complication usually follow longer time period, increasing the 
incidence with the time of the duration of the session. Such a small period should not be 
associated with skin lesions. The research team together with respiratory therapist will 
follow all the indications presented in literature in order to minimize the risk of this kind 
of complication. To avoid pressure sores, a non-rebreathing mask may be available. 
• Due to noise and the tightness of the facial mask the patient could experience a sensation 
of discomfort. The limited period should reduce the risk of patient’s discomfort. For 
better tolerance, a non-rebreathing mask is available. 
The patients will be informed for possible risks as indicated in the informed consent.  
 
Nitric Oxide:  
Breathing NO at 160 ppm for 30 minutes has been found to be safe in adults and children as 
shown in two scientific reports [14-15]. At MGH, we have been successfully delivered high dose 
of NO at 160 ppm to a minor over the past year (more than 70 sessions of NO gas treatments). 
The subject never had MetHb higher than 5% and NO2 was always below 5ppm. 
The theoretical risks of NO breathing include the following: pulmonary hypertension, pulmonary 
edema, methemoglobinemia, hypoxia, and hypotension. In the study in neonates breathing NO 
for up to 4 days, study subjects had hematuria (6%), hyperglycemia (7%), sepsis (2%), infection 
(3%), stridor (3%) and hypotension (10%). They all occurred in the placebo group as well and 
none of these findings was significantly higher in the NO group. In a small study of healthy 
volunteers, Frostell et al [16] report that there were no adverse clinical events in the group 
inhaling NO. Of note, the average baseline methemoglobin level prior to NO administration was 
0.61% and this increased to 0.77% after 10 minutes of inhalation of NO at 80 ppm. This was a 
statistically significant, although clinically irrelevant increase in methemoglobin levels. In our 

13 
Nitric oxide gas inhalation for healthcare providers  
to prevent COVID-2019 disease. Version 7.0 
 
May 22, 2020 
recent study [17], there were no side effects or drug toxicity associated to the use of NO at 80 
ppm for up to 24 hours. 
 
The inhalation of NO will be administered and monitored by a trained Respiratory Therapist 
(RRT). There will be dedicated therapists who also administer NO as part of their clinical 
responsibilities. They will be familiarized with the technical aspects of this protocol and 
supervised by some of the investigators (Dr. Lorenzo Berra or Dr. Robert Kacmarek or Dr. Ryan 
Carroll or Dr. Edward Bittner or Dr. Fumito Ichinose), who have significant experience with NO 
administration both clinically and as part of investigational protocols. The study staff will 
continuously monitor the NO, nitrogen dioxide (NO2) and oxygen concentration of the inhaled 
gas. The respiratory therapist will continuously monitor the oxygen saturation, via non-invasive 
oximetry during the procedure. Methemoglobin levels will be assessed using a non-invasive 
continuous co-oximetry monitor. If methemoglobin level rises more than 5%, NO will be halved 
and closely monitored until a reduction occurs. If methemoglobin persists at > 5%, NO will be 
halved until methemoglobin is < 5%. 
Since we will treat many subjects in the same room, we will monitor room nitric oxide 
concentration to keep it below 1 ppm.  
Any study subject who experiences a side effect which is felt to be related to the study drug will 
not be allowed to continue in the protocol.  
 
There are no known risks associated with the use of the electric NO generator device. The machine 
is not currently approved by the FDA for any medical purpose. It has been used safely in other 
small research studies at MGH however (Appendix A).  If any problems associated with the use 
of the device occur, the IRB will be notified immediately.   
 
There are no additional anticipated physical risks associated with the throat swab. 
 
Psychosocial risks 
We do not anticipate psychosocial risks to the study subjects from participation in this protocol. 
Each subject will personally sign the consent form, we will not ask for a surrogate’s permission 
to enter a patient into the study. The consent form will be obtained prior to the initiation of any 
study procedures, including those done for screening. Strict confidentiality will be maintained by 
the research team at all times, including keeping all data in a secure, locked cabinet with limited 
access.  
 
Radiation risks 
None. We do not request use of x-rays for research purposes. 
 
VIII. POTENTIAL BENEFITS 
 
Benefits to participating individuals:  
Subjects breathing NO might benefit from a decreased risk of COVID-19 disease development 
after professional exposure to SARS-CoV-2 positive patients.  
 

14 
Nitric oxide gas inhalation for healthcare providers  
to prevent COVID-2019 disease. Version 7.0 
 
May 22, 2020 
Benefits to society:  
There is no definitive drug available for the treatment of the disease or for the prevention of the 
development of COVID-19 disease. New information suggests that remdesivir may speed up a 
patient’s recovery and discharge from the hospital and can be used in the hospital for severe 
COVID-19 infection, however it has not been used in healthcare providers.   
 
One of the major benefits of NO might be the reduction of healthcare disease development and 
subsequent reduced need for quarantine. If administration of NO in this trial showed clinical 
benefits, the reduced number of quarantined healthcare providers will give a great support to the 
whole healthcare system in this world pandemic.   
 
IX. MONITORING AND QUALITY ASSURANCE 
 
Independent monitoring of source data 
Informed consent forms, case report forms, and data will be reviewed by the principal 
investigator following enrollment of every 50 subjects.  
 
Safety monitoring (e.g. Data Safety Monitoring Board) 
An interim safety report will be sent to IRB after the enrollment of the 30th subject. 
The Data Safety Management Board (DSMB) will perform an interim analysis for superiority 
after the 25th patient. In case of a significant decreased infection rate in the treated subjects, the 
trial will be stopped. 
Safety data include levels of Methemoglobin, NO2 levels, minor deviation from the protocol and 
minor adverse reactions to non-invasive ventilation. Other data to be reviewed includes 
maintenance of patient confidentiality throughout the study.  
Members of the Data Safety management Board (DSMB) consist of an intensivist, a virologist 
and an NO expert. Names and contacts of the DSMB members are:  
 
Richard Pino MD, PhD 
Associate Professor of Anesthesia at Harvard Medical School 
Department of Anesthesia Massachusetts General Hospital 
Email: RPINO@mgh.harvard.edu 
 
John Thomas MD, PhD 
Professor Emeritus in Microbiology at West Virginia University 
Email: jgthomas@hsc.wvu.edu 
 
Carl Nathan, MD 
R.A. Rees Pritchett Professor of Microbiology 
Weill Cornell Medicine 
Email: cnathan@med.cornell.edu 
 

15 
Nitric oxide gas inhalation for healthcare providers  
to prevent COVID-2019 disease. Version 7.0 
 
May 22, 2020 
Outcomes monitoring 
All the subjects will undergo an oro/nasopharyngeal swab with RT-PCR for SARS-CoV-2 at 
COVID-19 symptoms development. Follow up will continue for 14 days after recruitment as 
described in “V Study procedure”. 
 
Adverse event reporting guidelines 
In accordance with PHRC policy on Adverse Event Reporting and Unanticipated Problems 
Involving Risks to Subjects, the Principal Investigator (Lorenzo Berra, MD) will report adverse 
events or other unanticipated problems to the DSMB and to the PHRC within 5 working days/7 
calendar days of the date the investigator first becomes aware of the problems. Mild or moderate 
adverse events will be presented in progress reports at continuing reviews.  
 
Stopping rules. The review and decision regarding altering or stopping the protocol will be 
performed by the principal investigator together with the Data Safety Management Board 
(DSMB). Mild or moderate adverse events will be presented in progress reports at continuing 
reviews. Protocol exit criteria will be: 
a. Acute worsening hypotension: decrease in mean blood pressure of > 20 mmHg not 
attributable to other causes, such as: progression of the disease, hypovolemia, 
hemorrhage, sepsis, acute heart failure. 
b. Sudden hypoxemia defined as a 5% reduction of peripheral oxygen saturation (SpO2) of 
from the basal value. 
Or any life-threatening symptom potentially attributed to NO administration by the physician 
investigator. 
 
 
 

16 
Nitric oxide gas inhalation for healthcare providers  
to prevent COVID-2019 disease. Version 7.0 
 
May 22, 2020 
References 
1.        https://www.healthmap.org/covid-19/. 
2.        https://www.arcgis.com/apps/opsdashboard/index.html#/bda7594740fd40299423467b48e9ecf6. 
3.        WHO, Coronavirus disease 2019 (COVID-19) Situation Report - 37. 2020. 
4.         Wang, D., et al., Clinical Characteristics of 138 Hospitalized Patients With 2019 Novel Coronavirus-
Infected Pneumonia in Wuhan, China. JAMA, 2020. 
5.         Yang, X., et al., Clinical course and outcomes of critically ill patients with SARS-CoV-2 pneumonia 
in Wuhan, China: a single-centered, retrospective, observational study. The Lancet Respiratory 
Medicine, 2020. 
6.         Zhou, P., et al., Protecting Chinese Healthcare Workers While Combating the 2019 Novel 
Coronavirus. Infection Control & Hospital Epidemiology, 2020: p. 1-4. 
7. 
Privett, B.J., et al., Examination of bacterial resistance to exogenous nitric oxide. Nitric oxide : 
biology and chemistry, 2012. 26(3): p. 169-173. 
8. 
McMullin, B.B., et al., The Antimicrobial Effect of Nitric Oxide on the Bacteria That Cause 
Nosocomial Pneumonia in Mechanically Ventilated Patients in the Intensive Care Unit. 
Respiratory Care, 2005. 50(11): p. 1451-1456. 
9. 
Akerstrom, S., et al., Nitric oxide inhibits the replication cycle of severe acute respiratory 
syndrome coronavirus. J Virol, 2005. 79(3): p. 1966-9. 
10. 
Akerstrom, S., 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): p. 1-9. 
11. 
Keyaerts, E., 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(4): p. 223-6. 
12. 
Chen, L., et al., Inhalation of nitric oxide in the treatment of severe acute respiratory syndrome: a 
rescue trial in Beijing. Clin Infect Dis, 2004. 39(10): p. 1531-5. 
13. 
Deppisch, C., et al., Gaseous nitric oxide to treat antibiotic resistant bacterial and fungal lung 
infections in patients with cystic fibrosis: a phase I clinical study. Infection, 2016. 44(4): p. 513-
20. 
14. 
Regev-Shoshani, G., et al., Gaseous nitric oxide reduces influenza infectivity in vitro. Nitric Oxide, 
2013. 31: p. 48-53. 
15. 
Miller, C., et al., Gaseous nitric oxide bactericidal activity retained during intermittent high-dose 
short duration exposure. Nitric Oxide, 2009. 20(1): p. 16-23. 
16. 
Lenth, R.V., Java Applets for Power and Sample Size [Computer software]. Retrieved 02/24/2020 
from http://www.stat.uiowa.edu/~rlenth/Power. 2006-9. 
 
 
 

17 
Nitric oxide gas inhalation for healthcare providers  
to prevent COVID-2019 disease. Version 7.0 
 
May 22, 2020 
 APPENDIX A. NITRIC OXIDE GENERATOR 
 
 
As of April 27, 2020, we have enrolled 16 
HCWs, of which 8 receive NO treatments, for 
a total 64 administrations. We did not 
experience 
any 
adverse 
events 
and 
methemoglobin levels remained within safe 
range. Please see Table 2 for more details. 
 
We also measured inspiratory and expiratory 
concentrations of NO and NO2 gas in a health-
care worker enrolled in our present study 
breathing 160 ppm of NO from a NO/N2 tank 
mixed with air (please see Figure 1). Please 
note that the levels of NO2 remain below 1 ppm 
during the entire respiratory cycle both in 
inspiratory gas and in exhalation gas. 
 
Figure 1: Inspiratory and expiratory concentration of NO and NO2.  
 
 
 
Since our study opened enrollment, we have been challenged by the large numbers of healthcare 
workers willing to participate to our trial. Using only tanks, we have no means to deliver NO gas 
to more than two volunteers at each session, as each volunteer requires a NO tank setup, which is 
cumbersome, large, and needs additional equipment (oxygen flowmeter and a source of oxygen), 
and additional personnel. 
 
The use of an electric  NO-from-air generator would allow us to avoid (I) handling and transporting 
heavy tanks, (II) tank storage, (III) additional respiratory tubing and an  oxygen source (tank NO 
is mixed with nitrogen so to maintain the FiO2 at 21% (air) we require an external oxygen line).  
 
Table 1.  

18 
Nitric oxide gas inhalation for healthcare providers  
to prevent COVID-2019 disease. Version 7.0 
 
May 22, 2020 
In the Department of Anesthesia, Critical care research unit at MGH, we recently conceived, built 
and tested two electric NO generators that produces NO gas equivalent to NO commercial tanks. 
However, since it produces NO from ambient air it does not require an additional source of oxygen 
and allows us to treat at least 12 additional HCW per day. 
 
In awake sheep, we successfully tested the device to determine the vasodilator response of the 
pulmonary circulation at clinical concentrations of inhaled NO, from 20 to 80 parts per million 
(ppm) (please see manuscript, Yu et al. “Intratracheal injection of nitric oxide, generated from air 
by pulsed electrical discharge, for the treatment of pulmonary hypertension in awake ambulatory 
lambs” Nitric Oxide. 2020 Apr 1;97:11-15.) 
 
In three sheep, we also delivered higher 
concentrations of NO gas (up to 240 
ppm of gas) and measured the levels of 
nitric dioxide (NO2), which remained 
low, below 1 ppm (Table 2). 
 
The gas produced by our electric NO 
generator is pure and no particles were 
found in mice lungs after 28 days of 
breathing continuously NO at 50 ppm 
(Yu et al. Nitric Oxide. 2016. Detection 
and removal of impurities in nitric oxide 
generated from air by pulsed electrical 
discharge.) 
  
In a previous study in the MGH catheterization laboratory, (Partners IRB #2014P00253636), we 
studied an early prototype of the same device. In six patients with chronic pulmonary hypertension 
we found that the device is able to produce an equivalent vasodilator response of NO obtained 
from a NO/NO2 tank (Berra L, et al. Electric Plasma-generated Nitric Oxide: Hemodynamic 
Effects in Patients with Pulmonary Hypertension. Am J Respir Crit Care Med. 2016 Nov 
1;194(9):1168-1170.). 
 
This device has been inspected by the MGH Respiratory Care Dept N: 54923 (inspected  in 
12/2019). 
 
Table 2. Nitric oxide (NO) was delivered 
intratracheally to awake sheep. NO and NO2 were 
sampled and measured inside the lamb’s trachea. 
Awake sheep 
Nitric oxide 
(NO, ppm) 
Nitrogen dioxide 
(NO2, ppm) 
Sheep #1 
150 
0.61 
 
200 
0.80 
 
220 
0.91 
Sheep #2 
160 
0.62 
 
210 
0.87 
 
240 
0.90 
Sheep #3 
130 
0.52 
 
180 
0.65 
 
200 
0.76