Conditions: Depressive Disorder, Major Depressive Disorder, Treatment-Resistant
Keywords: Depression treatment-resistant
Allocation: RANDOMIZED | Intervention model: CROSSOVER
Enrollment: 24
Healthy volunteers: no

Brief summary:
The purpose of the study aims to determine whether different concentrations of nitrous oxide (N2O) have different antidepressant effects for adults with treatment-resistant major depression.

Detailed description:
Most clinical major depression responds to standard treatments (medication and psychotherapy); however, a significant subset of depressed patients (15-20%) do not respond to these treatments and are referred to as treatment-resistant major depression (TRMD). New treatments for TRMD are needed, and one promising line of research are drugs known as N-methyl-D-aspartate (NMDA) glutamate receptor antagonists. In a recent pilot study, our group demonstrated that the NMDA antagonist nitrous oxide is effective in TRMD.

All patients will receive 3 randomized, one hour nitrous oxide inhalations to placebo (0% N2O), low dose (25% N2O), and high dose (50% N2O). Inhalation sessions will be at least 4 weeks apart. Mood will be assessed at baseline, 2 and 24 hours, and 1, 2, and 4 weeks post-inhalation for each dose.

Arms:
arm 1: Participants will be studied for 14 visits over approximately 18 weeks. The participants will receive 1 hour-long gas inhalation mixtures at 3 different times, which are randomly assigned. arm 2: Participants will be studied for 14 visits over approximately 18 weeks. The participants will receive 1 hour-long gas inhalation mixtures at 3 different times, which are randomly assigned. arm 3: Participants will be studied for 14 visits over approximately 18 weeks. The participants will receive 1 hour-long gas inhalation mixtures at 3 different times, which are randomly assigned.

Interventions: intervention 1: Nitrous Oxide 25% intervention 2: Nitrous Oxide 50% intervention 3: Placebo Gas
Intervention descriptions: intervention 1: Nitrous oxide, an odorless, colorless gas typically used as an induction agent for general anesthesia or for dental sedation, is a known N-methyl-D-aspartate (NMDA) antagonist. It will be given at a concentration of 25% nitrous oxide/50% oxygen/25% nitrogen. intervention 2: Nitrous oxide, an odorless, colorless gas typically used as an induction agent for general anesthesia or for dental sedation, is a known N-methyl-D-aspartate (NMDA) antagonist. It will be given at a concentration of 50% nitrous oxide/50% oxygen. intervention 3: Placebo gas given at 50% nitrogen \[inert\]/50% oxygen.

Protocol document:
Prot_SAP_000.pdf: 
1 
 
Study Protocol 
 
Inhaled Nitrous Oxide for Treatment-Resistant Depression: Optimizing Dosing Strategies 
 
Protocol Version: 
6 
5/24/2019 
 
 
 
Principal Investigator: 
 
Ben Palanca, MD PhD 
Assistant Professor of Anesthesiology  
Department of Anesthesiology, Campus Box 8054 
Phone: 314-362-7823 
Email: palancab@wustl.edu 
 
 
Co-Investigator: 
Peter Nagele, MD, MSc 
Professor of Anesthesiology  
Department of Anesthesiology & Critical Care,  
University of Chicago 
Email: nagelep@uchicago.edu 
 
Charles Conway, MD 
Professor of Psychiatry 
Department of Psychiatry, Campus Box 8134 
Phone: 314-362-2463 
Email: conwayc@psychiatry.wustl.edu 


2 
 
ABSTRACT 
 
Most clinical major depression responds to standard treatments (medication and psychotherapy); however, a 
significant subset of depressed patients (15-20%) do not respond to these treatments and are referred to as 
treatment-resistant major depression (TRMD). New treatments for TRMD are needed, and one promising line 
of research are drugs known as N-methyl-D-aspartate (NMDA) glutamate receptor antagonists. In a recent pilot 
study, our group demonstrated that the NMDA antagonist nitrous oxide (N2O) is effective in TRMD, however, 
the dose concentration of nitrous oxide to have antidepressant effects is unknown. Therefore, we propose 
conducting a randomized, placebo controlled double-blind crossover (x2) study in which treatment-resistant 
depression patients will all receive 3 randomized, one hour nitrous oxide inhalation sessions of placebo (0% 
N2O), 25% N2O (low dose), and 50% N2O (high dose). We aim to compare nitrous oxide’s relative 
antidepressant efficacy of two doses of N2O by demonstrating that inhaled nitrous oxide, but not inhaled 
placebo, will significantly show rapid and sustained antidepressant effects. 
 
 


3 
 
SYNOPSIS 
 
Study Title 
Inhaled Nitrous Oxide for Treatment-Resistant Depression: Optimizing Dosing Strategies 
Objective 
This study aims to determine whether different concentrations of nitrous oxide (N2O) have 
different antidepressant effects for adults with treatment-resistant major depression.  
Study Period 
Planned enrollment duration: Approximately 2 years 
Planned study duration: Approximately 18 weeks 
Number of 
Patients 
40 
Study Drug 
Nitrous oxide (laughing gas). This is an FDA-approved general anesthetic and sedative agent used 
in hospitals and dentist offices. The use of nitrous oxide in this protocol is off-label and outside of 
the approved indication. Nitrous oxide will be administered under supervision of an attending-
level anesthesiologist and using standard anesthesia equipment (mobile anesthesia machine/FDA-
approved nitrous oxide delivery system, standard monitoring, oxygen, rescue equipment). 
Study Design 
Prospective, randomized, placebo-controlled, cross-over (x2) trial.  All patients will receive 3 
randomized, one hour N2O inhalations to placebo (0% N2O), low dose (25% N2O), and high dose 
(50% N2O).  Inhalation sessions will be at least 4 weeks apart. Mood will be assessed at baseline, 
24 hours, and 1, 2, and 4 weeks post-inhalation for each dose. 
Inclusion and 
Exclusion 
Criteria 
Inclusion Criteria 
a) Adults 18-75 years of age; 
b) Current diagnosis of unipolar major depressive disorder (MDD) without psychosis as 
confirmed by structured clinical interview for DSM-IV disorders; 
c)  A score of  ≥ 9 on the Montgomery-Åsberg Depression Rating Scale (MADRS); 
d) Documented (i.e., chart review) lifetime failure to respond to ≥3 adequate dose/duration 
antidepressant treatment trials, ≥1 medication failure in the current depressive episode; 
e) Good command of the English language. 
 
Exclusion Criteria  
a)  Meets criteria for any DSM-IV diagnosis for schizophrenia, bipolar, schizoaffective, 
obsessive-compulsive, personality, or panic disorders; 
b) Any recent (within past 12 months) history of substance dependence or abuse (except tobacco), 
determined by reported history and urine drug screen; 
c) Ability to become pregnant and not using effective contraception; 
d) Contraindication against the use of nitrous oxide: 
1) Pneumothorax 
2) Bowel obstruction 
3) Middle ear occlusion 
4) Elevated intracranial pressure 
5) Chronic cobalamin and/or folate deficiency treated with folic acid or vitamin B12 
6) Pregnant patients 
7) Breastfeeding women 
e) Inability to provide informed consent; 
f) Any other factor that in the investigators’ judgment may affect patient safety or compliance  
Primary Outcome 
Comparison of change in Hamilton Depression Rating Scale score at immediate (2 hrs) and 
sustained (24hrs) post nitrous oxide inhalation. Secondary depression measures will be assessed at 
the later time points for duration of sustained antidepressant effects. 
Measurements 
Hamilton Depression Rating Scale, HDRS-21 scale;  Montgomery-Åsberg Depression Rating 
Scale, MADRS; Quick Inventory of Depressive Symptomology-Self Report, QIDS-SR; Clinician 
Administered Dissociative States Scale, CADSS-28 item;  Brief Psychiatric Rating Scale, BPRS; 
Profile of Mood States, POMS-2, MINI International Neuropsychiatric Interview. 
Electroencephalography (EEG) during eyes open and eyes closed periods. 
Statistical 
Methodology 
Outcomes will be analyzed with a repeated measures mixed effects linear model using restricted 
maximum likelihood estimation. To adjust for the observed carryover effect, the model will 
include a randomization group term and a three-way interaction. Response and remission rates 
will be compared with an exact binomial test (and corresponding odds ratios calculated). 


4 
 
1. Specific Aims 
1.1.  To demonstrate that both low and high concentrations of nitrous oxide will show equivalent and 
superior antidepressant effects relative to placebo at immediate (2 hours) and sustained (24 hours) time 
points. 
1.2.  To compare the sustained antidepressant effect of low and high concentrations of inhaled nitrous oxide 
at 1, 2, and 4 weeks post-inhalation. We hypothesize that for both low and high doses, antidepressant 
effects will be attenuated at 1 and 2 weeks, and will dissipate by one month. 
 
2. Background 
It is estimated that as many as 16 million Americans suffer from major depressive disorder (MDD). Fifty 
percent fail an initial pharmacotherapy trial and ~30% fail a series of treatment trials [1], and are referred to as 
“Treatment-Resistant Major Depression” (TRMD). TRMD exacts a high toll on treatment resources and studies 
suggest that patients with TRMD place an unusually heavy burden on total MDD costs (suicide, personal 
financial costs, loss of work, medical co-morbidity [2, 3]). Unfortunately, knowledge regarding the best 
treatments for TRMD remains incomplete.  
 
Studies demonstrate that there are limited effective antidepressant treatments for TRMD. Evidence shows that 
only four treatment approaches are effective in MDD after two sequential ineffective antidepressant trials: 
lithium augmentation, monoamine oxidase inhibitors, electroconvulsive therapy, and atypical antipsychotics 
(e.g. aripiprazole, quetiapine; [3]). To address this problem, the NIMH sponsored the largest sequential 
antidepressant trial (STAR*D). Following four levels of adequate dose-duration medications, ~36% of patients 
failed to remit [1]. The STAR*D trial concluded that a significant proportion of MDD patients do NOT achieve 
remission and constitute TRMD. With few exceptions, the majority of existing antidepressant treatments act via 
mechanisms discovered over three decades ago. Hence, antidepressant treatments with alternate mechanisms 
of action are needed. 
 
Over the past 15 years, multiple studies have indicated that the NMDA antagonist ketamine is an effective, 
rapid onset antidepressant in TRMD [4]. Most noteworthy, these studies suggest that ketamine acts 
immediately, producing an antidepressant response in less than 24 hours- a distinct advantage to existing 
antidepressant medications, which typically take 4-6 weeks to effectively decrease depressive symptoms. 
Despite the enormous potential benefits of ketamine in TRMD, this compound carries significant limitations: it 
is associated with psychosis, high addiction potential, and potential cognitive impairment [5-8].  
 
Nitrous oxide (N2O), an odorless, colorless gas typically used in anesthesia and dentistry, is an NMDA receptor 
antagonist [9]. Our group recently completed the first study of N2O in TRMD patients [10]. Using a double-


5 
 
blind, crossover design, we found that TRMD patients receiving a one hour session of inhaled N2O (50% N2O 
in 50% oxygen) versus placebo (50% nitrogen in 50% oxygen) experienced a significant decrease in MDD 
symptoms at 2 and 24 hours after inhalation, using the Hamilton Rating Scale for Depression-21 item (HDRS-
21). Mean HDRS score differences at 2 hours: -4.8 points, p=0.002; and 24 hours: -5.5 points, p<0.001). Four 
patients (20%) had treatment response (HDRS reduction ≥50%) and three patients (15%) a full remission 
(HDRS ≤7 points) after N2O, compared to one patient (5%) and none after placebo (odds ratio [OR] for 
response 4.0, 95% CI 0.45–35.8; OR for remission 3.0, 95% CI 0.31– 28.8). No serious adverse events 
occurred; however, two patients experienced panic attacks/anxiety onset, suggesting that the inspiratory N2O 
concentration may have been too high. Importantly, the N2O dose in our pilot study (50% N2O in 50% oxygen) 
was based on its use in dentistry because no prior information about N2O in TRMD existed. 
 
Preclinical studies suggest that different doses of ketamine are associated with variable NMDA receptor and 
antidepressant activity. In single neurons, 50% N2O blocks >50% of NMDA receptors, whereas ketamine 
concentrations achieved in depression studies likely block ~25-35% of NMDA responses [11]. Thus, it is 
possible that a lower inspiratory concentration of N2O (25%) will have substantial antidepressant effect while 
also reducing the risk of paradoxical anxiety. 
 
We propose a pilot study using a double-blind, crossover (x 2), placebo-controlled design to determine 
whether different concentrations of N2O have different antidepressant effects. We hypothesize that 25% and 
50% N2O, as compared to placebo, will have equivalent antidepressant effects. Further, we predict that these 
effects will occur rapidly (2 hours) and be sustained (24 hours, one and two weeks), but will attenuate by one 
month. 
 
3. Drug Information 
Nitrous oxide (laughing gas) is a colorless, odorless gas. Nitrous oxide is the oldest and most widely used FDA-
approved anesthetic gas. It is commonly used as a component of general anesthesia and as a sedative/analgesic 
agent used in hospitals and dentist offices. The onset as well as offset of effect is within a few minutes. Nitrous 
oxide is the least potent of inhalational anesthetics. Concentrations above 1 atm are theoretically needed to 
produce complete general anesthesia when nitrous oxide is used as a sole agent. Therefore, concentrations 
typically used (50%) in dentistry and pediatrics achieve only mild to moderate sedation (but potent analgesia) 
and are not sufficient to produce general anesthesia. Well-known side effects include euphoria, sedation, nausea 
and vomiting, inactivation of vitamin B12 (commensurate with the duration of exposure and concentration used). 
In general, exposure to 25-50% nitrous oxide for 1 hour is considered extremely safe. The use of nitrous oxide 
in this protocol is off-label and outside of the approved indication. 
 
 
 


6 
 
4. Eligibility 
4.1. Inclusion Criteria 
a) Adults 18-75 years of age; 
b) Current diagnosis of unipolar major depressive disorder (MDD) without psychosis as confirmed by 
structured clinical interview for DSM-IV disorders; 
c) A score of ≥ 9 on the Montgomery-Åsberg Depression Rating Scale (MADRS); 
d) Documented (i.e., chart review) lifetime failure to respond to ≥3 adequate dose/duration antidepressant 
treatment trials, ≥1 medication failure in the current depressive episode; 
e) Good command of the English language. 
 
Exclusion Criteria  
a) Meets criteria for any DSM-IV diagnosis for schizophrenia, bipolar, schizoaffective, obsessive-compulsive, 
personality, or panic disorders; 
b) Any recent (within past 12 months) history of substance dependence or abuse (except tobacco), determined 
by reported history or urine drug screen; 
c) Ability to become pregnant and not using effective contraception; 
d) Contraindication against the use of nitrous oxide: 
1) Pneumothorax 
2) Bowel obstruction 
3) Middle ear occlusion 
4) Elevated intracranial pressure 
5) Chronic cobalamin and/or folate deficiency treated with folic acid or vitamin B12 
6) Pregnant patients 
7) Breastfeeding women 
e) Inability to provide informed consent; 
f) Any other factor that in the investigators’ judgment may affect patient safety or compliance. 
 
5. Enrollment 
Participants will be recruited in part through the Volunteer for Health registry at Washington University School 
of Medicine and the Washington University TRMD Database (120+ individuals with TRMD carefully screened 
over the past 8 years, maintained by Dr. Conway; IRB# 201102247). Eligible and interested participants will be 
consented for participation and enrolled in the study by study personnel. We will recruit a total of 40 
participants currently in an episode of major depressive disorder, medication-free or on psychotropic 
medications. To complete the study, we will enroll up to 40 participants to achieve adequate sample size.   
 


7 
 
6. Methods 
6.1. Study Design 
This is a double-blind, randomized placebo-controlled crossover trial in which all subjects will undergo three 
randomized, one hour N2O inhalation sessions each separated by at least 4 weeks. Inhalations will include 
exposure to placebo (0% N2O), 25% N2O (low dose), and 50% N2O (high dose). Mood will be assessed at 
baseline (immediate pre-treatment), immediate (1 hour post-inhalation), sustained (24 hours) and at one and two 
weeks, and one month. We will also assess for potential psychiatric side effects including psychosis and 
disassociation. 
 
There will be up to 14 study visits involved with participation. The screening visit (Visit 1) will be used to 
verify eligibility and collect background information.  
 
Specifically, we will collect patient demographic information, medical history, vital signs and a research team 
member will perform a physical exam.  A urine sample will be collected for drug, and an optional blood draw 
may be collected if the study physician requests tests to confirm participants’ safety to participate.  Psychiatric 
assessments (completed by the participant and administered by the research team) will be performed to assess 
for major depression, other mental health diagnoses including significant anxiety disorder, post-traumatic stress 
disorder, and psychosis or dissociation. 
 
Visit 2 may directly follow Visit 1 on the same day, if needed.  Visit 2 (Baseline visit) will ask participants to 
fill out several mood-based questionnaires, pass a urine pregnancy screen (Baseline visit only), receive a total of 
60 minutes gas inhalation, and after the observation period, will be asked several mood-based questionnaires 
again. Visit 3 is scheduled approximately 22-28 hours after completion of the inhalation session, and 
participants will have vital signs taken and mood questionnaires asked. Visits 4 and 5 will occur 1 and 2 
week(s), respectively, after inhalation session one, and involve asking participants about their mood and mental 
state. Visit 6 will occur 4 weeks after inhalation session one, and will include questionnaires to account for a 4 
week follow up and also a baseline visit for the next gas concentration.  The procedures for Visit 6 will be 
identical to Visit 2 (besides urine pregnancy screen) with gas inhalation and mood-based questionnaires before 
and 1 hour after inhalation.  The remaining follow up visits will follow the same outline until all 3 gas 
concentrations have been given to each participant and follow up visits 24 hours, and 1, 2, and 4 weeks after 
each inhalation session have been completed.  If necessary, the 1, 2, and 4 week post inhalation session visits 
may be conducted over the phone with Principal Investigator approval. 
 


8 
 
Participants will be monitored after each inhalation session before being allowed to leave. Optional labs may be 
ordered by the screening physician to confirm the safety of a patient.  These screening labs might include but 
are not limited to a comprehensive blood panel and a complete blood count. 
Patients will be reimbursed with $5 + parking for each visit completed after the Screening Visit for their 
expenses and time commitment.  If a participant completes all visits they will receive $65.  Otherwise if the 
participant ends participation before the study visits are complete, the participant will be compensated $5 for 
each visit they did complete. 
 
6.2. Randomization 
The order of the 3 inhalation sessions will be randomly assigned for each patient by a random number 
generator.  
 
6.3. Blinding 
Patients will be blinded to the group assignment. Nitrous oxide is a colorless and odorless gas which makes it 
unlikely for patients to identify the group assignment. Likewise, the study setup will be identical for both 
sessions, which will make an inadvertent unblinding of the study unlikely. For the team responsible for the 
administration of the nitrous oxide treatment, the group assignment will be known. This team is completely 
separate from the team assessing the study outcomes which will be blinded to the group assignment. Treatment 
administration and outcomes assessment may also be physically separated in different rooms within the study 
facility.   
  
In addition, after each inhalation session, subjects will be asked whether they thought they received the active 
nitrous oxide gas or the placebo gas. Responses will be recorded with an assessment (5 point scale) asking 
participants to rate the extent to which they knew they were exposed to nitrous oxide; 1) strongly believe the 
treatment was nitrous oxide, 2) somewhat believe the treatment was nitrous oxide, 3) somewhat believe the 
treatment was placebo, 4) strongly believe the treatment is placebo, and, 5) don’t know. 
 
6.4. Setting 
The inhalation sessions will be performed in Barnes-Jewish-Hospital, or the Clinical Research Unit (CRU) suite 
within Washington University. The procedure room used is equipped with vital sign monitoring, resuscitation 
equipment and devices, and has oxygen wall outlets. As part of the standard setup, a mobile FDA-approved 
nitrous oxide delivery system will be used or an anesthesia machine.    
 
 
 


9 
 
6.5. Study Procedures 
Except for the choice of gas (either nitrous oxide concentration or nitrogen [inert]; all mixed with 50% oxygen) 
treatment sessions will be identical. The gas mix will be administered via a standard anesthesia facemask 
through tubing connected to the anesthesia machine or via a facemask which is connected via hose to an FDA-
approved Porter/Praxair MXR breathing circuit. A small sample connector line will be inserted into the 
facemask which allows the measurement of inhaled and exhaled gas concentrations. Inhaled and exhaled gas 
concentrations of nitrous oxide, oxygen and nitrogen will be monitored and adjusted according to protocol. 
Total gas flow will be 2-8 L/min.  
 
Patients will be monitored during and after the treatment according to American Society of Anesthesiologists 
standard which include continuous 3-lead ECG, pulsoximetry, non-invasive blood pressure and endtidal CO2 
under the supervision of an attending-level anesthesiologist. After the one hour treatment session, patients may 
be transferred if needed, and monitored in a recovery room for approximately 2 hours. A study team physician 
will determine if the patient meets criteria for discharge before the patient will be allowed to leave the suite.  
 
 
7. Data Collection 
7.1. Follow-Up 
Data will be examined from baseline (prior to each inhalation session) to immediate (1 hours post-inhalation) 
and sustained (24 hours post-inhalation) effects versus compared to each other and the placebo gas.  Additional 
follow-up visits will be scheduled approximately 1, 2, and 4 weeks following completion of each gas inhalation 
session (to assess for treatment efficacy and also duration of efficacy of nitrous oxide effects).   
 
7.2. Assessment of Treatment Efficacy 
The effects of the inhalation gases on behavioral and mood state will be assessed using standard scales used in 
clinical psychiatry:  
 
Hamilton Depression Rating Scale  21 items (HDRS-21) scale (Primary Outcome) 
 
Montgomery-Åsberg Depression Rating Scale (MADRS) 
 
Quick Inventory of Depressive Symptomology-Self Report (QIDS-SR) 
 
Clinical Administered Dissociative States Scale 28 item (CADSS-28) 
 
Brief Psychiatric Rating Scale  (BPRS) 
 
Profile of Mood States 2nd Edition (POMS-2) 
Patients will be assessed at all time points by one of the psychiatry study team members who will be blinded to 
the study group allocation.  


10 
 
Additionally, adequacy of the blind will be assessed by a Blinding Questionnaire given after completion of each 
inhalation session (see Section 6.3).  
 
7.3. Assessment of Treatment Safety 
Treatment safety will be monitored at several levels: adverse events (AE) related to (i) cardiovascular status; (ii) 
respiratory function; (iii) central nervous system; and (iv) psychiatric symptoms, particularly the presence of 
psychotic symptoms.  
(i) Cardiovascular, respiratory, and other AEs will be identified clinically by continuous monitoring during 
and after the treatment. Follow up recommendations will be given as needed, if necessary, by study 
personnel. 
 (ii) Psychiatric AEs, such as psychotic and dissociative symptoms, will be assessed via clinical interview as 
well as via standard scales before and after inhalation sessions. Emergence of psychosis will be identified 
by an increase in psychotic symptoms on the BPRS (for psychotic symptoms); suicidal ideation/intention 
will be assessed clinically and via the suicide questions on the QIDS-SR (question #3) and the HDRS-21 
and recommended for additional follow-up treatment per Psychiatry P.I. 
 
7.4. Sources of Research Material 
Data may be obtained from medical records and hospital charts, as well as from data collected at each study 
visit from the research assessments administered either by study personnel interview or by participant self-
report.  
 
8. Data and Safety Monitoring 
8.1. 
In general, the PI has developed a specific set of Standard Operating Procedures (SOPs) for clinical 
research. All individuals working under the PI are required to read and be familiar and compliant with the 
SOPs. The PI's SOPs are in part developed from and are compliant with the Institutional guidelines, including 
those for a) Interactions with the Washington University Human Subjects Review Committee, b) Informed 
Consent Development and Implementation, c) Subject Recruitment and Screening, d)  Subject Management 
While on Study, e) Adverse Event Reporting. 
 
8.2. 
The specific monitoring plan for this study is commensurate with the risks and the size and complexity 
of the investigations planned.  The PI will review all study data on an ongoing basis. The potential risks are 
attributable to the use of the gas mixtures or emotional discomfort while completing the 
assessments/questionnaires.   
 
 
 


11 
 
9. Data Analysis  
Major depressive disorder symptoms will be evaluated at baseline (pre-inhalation), at 1 and 24 hours, at one and 
two weeks, and one month post-inhalation. The primary MDD measure is the change in HDRS-21 scale at 2 and 
24 hours post-inhalation.  Later time points will assess for duration of sustained antidepressant effects (Aim 2).  
 
Secondary MDD measures taken at the same time points, will include the MADRS, the QIDS-SR, and the 
POMS 2 (immediate subjective mood change). At the same time points, euphoria, psychosis (BPRS), and 
disassociation (CADSS) will also be measured.  Outcomes will be analyzed with a repeated measures mixed 
effects linear model using restricted maximum likelihood estimation.  To adjust for the observed carryover 
effect, the model will include a randomization group term and a three-way interaction (treatment x time x 
randomization group). To compare response and remission rates (≥50% decrease, ≤7 on HDRS-21, 
respectively), an exact binomial test will be used (and corresponding odds ratios [ORs] calculated). Symmetry 
and spectral power will be assessed from EEG acquired during periods of eye closure or opening. 
 
Power Analysis 
Our original pilot study in TRMD patients found statistical significance with 20 subjects. Based on the 
assumption that the low N2O concentration (25%) is non-inferior to the high N2O concentration, 24 patients 
will be required with a power of 90% to show that the lower limit of a one-sided 95% confidence interval (or 
equivalently a 90% two-sided confidence interval) will be above the non-inferiority limit of -3 points on the 
HDRS with an expected SD of 2.5. 
 
10. Risk Assessment 
10.1. Nitrous Oxide 
The side effects of nitrous oxide are well known to all anesthesiologists. The dose (25% or 50%) and duration 
(1 hour) used in this protocol are considered extremely safe and of low risk. Similar doses and durations are 
used in everyday dental practice without vital sign monitoring or supervision by a resuscitation-trained 
physician. The gas inhalation will be administered under supervision of an attending-level anesthesiologist and 
according to standards set by the American Society of Anesthesiologists, which includes monitoring via: 3-lead 
ECG, pulsoximetry, non-invasive blood pressure, and end-tidal CO2. With this dose of nitrous oxide, sedation 
may occur but not general anesthesia or respiratory depression. Previous evidence shows that the inactivating 
effect of nitrous oxide on vitamin B12 is dose and duration-dependent and are minor when used as in this 
protocol. An increase in plasma homocysteine by +25% can be expected which is expected to revert to baseline 
within the next 24 hours. Minor side effects after nitrous oxide exposure such as nausea and vomiting may 
occur which are typically self-limited and short. If a patient develops moderate to severe nausea and vomiting, 
we may administer 4mg of ondansetron. After the inhalation treatment sessions, clinical and nursing staff will 


12 
 
continue to be present to monitor patients’ symptoms and state before discharging the patient. Blood pressure 
and heart rate are also checked at the 22-28 hour after inhalation session follow-up visit.  
 
10.2. Major depression and suicide risk 
Because this is a novel study of nitrous oxide in the treatment of treatment-resistant depression, a worsening of 
depression symptoms and an increased risk for suicide cannot a priori be excluded. After each treatment, an 
expert research team member and, if needed, psychiatrist will determine the suicide risk and if indicated, take 
the necessary precautions to mitigate this risk including recommendation of treatment, hospitalization and 
withdrawal of subject from the study to preserve safety. 
 
10.3. Clinical Interview and Assessment 
All clinical assessors have extensive experience in clinical psychiatric assessment and will make every effort to 
implement protocol procedures in a sensitive and supportive manner.  Subjects will be given these assessments 
in a quiet, private setting with breaks given as needed. Other measures to minimize risks include the careful 
assessment of each subject before the study, and close clinical scrutiny during all aspects of the study.   
 
 
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