A phase 2 trial of inhaled nitrous oxide for treatment-resistant major depression

This randomized, within-subjects, placebo-controlled, phase 2 clinical study (n=24) investigated the efficacy of nitrous oxide (25% air concentration) treatment for patients with depression and found that 25% nitrous oxide has comparable antidepressant efficacy to 50% nitrous oxide but with fewer adverse side effects.


Introduction: Nitrous oxide at 50% inhaled concentration has been shown to improve depressive symptoms in patients with treatment-resistant major depression (TRMD). Whether a lower concentration of 25% nitrous oxide provides similar efficacy and persistence of antidepressant effects while reducing the risk of adverse side effects is unknown.

Methods: In this phase 2 clinical trial (NCT03283670), 24 patients with severe TRMD were randomly assigned in a crossover fashion to three treatments consisting of a single 1-hour inhalation with (i) 50% nitrous oxide, (ii) 25% nitrous oxide, or (iii) placebo (air/oxygen). The primary outcome was the change on the Hamilton Depression Rating Scale (HDRS-21).

Results: Whereas nitrous oxide significantly improved depressive symptoms versus placebo (P = 0.01), there was no difference between 25 and 50% nitrous oxide (P = 0.58). The estimated differences between 25% and placebo were −0.75 points on the HDRS-21 at 2 hours (P = 0.73), −1.41 points at 24 hours (P = 0.52), −4.35 points at week 1 (P = 0.05), and −5.19 points at week 2 (P = 0.02), and the estimated differences between 50% and placebo were −0.87 points at 2 hours (P = 0.69), −1.93 points at 24 hours (P = 0.37), −2.44 points at week 1 (P = 0.25), and −7.00 points at week 2 (P = 0.001). Adverse events declined substantially with dose (P < 0.001).

Discussion: These results suggest that 25% nitrous oxide has comparable efficacy to 50% nitrous oxide in improving TRMD but with a markedly lower rate of adverse effects.”

Authors: Peter Nagele, Ben J. Palanca, Britt Gott, Frank Brown, Linda Barnes, Thomas Nguyen, Willa Xiong, Naji C. Salloum, Gemma D. Espejo, Christina N. Lessov-Schlaggar, Nisha Jain, Wayland W. L. Cheng, Helga Komen, Branden Yee, Jacob D. Bolzenius, Alvin Janski, Robert Gibbons, Charles F. Zorumski & Charles R. Conway



Treatment-resistant major depression is a severe form of major depressive disorder, and there are about 17 million adults with TRMD in the United States alone.

A proof-of-principle study demonstrated that nitrous oxide has rapid antidepressant effects in patients with TRMD. However, the study had two important limitations and used a high concentration of nitrous oxide (50%).

In this current trial, we sought to determine whether a lower concentration of nitrous oxide (25%) had comparable antidepressant efficacy in TRMD as 50% nitrous oxide.

Recruitment and characteristics of enrolled patients

28 patients were enrolled between November 2016 and October 2019. Twenty patients completed all three inhalation sessions, one patient withdrew after two sessions, three patients withdrew after one session, and four patients withdrew after screening but before any treatment.

Patients had sustained and refractory depressive illness, with a median HDRS-21 score of 20.5 and a median MADRS score of 30, indicative of severe TRMD.

Primary and secondary study outcomes

In the intention-to-treat analysis, nitrous oxide had a significant effect on the primary outcome (HDRS-21) compared to placebo over the course of 2 weeks. However, there was no significant difference between 25 and 50% nitrous oxide (P = 0.58).

At 2 hours, 0.81 points were scored, 1.67 points were scored at 24 hours, 3.35 points were scored at week 1 and 6.1 points were scored at week 2. The effects of the active treatment groups increased over time and a dose-response relation was found.

Results on the MADRS, QIDS, and POMS scales were similar for 50% and 25% nitrous oxide, but not for 25% nitrous oxide.

Patients experienced a clinically significant improvement in depressive symptoms over the course of treatment, with a median change of 11.0 points (IQR, 3.3 to 14.0 points; P 0.0001) after the 3-month study period.

Figure 3 shows that 11.1% of patients had a treatment response after placebo treatment, 33.3% had a treatment response after 25% nitrous oxide treatment, and 41.7% had a treatment response after 50% nitrous oxide treatment.

We assessed the quality of blinding by including a questionnaire after each inhalation session in which patients were asked whether they were receiving active treatment or placebo.

Safety and adverse effects of inhaled nitrous oxide

We observed a statistically significant difference in adverse events between treatments: 47 after 50% nitrous oxide, 11 after 25% nitrous oxide, and 6 after placebo.


In this randomized controlled phase 2 crossover trial, nitrous oxide provided rapid antidepressant efficacy in patients with severe TRMD. The antidepressant effects increased in magnitude over time, lasting up to 4 weeks in some patients, and demonstrated a high rate of response, remission, and symptom improvement.

Although most patients saw a marked improvement of their depressive symptoms after nitrous oxide inhalation, some patients had minimal or no improvement after nitrous oxide and placebo. Furthermore, some patients had a strong placebo response, which, in some instances, mirrored the response to nitrous oxide.

A study of 37 adult and geriatric placebo-controlled double-blind randomized trials of fluoxetine and venlafaxine found a separation of 2.55 HDRS-17 units between active treatment and placebo control at 6 weeks, and smaller effects were observed at 2 weeks.

This phase 2 clinical trial of 50% nitrous oxide for treatment-resistant MDD suggests that it may be reasonable to start treatment with a lower dose of 25% nitrous oxide and escalate to 50% nitrous oxide if a stronger treatment effect is desired.

25% nitrous oxide had a fourfold decrease in adverse events compared to 50% nitrous oxide. Adverse events were related to sedation, mild dissociative effects, and nausea and vomiting.

There are several limitations to this study, including the small sample size, short follow-up, and possibility of bias due to patients correctly guessing their treatment. However, a median reduction of 11 HDRS-21 points was observed for the entire 3-month treatment regimen, which involved two treatments with nitrous oxide.

Given the calming effects of nitrous oxide, it is extremely difficult to completely blind patients to nitrous oxide versus placebo. However, a small placebo effect was observed, and some patients changed the dosage or choice of their antidepressant medication, which may have influenced some study results.


A single-center, double-blind, randomized placebo-controlled crossover trial was conducted on patients with major depressive disorder. The study used N2O inhalation treatments and was approved by the Washington University in St. Louis Institutional Review Board.

Patient enrollment

Patients with unipolar MDD without psychosis were recruited from an existing database and the Volunteers for Health patient pool at Washington University School of Medicine. They were excluded if they had a history of substance dependence or abuse, or were unable to provide informed consent. Patients were instructed to continue their current standard-of-care MDD treatment and not to modify their antidepressant treatments during the 3-month course of the trial.

Study procedures

The 14 planned study visits included a screening visit, a clinical interview, a urine sample collection, a blood draw, and other psychiatric assessments to measure baseline depression severity.

Inhalation sessions

Patients underwent 1 hour of inhalational anesthesia with either placebo, 25% nitrous oxide in oxygen, or 50% nitrous oxide in oxygen. They were monitored during and after the treatment with continuous three-lead electrocardiography, pulse oximetry, noninvasive blood pressure, and end-tidal CO2 measurement.


Data were collected at baseline, 2 hours, 24 hours, 1 week, and 2 weeks after inhalation to assess efficacy and safety of the inhalation treatment on mood, dissociation, and the emergence of psychosis.

Statistical analysis

Intention-to-treat analysis was performed using mixed-effects linear regression models to accommodate correlation produced by the within-subject crossover design. The severity of depressive symptoms was compared between the 25% and 50% treatment groups and placebo over time, and a linear dose-response relation was also tested.

Study details

Compounds studied
Nitrous Oxide

Topics studied

Study characteristics
Placebo-Controlled Double-Blind Within-Subject Randomized