Study design

All study procedures were approved by institution review boards at Yale and Connecticut Veteran Affairs Hospital, and all participants completed an informed consent process prior to enrollment (ClinicalTrials.gov: NCT02487485). A Data and Safety Monitoring Board (DSMB) oversaw the study protocol and monitored the study progress. The clinical trial included two study phases (I and II).

In phase I (September–December 2015), three participants received open-label oral rapamycin (a.k.a. sirolimus) followed 2 h later by open-label intravenous ketamine. Participants remained on the research unit for at least 10 h following the administration of rapamycin and were discharged upon clearance by the covering physician. The aim of phase I was to qualitatively assess the safety and feasibility of the co-administration of rapamycin and ketamine. The three participants tolerated the rapamycin + ketamine combination well, without any residual symptoms or unexpected adverse events. Therefore, the study proceeded to phase II.

In phase II (June 2016–February 2018), 23 participants were randomized to first receive either rapamycin or placebo, followed 2 h later by open-label ketamine (see CONSORT Diagram in Supplementary material). Phase II was a double-blind, placebo-controlled, cross-over design with at least 2 weeks between Infusion 1 (i.e., 1st treatment day) and Infusion 2 (i.e., 2nd treatment day). Randomization assignments were generated and assigned by the research pharmacist according to a block randomization with block size of six. Individuals, clinicians, investigators, and research staff were blinded to randomization assignments. Depression severity no less than 80% of baseline was required prior to proceeding with Infusion 2. If the participant did not meet this severity criteria, the infusion 2 was rescheduled until the following week. Participants who received placebo on Infusion 1 received rapamycin on Infusion 2, and vice-versa. Both study phases used an oral single-dose of 6 mg rapamycin in liquid form, diluted in orange juice to maintain the blinding, and ketamine 0.5 mg/kg intravenously infused over 40 min. Ketamine administration and monitoring was comparable to previous studies [1, 2, 30]. Preplanned interim analysis of the first six subjects confirmed that the rapamycin level is reaching therapeutic levels. Hence, the study continued randomization without rapamycin dose adjustments. Participants were assessed up to 2 weeks following each Infusion.

Study criteria

The study enrolled subjects between the age of 21 and 65 years, recruited through advertisement and referrals from outpatient clinics. Participants were (1) diagnosed with current major depressive episode, (2) had a history of non-response to at least one adequate antidepressant trial, (3) were unmedicated or on a stable antidepressant or psychotherapy for at least 4 weeks prior to randomization, then during the study, (4) had a MADRS ≥ 18 prior to randomization, (5) females were not pregnant or breastfeeding and were on a medically acceptable contraceptive method, (6) were able to read, write, and provide written informed consent, (7) did not have psychotic disorder or features, or current manic or mixed episodes, (8) did not have an unstable medical condition, (9) did not require prohibited medications (see Table S1 in Supplementary material), (10) did not have urine drug screen positive for cannabis, phencyclidine, cocaine, or barbiturates, (11) had no substance dependence within 3 months, (12) had no known sensitivity to rapamycin, ketamine, or heparin as reported by the subjects, and (13) had resting blood pressure higher than 85/55 and lower than 150/95 mmHg, and heart rate higher than 45/min and lower than 100/min.

Outcomes

Assessment measures included: (1) the Mini International Neuropsychiatric Interview (MINI) to determine the diagnosis, (2) Montgomery Åsberg Depression Rating Scale (MADRS) as primary outcome of depression severity, (3) Quick Inventory of Depressive Symptoms Self-Report (QIDS-SR) and Hamilton Anxiety Rating Scale (HAMA) as secondary measures of depression and anxiety severity, respectively, (4) Clinician Administered Dissociative States Scale (CADSS) and Positive and Negative Symptom Scale (PANSS), as safety measures of the psychotomimetic effects of ketamine, (5) rapamycin level immediately before starting ketamine and ~4 h later, (6) ketamine level before the end of each infusion and (7) high-sensitivity C-reactive protein (CRP) and erythrocyte sedimentation rate (ESR) prior to randomization to examine whether baseline inflammatory markers affect the antidepressant response to ketamine.

The study a priori primary outcome was MADRS. Response was defined as 50% improvement, and remission was defined as MADRS < 10 [31]. MADRS scores were measured immediately prior to rapamycin and placebo administration, and after starting ketamine infusion at 1, 2, 4 h, 3 days, 5 days, 1 week, and 2 weeks.

Statistics

Descriptive statistics (means, standard deviations, and frequencies) were calculated prior to statistical analysis. Data distributions were checked using normal probability plots. Outcome variables were analyzed using mixed models with fixed effects of treatment (rapamycin vs. placebo), time (appropriate time points during Infusions 1 and 2), the interaction between treatment and time, and order (placebo first vs. rapamycin first). The best-fitting variance–covariance structure for each model was selected based on the Schwartz’ Bayesian Information criterion. Interactions between order and the other factors were checked for significance but not included in the final models for parsimony. Similarly, the effects of the variables CRP and ESR (log-transformed) were checked for significance, but since these were non-significant, they were removed from the final models. Post-hoc tests were used to interpret significant effects in the models: comparisons of treatment conditions by time-point for significant rapamycin by time interactions, and pairwise comparisons of time points for significant main effects of time. Least-square means and standard errors by treatment and by time were used for visualization of results. The response and remission rates were compared between treatments using McNemar test. Effect size (Cohen’s d′) was calculated as the mean of the within-subject difference over its standard deviation. Correlation analyses explored the relationship between rapamycin level and improvement in depression severity. All tests are two-tailed with significance set at p < 0.05.

The sample size was targeted based on feasibility within the 3-year funding available for this discovery phase project. Initially, we aimed to randomize 30 subjects in 3 years. The targeted sample provides 80% power for detecting ketamine–rapamycin differences of moderate size (Cohen’s d′ = 0.55), assuming a two-tailed alpha = 0.05. However, we had a 1-year delay in starting randomization due to the addition of Phase 1 and the need for an investigational new drug exemption, both of which were requested by the institution review board. Thus, we were able to randomize a total of 23 patients in 2 years, 20 of them were included in the analysis. With 20 individuals, under the same assumptions, the detectable effect size is d′ = 0.68. Following randomization, one participant was excluded from the primary analysis due to receiving high dose hydrocortisone the night before randomization and the DSMB was informed accordingly. The decision to exclude the participant was made prior to compiling and unblinding the study data. However, for full transparency, a secondary analysis including this participant was conducted and reported in the Supplementary material. The results were found to be comparable to those of the primary analysis.