This study was a 12-week, randomized, double-blind, SPCD [28,29,30] trial of NSI-189 monotherapy for MDD (ClinicalTrials.gov registration number NCT02695472). The study screened 353 recurrent MDD patients and randomized 220 subjects from 12 US, non-academic sites over an approximate 9-month period. The detailed study schedule of activities is shown in Supplemental Table 1. Institutional review board-approved written informed consent was obtained from all study patients before any study procedures were conducted. Eligibility was assessed during a site screen visit, followed by a remote assessment involving the SAFER interview conducted by Massachusetts General Hospital Clinical Trials Network and Institute (MGH CTNI) clinicians [31, 32] and, finally, by a site baseline visit. Safety was monitored by an independent MGH CTNI clinician who served as medical monitor for the study (GIP); a data safety and monitoring board was not involved.

Patient inclusion and exclusion criteria were as follows:

Inclusion criteria: Patients were eligible for study participation if they were between the ages of 18–60 years, with current MDD of at least 8 weeks duration according to the fifth version of the Diagnostic and Statistical Manual for Mental Disorders (DSM-5), as diagnosed by the Structured Clinical Interview for the DSM-5 clinical trial version (SCID-5-CT) (http://scid5.org/info/) during the screen and remote assessment visits, and if they were scored at least 20 at screen, remote assessment, and baseline visits on the Montgomery-Asberg Depression Rating Scale (MADRS [33])

Exclusion criteria: The following patients were excluded from being randomized in the study: (1) pregnant or lactating women or women with a positive serum or urine pregnancy test administered at screening and baseline, (2) women of childbearing potential not on a medically acceptable form of birth control or who did not agree to continue such birth control for the duration of the study, (3) clinically significant history or evidence of cardiovascular, respiratory, hepatic, renal, gastrointestinal, endocrine, neurological, immunological, or other major disease as determined by the site investigator such that participation in the study would place the subject at increased risk for a serious adverse event, (4) lifetime history of mania, hypomania, or psychosis, (5) a primary psychiatric diagnosis currently other than MDD, (5) non-response to at least three antidepressant trials of adequate dose and duration during the current major depressive episode as defined by the MGH Antidepressant Treatment History Questionnaire (MGH-ATRQ; Fava, 2003 [34]) and administered both by site investigators and MGH CTNI raters remotely (meeting this criterion during either assessment was sufficient for exclusion), (6) subjects with significant suicidal ideation, (7) subjects with an alcohol or drug use disorder active within the past 12 months, or a positive urine drug screen for drugs of abuse at either screening or baseline, and (8) patients on an excluded medication (antidepressants, antipsychotics, buspirone, and lithium were excluded, while anticonvulsants, dopamine agonists, psychostimulants, modafinil, T3, benzodiazepines, zolpidem, zaleplon, eszopiclone, melatonin, and low-dose trazodone were allowed if stable in dose for at least 4 weeks prior to the screen visit).

Study procedures

Enrolled patients were randomized in a 1:1:3 fashion to receive fixed-dose treatment with NSI-189 40 mg daily during Stages 1 and 2, NSI-189 80 mg daily during Stages 1 and 2, or to receive placebo during Stage 1. The higher probability of randomization to placebo in Stage 1 is to generate a sufficient sample size for Stage 2 (since Stage 1 placebo non-responders comprise the entire Stage 2 efficacy sample). Hence, placebo-treated patients who completed Stage 1 and met the specific criteria for non-response (see below) were then re-randomized in a 1:1:1 fashion to receive either placebo, NSI-189 40 mg daily, or NSI-189 80 mg daily during Stage 2. Specifically, the criteria for re-randomization were as follows: (1) <50% reduction in MADRS scores from baseline during Stage 1 and (2) MADRS score >15 during the baseline visit of Stage 2. The sixth post-randomization visit served as the final visit for Stage 1, the re-randomization visit for placebo-treated subjects who completed Stage 1 and who met criteria the criteria listed above, and the baseline visit for Stage 2. The following scales were administered during the randomization and several (either 6 or 8) post-randomization visits: MADRS, the clinician-rated 17-item Hamilton depression rating scale (HAMD-17 [35]), clinical global impressions-severity and improvement (CGI-S/I [36]), the self-rated MGH Cognitive and Physical Functioning Questionnaire (MGH CPFQ [37]), and the self-rated Symptoms of Depression Questionnaire (SDQ [38]), which also includes the seven items of the CPFQ. The self-rated version of the quick inventory for depressive symptomatology (QIDS-SR [39]) was administered during the randomization, re-randomization, and study final visits (end of Stage 2).

Objective cognitive measures

In addition to the CPFQ, two objective cognition instruments were used in the study: Cogstate and CogScreen. Both were administered using a computer interface. The Cogstate battery consisted of the following tests: (1) Detection, (2) Identification, (3) One Card Learning, and (4) One Back. The CogScreen battery consisted of the following subtests: (1) Previous Number Alone, (2) Shifting Attention Test Arrow Direction, (3) Shifting Attention Test Arrow Color, (4) Shifting Attention Test Instruction, (5) Shifting Attention Test Discovery, (6) Symbol Digit Coding, and (7) Symbol Digit Coding Delayed Recall.

General statistical considerations

Efficacy analysis was performed on the full analysis set (FAS) who received at least one dose of study drug at the visit subsequent to randomization (or re-randomization in Stage 2), and had at least one post-randomization (or re-randomization in Stage 2) MADRS assessment. Safety evaluation was performed on all subjects randomized in the study who received at least one dose of study drug (safety dataset), separately for Stages 1 and 2.

The primary efficacy analysis used Stage 1 and 2 FAS using a mixed model repeated measures (MMRM). The effect within each treatment was measured as the change in the MADRS total score from baseline to the end of treatment, and was calculated by stage. An unstructured variance/covariance structure was used for the statistical modeling. The weighted restricted maximum likelihood (REML) estimate for differences between active and placebo groups was calculated for each active treatment group for each stage. In order to account for multiplicity due to the two pairwise comparisons of NSI-189 (80 and 40 mg) versus placebo, the sequentially rejective Hommel procedure [40] was applied to all overall p values with the exception of CogScreen and Cogstate, which were exploratory analyses. An analysis of covariance (ANCOVA) model was used to assess the sensitivity of the primary analysis of MADRS to statistical assumptions behind the MMRM. Responder and remitter analyses were performed stage wise using the logistic regression model to assess the robustness of the results. All analyses described for the primary efficacy endpoint were also applied for the HAMD17, SDQ, and MGH CPFQ. The CGI-S and CGI-I were analyzed using the FAS for each stage using a proportional odds logistic regression. As an exploratory analysis, the frequencies of CGI-I and CGI-S outcome were summarized by visit for all subjects who received the same treatment in both stages.

Safety analyses: All safety parameters were summarized separately for Stages 1 and 2 as well as overall, using the safety set. Differences in the incidence of treatment emergent adverse events (TEAEs) between treatment groups were presented using descriptive statistics. A summary of TEAEs was also presented by Medical Dictionary for Regulatory Activities system organ class and preferred term. Vital sign measurements, electrocardiogram results, laboratory assessments, and physical examination findings were presented using descriptive statistics.

Analysis of efficacy (SPCD): The SPCD test statistic is based on a weighted combination of the estimated treatment effects [41]. The treatment effects in Stage 1 and the variances of the estimated treatment effect were obtained from a linear mixed model. The treatment effects in Stage 2 and their variances were estimated in a similar way. Only data from Stage 1 placebo non-responders who were re-randomized were used to estimate Stage 2 treatment effects. The treatment estimates were weighted means of the estimated effects from the two stages: \(\widehat {\theta _j} = w\widehat \theta _{1,j} + \left( {1 - w} \right)\widehat \theta _{2,j}\), and the variance of the treatment estimate under the null hypothesis is \({\mathrm{Var}}( {\widehat {\theta _j}} ) = w^2{\mathrm{Var}}( {\widehat \theta _{1,j}} ) + ( {1 - w} )^2{\mathrm{Var}}( {\widehat \theta _{2,j}} )\). Here j = 1,2 denotes the 40 day and 80 mg/day groups. Note that \({\mathrm{Var}}( {\widehat \theta _{1,j}} ),\;j = 1,2\) is simply the square of the standard error estimates of the treatment effects given in the statistical output of the MMRM.

Estimated treatment effects and their variances were then combined into the SPCD test statistic:

$$T_j = \frac{{w\widehat \theta _{1,j} + \left( {1 - w} \right)\widehat \theta _{2,j}}}{{\sqrt {w^2{\mathrm{Var}}\left( {\widehat \theta _{1,j}} \right) + \left( {1 - w} \right)^2{\mathrm{Var}}\left( {\widehat \theta _{2,j}} \right)} }},\; j = 1,2.$$

T j is asymptotically standard normally distributed and therefore the p values were computed as p j = 2(1 − Φ(|T j |)) for j=1,2, there Φ(x) is the standard normal cumulative distribution function [41]. For this study, w = 0.5.

The 95% confidence interval for the overall treatment effect was calculated as:

$$ \left(\vphantom{\sum^{x}}\right.{w\hat \theta _{1,j} + \left( {1 - w} \right)\hat \theta _{2,j} - {\mathrm{Z}}_{0.025}}\\ \hskip 10pt \cdot \sqrt {w^2{\mathrm{Var}}\left( {\hat \theta _{1,j}} \right) + \left( {1 - w} \right)^2{\mathrm{Var}}\left( {\hat \theta _2} \right)} ,\\ w\hat \theta _{1,j} + \left( {1 - w} \right)\hat \theta _{2,j} + {\mathrm{Z}}_{0.025}\\ \hskip 10pt { \cdot \sqrt {w^2{\mathrm{Var}}\left( {\hat \theta _{1,j}} \right) + \left( {1 - w} \right)^2{\mathrm{Var}}\left( {\hat \theta _{2,j}} \right)} } \left.\vphantom{\sum^{x}}\right).$$

For each comparison of dose group versus placebo, the sum of the equally weighted stage-wise weighted REML difference estimates and the corresponding test statistics were used to summarize the estimates and perform inference integrating the data from the two stages.