The colonization of bacteria in the gut begins at birth and significantly influences gut health throughout the lifespan [11]. In adults, the gut microbiome comprises more than 100 trillion commensal bacteria essential for the normal development and regulation of the immune system, central nervous system circuitry, and gastrointestinal (GI) functioning [12]. Under certain circumstances, environmental risk factors such as diet, lifestyle, stress, medications, and genetics can alter microbiota composition and deviate the homeostatic balance within the microbiome [13].

Previous studies have demonstrated that these alterations in microbiota composition owing to external factors may be mediated by the gut-brain axis (GBA). The GBA is a bidirectional, biochemical, and neural signaling pathway between the GI tract and the brain [14]. Although it has been long established that there is communication between the brain and the gut, only recently has there been a surge of interest in the GBA for its potential influence on depression and other symptoms of psychiatric illnesses [12]. Owing to the interaction between the gut and environmental risk factors of anxiety and depression, some interventions target the gut microbiome to aid in alleviating symptoms of anxiety and depression [15].

Although alterations of gut microbiota may play a role in the etiology of psychiatric illnesses, the pathophysiology of MDD and how the GBA directly affects mood is complex. One of the most well-known hypotheses for the pathophysiology of depression is the monoamine hypothesis. It predicts that a deficiency in the levels of dopamine, serotonin, and norepinephrine in the central nervous system may be a key factor in the underlying mechanisms of depression [16]. Other central mechanisms that are also hypothesized to be involved in the pathophysiology of MDD include inflammation of the immune system and microbial imbalance [7].

Several studies have documented that patients diagnosed with MDD often present with elevated levels of proinflammatory cytokines, such as interleukin-6, and decreased levels of anti-inflammatory cytokines, such as interleukin-10 [7,17,18]. This proinflammatory state observed in patients with depression reaffirms the neuroinflammation hypothesis, which suggests that an increase in systemic inflammation is likely to be involved in the pathophysiology of depression by decreasing the synthesis and production of monoamines such as serotonin, resulting in a reduction of serotonin and an increase in depressive behavior [19].

Recent literature documenting the relationship between gut microbiota and symptoms of psychiatric illness has uncovered interesting findings. Microbial balance in the gut is an integral part of gut health; however, when one experiences an imbalance, the integrity of the protective epithelial and mucosal gut barrier can be compromised. This compromise might result in increased intestinal permeability of the GI tract, referred to as leaky gut. Leaky gut has been associated with various conditions, such as irritable bowel syndrome, autoimmune disorders, and multiple psychiatric illnesses [15]. Maes et al [20] demonstrated that bacterial translocation from the gut, also known as leaky gut, could activate immune cells to evoke specific IgA and IgM responses. Interestingly, patients diagnosed with depression often exhibit the same elevated levels of IgA and IgM responses, which may be owing to the progressive amplification of the immune pathways and overall increased systematic inflammation [20].

Despite these relevant advances in recent literature, a complete understanding of microbial changes in the gut and their associated outcomes is still required. Previous studies have demonstrated that patients diagnosed with depression often display altered gut microbiota composition compared with healthy controls [12,21,22], which may result from the decreased abundance and lack of diversity among healthy gut microbiota [21]. In an attempt to address this problem, related research has turned to fecal microbiota transplantation (FMT) as a potential treatment option to improve symptoms of depression. FMT is a therapeutic approach that aims to repopulate the gut of an individual with fecal bacteria from a healthy donor via colonoscopy, nasogastric tube, or oral administration [23,24]. A potential advantage of discovering this link between symptoms of depression and microbiota in the gut is the greater accessibility and modifiability of microbiota compared with the human genome, giving microbial therapies a greater opportunity for aiding in the personalization of treatments for psychiatric illnesses [25]. Furthermore, given the adaptable nature of the microbiome, it may be a good representation of the individual’s history and could better explain the differences in the risk of illness, disease course, and response to treatment.

The 0.5-g MET-2 and placebo will be supplied as capsules for oral administration. Placebo capsules will be filled with cellulose and will be identical in appearance. The participants will be provided with a 4-week supply of either MET-2 or placebo capsules at the baseline visit and a 2-week supply at the week 4 visit. Loading or booster doses will be kept separate from daily maintenance doses.

The 0.5-g MET-2 capsules are produced under conditions compatible with good manufacturing practices at the University of Guelph and are shipped at room temperature and sealed under anaerobic conditions. All capsules are to be stored at room temperature.

Participants who meet the inclusion criteria and pass screening will be scheduled to start treatment at baseline. At the baseline visit, all participants will receive a 10-capsule orally administered loading dose of 5 g of either MET-2 or placebo on both days 1 and 2 (Table S2 in Multimedia Appendix 1). The participant will consume the initial loading dose in the clinic and receive the second loading dose to be taken at home on day 2. The participant will also remain at the clinic for 30 minutes after receiving the first loading dose to ensure there are no adverse reactions to the medication. A 3-capsule maintenance dose containing 1.5 g of MET-2 or placebo will be administered for the remainder of the 2-week interval from 3 to 14 days. At the week 2 visit, participants will be asked to take a 10-capsule booster dose on days 15 and 16. The 3-capsule maintenance dose will be administered for the remainder of the study for a total of 6 weeks of treatment.

If a participant is a nonresponder (a participant whose MADRS total score is not reduced by at least 50% from baseline by week 4 visit), an additional 10-capsule booster dose is to be administered at week 4 for 2 days (days 29 and 30) followed by a maintenance dose of 3 capsules for the remainder of the study (Table S2 in Multimedia Appendix 1). Responders (participants whose MADRS score is reduced by ≥50%) will not receive a booster dose at week 4. Participants will not take the maintenance dose during the loading or booster dose periods.

Compliance with treatment will be assessed by reviewing the participant’s personal logs and charts while documenting any missed and unused treatment material during the 6 weeks of intervention.

At the first endpoint, we will assess changes in symptoms of depression from baseline to week 6 as measured by MADRS scores.

The secondary endpoints are:

Mood will primarily be assessed using the MADRS, a 10-item clinician-rated questionnaire used to evaluate the severity of depression.

Anxiety will primarily be assessed using the HAM-A, a clinician-rated questionnaire that measures the severity of anxiety symptoms. The GAD-7, a 7-item questionnaire, will be used to measure self-reported anxiety symptoms and severity. The CGI scale is a 2-item clinician-rated scale that will be used to assess the severity of illness and improvement of symptomology over time. The SHAPS is a 14-item self-rated instrument used to assess the presence of anhedonia. The QIDS-SR16 is a 16-item questionnaire that measures self-reported depressive symptom severity. The Pittsburgh Sleep Quality Index is a 19-item self-report questionnaire used to assess sleep quality and disturbances. Early life stress will be assessed using the Childhood Experience of Care and Abuse questionnaire to determine the effects of maltreatment and upbringing on treatment response and MDD biomarkers.

Participants will also use a personal log to track any newly emerging GI symptoms since the beginning of treatment to assess their tolerability to treatment and keep track of their mood symptoms and sleep. Tolerability and GI effects will be assessed using the Toronto Side Effects Scale and the GI Symptom Rating Scale during all treatment-related visits.

Stool samples will be collected and analyzed using 16S ribosomal RNA sequencing at the University of Guelph. These data will be used to assess the relative abundance of microbial species, level of engraftment of MET-2 species, and α and β diversity of the gut microbiome. Urine samples will be collected to analyze soluble metabolites at the University of Guelph, whereas blood samples will be collected for clinical chemistry, hematology, and additional biomarker testing for safety and discovery purposes at Life Labs and Queen’s University, respectively. Statistical analyses will be performed to assess the relationship between immune biomarkers and clinical symptom improvement. Additional safety blood samples may be required in the event of abnormal values or analysis failure. The blood samples collected for safety laboratory analysis will be destroyed after the analyses have been completed.

The specific parameters being assessed are presented in Table S3 in Multimedia Appendix 1, whereas collection time points can be found in Table S1 in Multimedia Appendix 1.

The primary safety analysis for this study will include the total number of treatment-emergent adverse events (TEAEs) while also categorizing TEAEs by causality, severity, and seriousness assessments made by the investigator by comparing study drug exposure to placebo.

Trends in safety will also be evaluated for the following assessments: physical examinations, vital signs, laboratory results (hematology, lipid levels, and serum chemistry), pregnancy tests, and discontinuations because of adverse events (AEs).

Vital signs, including temperature, blood pressure (systolic and diastolic), pulse, and body weight (using a calibrated weight scale), will be measured at screening, baseline, week 4, and week 8. Blood pressure will be measured with a cuff size appropriate to the participant after the participant has been sitting for 5 minutes.

A complete physical examination will be conducted at the baseline visit. The physical examination will include general appearance, skin, head, ears, eyes, nose, throat, lungs, cardiovascular, abdomen, musculoskeletal and extremities, lymph nodes, and neurological. Other body and organ systems may be examined if clinically relevant.

A pregnancy test will be performed at baseline. If pregnancy is discovered in a female participant enrolled in the study before the end of dosing, the study drug will be permanently discontinued, and an end-of-treatment visit will be scheduled. If the pregnancy is discovered in a female participant enrolled in the study after the end of dosing, the participant will continue in the study per protocol. If pregnancy occurs in a male participant’s partner at any time during the study, the pregnancy is to also be reported and followed.

An AE is any untoward medical occurrence in a participant administered an investigational treatment, which does not necessarily have a causal relationship with this treatment. An AE can therefore be any unfavorable and unintended sign or symptom temporally associated with the use of the investigational treatment, regardless of whether it is related to the treatment. Each participant will be carefully monitored for the development of AEs during the safety reporting period. Both the frequency and severity of AEs will be collected at the time of consent throughout the study until the 2-week follow-up. AEs will be assessed and recorded at all in-hospital visits or through the phone using questionnaires and probing via discussion; these AEs will be categorized by frequency, severity, and causality [7]. Any clinically relevant abnormalities will be noted by the investigator during the follow-up interviews. All AEs will be recorded in an AE log for each participant. Any instances that lead to serious AEs (SAEs) and any untoward medical occurrences at any dose will be reported to the research ethics board (REB).

A TEAE is an AE that begins during the treatment or worsens a pre-existing medical condition (eg, worsening diarrhea). The treatment period is the period during which a participant receives the investigational treatment.

An SAE is a life-threatening adverse medical event that results in death, requires inpatient hospitalization or prolongation of existing hospitalization, results in persistent or significant disability or incapacity, and results in a congenital anomaly or congenital disability [7]. The study site will document all SAEs that occur (whether related to the study drug) and report them to the sponsor within 24 hours of the site’s first knowledge of the event. The collection period for all SAEs will begin after informed consent is obtained and will end after the procedures for the final study visit have been completed.

All SAEs will be followed until resolution. SAEs that remain ongoing past the participant’s last protocol-specified follow-up visit will be evaluated by the principal investigator and NuBiyota LLC. If both parties agree that the participant’s condition is unlikely to resolve, the principal investigator will determine the follow-up requirement.

A suspected unexpected serious adverse reaction (SUSAR) is an SAE that occurs in a participant, the nature or severity of which is not expected per the applicable product information (ie, the investigator’s brochure).

The sponsor and investigator will report any SUSARs concerning the drug that has occurred in the study to regulatory authorities:

The sample size for this clinical investigation was calculated based on the results from the pilot study and the change from baseline to week 8 of the first 13 participants for the MADRS (primary endpoint) and GAD-7 scores (first secondary endpoint). The change from baseline to week 8 was used for the two endpoints, mirroring the time frame for the evaluation of the primary and first secondary endpoints for this clinical investigation. Two series of estimates were generated: change from baseline for the participants with scores at baseline and week 8, and a second imputed data set for participants who did not have a week 8 value using the last observation carried forward. Although the final analysis from this study will use multiple imputation to address missing data, using the last recorded observation results in a higher SD and is considered a reasonable approach to derive a range of estimates. Estimates from the pilot investigation are presented in Table S4 in Multimedia Appendix 1.

For estimation purposes, it is assumed that the placebo participants will have a response that is 50% of the active treatment group, and the SD will be the same between the two treatment groups. The randomization will be in a 1:1 ratio (active:placebo), and the type 1 error rate will be 5%. The resulting sample sizes for 80%, 85%, and 90% power are presented in Table S5 in Multimedia Appendix 1.

If the true mean difference between the active and placebo groups is the midpoint between the observed and imputed results, a minimum target sample size of 50 participants will be required for the primary endpoint (MADRS: change at 8 weeks) and 79 participants (GAD-7: change at 8 weeks) for the other endpoints. On the basis of these estimates, the maximum number of participants to be enrolled in this clinical investigation is 80, and the target sample size is 60 participants. An interim assessment will be performed after 30 participants have completed the 8-week evaluation or withdrawn prematurely.

This study will be conducted in compliance with the current International Council for Harmonization of Technical Requirements for Pharmaceuticals for Human Use (ICH) Good Clinical Practice (GCP) guidelines. The sponsor shall ensure that all sites have the necessary standard operating procedures to ensure that the study is conducted and data are generated, documented, and reported in compliance with the protocol, ICH GCP, and applicable regulatory requirements. The investigator may not deviate from the protocol without a formal protocol amendment being established and approved by an appropriate competent authority and REB, except when necessary, to eliminate immediate hazards to the participant or when the changes involve only logistical or administrative aspects of the study. Any deviations may result in the participant being withdrawn from the study and render the participant nonevaluable. The sponsor is responsible for the distribution of protocol amendments to the principal investigators and those concerned with the conduct of the study. The principal investigator is responsible for the distribution of all amendments to the REB and all the staff concerned at their center.

All monitoring visits will be conducted by PhaseAdvance, a contract research organization appointed by NuBiyota LLC, to ensure compliance with the ICH guidelines for GCP (E6). The study monitors will conduct an initiation site visit to the institution to review the protocol and its requirements with the investigators, inspect the drug storage area, and fully inform the investigator of their responsibilities and the procedures for assuring adequate and correct documentation. During the study, the monitor will make regular site visits to review protocol compliance and individual participants’ medical records and ensure that the study is being conducted according to pertinent regulatory requirements. The review of medical records will be conducted in a manner that ensures confidentiality is maintained. No information in these records regarding the identity of the participants will leave the study center. Sponsor monitoring standards require full verification of the presence of the signed informed consent form (ICF), adherence to the inclusion and exclusion criteria, documentation of SAEs, and recording primary efficacy and safety variables. The clinical research associate will review source data compared with the case report forms (CRFs) and verify source data according to the study-specific monitoring plan. The study design, frequency of participant visits, and site enrollment rate will determine the frequency of monitoring visits. Upon study completion, the clinical research associate will visit the site to conduct a study termination visit, which will include the collection of any outstanding documentation.

To obtain and document informed consent, the investigator will comply with the applicable regulatory requirements and adhere to ICH GCP and the ethical principles that have their origin in the Declaration of Helsinki.

Informed consent shall be documented using a written consent form approved by the REB. The ICF must be signed and dated by the participant and investigator (or designated research professional) before protocol-specific procedures (screening or treatment) are performed. A consent form template will be provided by the sponsor and adapted by the investigator to meet the center, state, and country ethical guidelines, as appropriate.

Participants will be given a copy of the fully executed consent form, and the original will be maintained with the participant’s records.

This study will not commence until review and approval of the study protocol within each site and ICF has been obtained by local REBs. Any amendments to the study will be submitted concurrently to all REBs for review and approval unless they are required to eliminate immediate hazards to study participants.

Clinical data will be collected by the study coordinator as a source document and transcribed into REDCap (Research Electronic Data Capture), a web-based data collection software. REDCap will only contain the participant’s ID but no personal data.

Essential documents, as defined by ICH GCP, include the signed protocol and any amendments, copies of the completed CRFs (for site archiving, digital versions of e-CRF data for specific participants will be provided), signed ICFs, hospital records and other source documents, REB approvals and all related correspondence including approved documents, drug accountability records, study correspondence, and a list of participants’ names and addresses. Essential documents should be retained for at least 5 years.