Pharmacology and medical properties of cannabis

Cannabinoids, found in the hemp plant Cannabis sativa, have been recognised for centuries for their analgesic, anticonvulsant, bronchodilatory, sedative, hypnotic and antispasmodic properties.22 23 Their biological activity is conferred by cannabinoid receptors CB1 and CB2 through activation of heterodimeric G-proteins that function as signalling and regulatory proteins to operate or modulate intracellular signalling pathways.24 25 While CB1 receptors are expressed predominantly in the central nervous system, they are also found in the lung, liver and kidneys. The endocannabinoid system also plays a key role in the gastrointestinal tract’s neural and molecular control mechanisms. Indeed, the endocannabinoid system plays a role in normal physiological functions of the gastrointestinal tract including motility, gut–brain mediated fat intake, hunger signalling, inflammation and gut permeability.26 Furthermore, there is some evidence for and interactions between the endocannaboid system and the gut microbiota.26 In lean mice who received 4 weeks of a cannabinoid-receptor agonist HU-210, plasma lipopolysaccharides (LPS) were significantly increased.26 27 When obese mice with disrupted gastrointestinal mucosa were treated with rimonabant, an inverse CB1 agonist, for nearly 2 weeks, reductions in plasma LPS were observed.26 28 Furthermore, improvements in localisation of tight junction proteins, occluding and zonula occludens-1 were measured, suggestive of improvement in endothelial barrier function.26 28

The presence of cannabinoid receptors in the central nervous system accounts for the psychoactive effects of cannabis.24 25 In contrast, CB2 receptors are abundant on immune cells including T and B cells, natural killer cells, monocytes and neutrophils as well as the liver.24 29 Two primary active constituents found in hemp plants include Δ9-tetrahydrocannabinol (THC) and cannabidiol (CBD). Δ9-THC is a partial agonist at both CB1 and CB2,3 30 31 while CBD is thought to mediate its effects through a variety of signalling mechanisms and act as a negative allosteric modulator at CB1.32 33

Cannabis is perhaps best known for its effect on stimulating appetite via CB1 receptor activity and was historically used for this purpose in AIDS wasting syndrome. A variety of randomised controlled trials have shown that smoked cannabis is efficacious in the management of chronic pain, including in painful HIV-associated sensory neuropathy.34–36 In animal models, Δ9-THC prevents atherosclerosis by inhibiting macrophage migration into atheromas through CB2 activation.37 38 Endocannabinoids may also play a role in liver disease, where CB1 receptors are present in endothelial cells and hepatocytes and CB2 receptors are distributed in Kupffer cells.39 40 CB1 receptors have an important role in non-alcoholic fatty liver disease and alcoholic liver disease, while anti-inflammatory action of CB2 receptors can be useful in inflammatory liver disease. CB2 receptors show antifibrinogenic properties and administration of its agonists in fibrotic rats resulted in improvement in liver fibrosis, decreased inflammation and increased apoptosis of hepatic myofibroblasts.40–44

In vitro studies

Cannabinoids have been shown to inhibit productive HIV infection in primary human T cells, and a CB2 antagonist blocked this effect.45 Interference with the signal transduction of chemokine receptor CXCR4 is thought to lead to reduced F-actin accumulation. This in turn prevents movement of viral preintegration complexes to the nucleus. It has also been postulated that CB2 agonists may inhibit T cell activation induced by anti-CD3/anti-CD28.45 Along with reduced HIV production, immunological effects of cannabinoids include induction of immunosuppressive cytokines including interleukin (IL)-10, tumour growth factor (TGF)-β and inhibition of IL-2, which stimulates T cell division and expansion. They have also been shown to decrease adhesion capacity of leukocytes to extravasate into sites of inflammation.46–48 Apoptosis and induction of Treg may potentially add to the pathways of cannabinoid-mediated immunosuppression.49

Animal studies

Mice treated with Δ9-THC experience persistent inhibition of IL-2 production even 7 days after treatment.50 Rhesus macaques who were administered Δ9-THC for 28 days prior to simian immunodeficiency virus (SIV) inoculation had reduced mortality and reduced SIV viral load in the cerebrospinal fluid and plasma. In another study involving macaques infected with SIV for 17 months of chronic THC administration,51 Δ9-THC resulted in trends towards a decreased viral load. The lack of statistically significance in some parameters can be attributed to the small number of animals in the vehicle versus the Δ9-THC groups (n=4).30

Human studies

To date, seven randomised controlled studies have examined the medicinal use of cannabis for reducing morbidity and mortality in people living with HIV.52 Interventions included smoked marijuana or hashish, ingested marijuana or hashish or ingested THC (dronabinol or other synthetic cannabinoid). Studies ranged from 21 to 84 days. Primary outcomes included HIV-related mortality (all cause) and morbidity (including type and duration of episodes of opportunistic infections, malignancies, incidence of AIDS and hospital admissions). Secondary outcomes included change in appetite, nausea, mood, pain, quality of life, body composition, haematological and nutritional markers, cognitive function, respiratory function and effect on pharmacokinetics of antiretrovirals and development of dependence or adverse sociological effects. The use of cannabis posed challenges for blinding due to the psychoactive effects.52

Only one study involving 62 patients examined the effects of cannabinoids on immune function and parameters associated with HIV infection. Participants in this study were assigned to either a 3.95% THC marijuana cigarette, a 2.5 mg dronabinol capsule or a place capsule three times per day for 21 days. For the marijuana group, there was a statistically significant increase in CD4 counts from baseline versus placebo, and for the dronabinol group, there was a trend towards statistical significance. Neither CD4 nor CD8 cell counts were adversely affected, and the pharmacokinetic component of the study did not reveal any clinically significant interactions that would require dose adjustments of protease inhibitors (PIs).22 Adverse effects across studies included concentration difficulties, fatigue, sleepiness or sedation, increased duration of sleep, reduced salivation and thirst that improved on discontinuation. The authors of a systematic review on the use of cannabis for reducing morbidity and mortality in HIV concluded that: (1) evidence for substantial effects on morbidity and mortality is currently limited and (2) evidence for safety and efficacy of cannabis is lacking. Studies have been of short duration, in small numbers of patients and have focused on short-term measures of efficacy.52 Furthermore, no study examined the effects of cannabinoids on inflammatory markers or HIV reservoir markers through a randomised trial.

More recently, Manuzak et al published an observational study assessing the effect of cannabis use on peripheral immune cell frequency, activation and function in 198 people living with HIV.53 Individuals were grouped into heavy, medium or occasional cannabis users or non-cannabis users as determined by the quantify cannabis metabolite 11-nor-carboxy-tetrahydrocannabinol detected in plasma by mass spectrometry. They found that persons with heavy cannabis use had lower frequencies of HLA-DR+CD38+CD4+ and CD8+ T cell frequencies compared with people who did not consume cannabis.53 Furthermore, heavy cannabis use was associated with decreased frequencies of proinflammatory intermediate (CD14++CD16+) and non-classical (CD14+CD16+) monocyte subsets.53 They also documented a reduction in antigen-producing cells secreting proinflammatory cytokines IL-23 and tumour necrosis factor-α.53 Rizzo et al also demonstrated that levels of circulating CD16 monocytes and interferon-gamma-induced protein (IP)-10 from people living with HIV who either were or were not cannabis users.54 Lower levels of CD16+ monocytes and plasma IP-10 were found in cannabis users compared with non-cannabis users.54 However, this study did not quantify the level of cannabis exposure in the two groups. Although these studies demonstrated favourable associations between inflammation and cannabis use, it must be borne in mind that both of these studies were observational only. As these studies were not randomised controlled trials, it is possible that people living with HIV who used cannabis in these studies differed in other significant ways from PLWHIV who did not use cannabis.

Study rationale

Cannabis may hold many potential therapeutic benefits for people living with HIV due to its promising anti-inflammatory and antifibrotic effects. Before adequately powered interventional studies can be designed to study cannabis as a therapy for specific conditions associated with chronic inflammation and fibrosis, a key first step will be to demonstrate that cannabinoid use in a clinical trial is feasible and that they have a favourable safety and tolerability profile. As such, we propose a proof-of-concept pilot study to examine the feasibility, safety and tolerability of cannabinoid oils consumed orally in people living with HIV on effective ART. As a secondary objective, we will examine the effect of cannabinoid oils on immune profiles, including levels inflammatory markers associated with HIV disease progression and frequencies of activated and senescent CD4 and CD8 T cells. Frequencies of regulatory T cells and various subsets of Th17 will also be assessed. Finally, an exploratory objective will be to study the effect of cannabinoid oils on markers of HIV persistence and the composition of the gastrointestinal microbiome.

We propose to use combination therapy of THC:CBD oils in capsule format (TN-TC11LM and TN-TC19LM capsules) ingested orally to examine these outcomes. Although research to date involving HIV/SIV has examined THC, data from in vitro, animal and human studies suggests that CBD has favourable anti-inflammatory effects, and the combination of CBD with THC increases tolerability.55–59

Study design

This is a randomised, open-label, interventional study (the Canadian HIV Trials Network (CTN) number PT028) whereby capsules containing CBD:THC oils are consumed for 12 weeks to assess safety and tolerability. Their ability to reduce immune activation (as determined by percentage of activated CD8+CD38+HLA-DR+ T cells), size of the peripheral HIV reservoir and change in gastrointestinal microbiome composition will also be examined. Participants will continue to take their ART treatments as prescribed throughout the study.

Setting

Recruitment of participants will occur at the Chronic Viral Illness Service (CVIS), Royal Victoria Hospital (Glen campus) of the McGill University Health Centre (MUHC), the largest academic HIV clinic in Canada.

Recruitment and enrolment

Study staff at the CVIS will conduct chart reviews of prospective people living with HIV ahead of their clinic visits to determine which persons have had suppressed viral load for at least 3 years on ART. The patient chart will be flagged, and if the treating HIV physician believes the person to be suitable for the study, the physician or study staff will approach potential trial participants at their clinic visit. The trial staff will inform the patients about the trial and invite him or her for eligibility screening and possible trial enrolment. Participant eligibility will be documented and written informed consent obtained for eligible patients by the study coordinator. The study coordinator will systematically document all individuals who have been approached for the study in addition to reasons for acceptance and refusal to participate in the study. Individuals who wish to discuss their participation in the study with their treating physician and/or family and friends will have the opportunity to do and may enrol their next scheduled clinic visit. Following enrolment, participants will be followed during the study by the principal investigator and study coordinator at the CVIS.

Inclusion criteria

Eligible participants must meet the following criteria within 4 weeks prior to beginning the cannabinoid capsules to be considered eligible for study entry: (1) documented HIV infection by western blot, enzyme immunoassay or viral load assay; (2) aged 18 years or older; (3) viral load <40 copies/mL for at least the last 3 years (maximum two blips <500 copies/mL allowed); (4) no cannabinoid use for at least 1 month prior to enrolment with negative baseline cannabinoid urine screen; and (5) able to communicate in either English for French. We will attempt to enrol equal proportions of men who have sex with men (MSM) and heterosexual individuals.

Exclusion criteria

Individuals who meet any of the following criteria will be ineligible to participate: (1) using cannabinoid-containing products outside of the study or within 4 weeks of study commencement; (2) pregnant, breast feeding or planning to become pregnant during the course of the study; (3) enrolled in a separate study involving administration of medication, vitamin, supplement or herbal product; (4) active intravenous drug use; (5) active substance dependence; (6) prior history of hypersensitivity to cannabis or cannabis-containing products; (7) known or suspected allergy to sunflower lecithin oil; (8) active opportunistic infection or malignant condition; (9) unintentional weight loss of 10% or more of body weight in the last 6 months; (10) unstable angina or acute cardiac event in the past year; (11) active psychiatric disorder or history of psychiatric depression (other than mild depression or anxiety); (12) on antipsychotic medication; (13) known or suspected family history of schizophrenia or severe personality disorder; (14) serious cardiovascular disease such as ischaemic heart disease, arrhythmias, poorly controlled hypertension or severe heart failure; (15) anaemia (haemoglobin <100 g/L); (16) active liver disease or unexplained persistent elevations of serum transaminases; (17) coinfection with hepatitis B or C (positive Hepatitis B Surface antigen (HBsAg) or positive anti-HBc (Hepatitis B Core) antibodies with a detectable Hepatitis B virus (HBV) DNA viral load or positive anti-HCV antibodies with a detectable Hepatitis C virus (HCV) RNA viral load); (18) alanine aminotransferase (ALT) or aspartate aminotransferase (AST) or alkaline phosphatase (ALP) >2.5 × upper limit of normal; (19) opportunistic infection in the last month as determined by the treating physician; (20) renal dysfunction; (21) unstable psychological or psychiatric condition as determined by the treating physician; (22) holding employment that requires operation of heavy machinery or which requires undergoing drug screening (ie, pilot or police officer); and (23) concurrent use within the past 8 weeks of anabolic hormones, prednisone, IL-2 or other agents known to alter immune function.

The investigators will review potential participants’ current medication lists at the screening visit. If any concomitant therapy interacts with the study medication, and if this therapy cannot be substituted, that participant will not be eligible to enrol in the study. Although some HIV antiretroviral drugs/pharmacokinetic ‘boosters’ are metabolised by the CYP 1A2 and CYP3A4 (eg, ritonavir and cobicistat-boosted PIs), suggesting that a drug interaction may occur, in real practice at the CVIS, no clinically significant drug interactions have been observed in patients reporting heavy cannabis use. Therefore, individuals will not be precluded to participate based on their specific antiretroviral regimen.

Study intervention

The study medications are TN-TC11LM and TN-TC19LM capsules that contain THC:CBD in a ratio of 1:1 (2.5 mg/2.5 mg) and 1:9 (5 mg/45 mg), respectively. These study drugs are being provided by Tilray, and the active pharmaceutical ingredients are extracted from the cannabis plant and purified according to pharmaceutical standards (>98%). Participants will be advised to gradually increase the number of capsules they take based on the suggested titration scheme presented in tables 1 and 2, until a daily maximum is reached. These maximum amounts are composed of 10 capsules of TN-TC11LM (25 mg THC/25 mg CBD total per day) or 3 capsules of TN-TC19LM (15 mg THC: 135 mg CBD for TN-CT19L) per day. These doses were selected as in a clinical trial for neuropathic pain; doses equivalent to 2.5 mg of THC were well tolerated.34 More recently, among patients (ages 2–55 years) with the Lennox-Gastaut syndrome, CBD at a dose of 10 or 20 mg per kg per day resulted in greater reductions in the frequency of drop seizures than placebo and was well tolerated overall other than for an increase liver aminotransferase concentrations.60 Due to person-to-person variability in the ability to metabolise and tolerate cannabinoids,61 we have opted for patients to titrate their dose of medication to a range where they are comfortable as the titration method of dosing has proven successful in other clinical trials involving cannabinoids.34

Randomisation

After eligibility is confirmed and written informed consent obtained, participants will be randomised to either TN-TC11LM (group 1) or TN-TC19LM (group 2) capsules, which contain THC:CBD in a ratio of 1:1 and 1:9, respectively. Prior to study commencement, a statistician unassociated with the study will develop a randomisation scheme using SAS and input into a password-protected web-based randomisation system. Variable block sizes of 2 and 4 will be used. Participants will be assigned to either group 1 versus group 2 based on the predesignated allocation code. As this is an unblinded study, participants and study staff will be aware of the group to which the participant has been randomised. A computerised audit trail will track date and time of allocation, patient study identification number and treatment allocation. The randomisation group will be recorded in the study log, which will be accessible to the sponsor/medical manager and study coordinator.

Measurements

At the screening visit, clinical information will be collected from each participant including age, ethnicity, sexual orientation, list of current medications, dosage, date of treatment initiation, any antimicrobials taken in the previous 6 months, psychiatric disorders, duration of HIV infection, current ART regimen and duration of ART regimen months, ART history, CD4+ T cells count within the past 3 months, nadir CD4+ T cells count, CD4/CD8 ratio, duration of plasma viral load suppression and any pre-existing medical conditions, signs or symptoms. Information will also be collected on whether the individual consumed cannabis in the past, the form(s) in which it was consumed, frequency of use and reasons for use.

Clinical parameters

Scheduled visits will occur to monitor safety and tolerability, as per the visit schedule depicted in table 3. Visits will include physical exam with vital signs, weight, occurrence of adverse events (AEs; and concomitant medications) and the presence of common symptoms associated with cannabinoids including dizziness, nausea, headaches, appetite or mood changes. At visits, blood for some or all of the following will be collected: CD4+ T cells count, CD8+ T cells, CD4/CD8 ratio, plasma viral load, complete blood count, AST, ALT, ALP, total bilirubin, urea, creatinine and blood glucose, T cell activation and inflammatory markers and testing for syphilis if the participant tested positive during the 4 weeks prior to beginning consuming the study capsules. A stool sample for analysis of the bacterial and fungal microbiome assessment will also be collected prior to beginning the study capsules. Participants will be enrolled in the study for up to 15 weeks but will consume capsules for a period of 12 weeks. Participants will undergo screening tests and eligibility assessment within 4 weeks prior to initiating study capsules. Participants will then undergo assessments after the first week of capsule consumption and every 2 weeks thereafter. A second stool sample for bacterial and fungal microbiome analysis will be collected during the final week of capsule consumption. The final visit will occur 2 weeks after study drug cessation.

Medication adherence

Participants will be asked to keep a log book in which they enter the number of TN-TC11LM or TN-TC19LM capsules consumed, the time, as well any AEs they noted and the timing of these AEs relative to capsule intake. Furthermore, individuals will be asked to record whether they took their ART that day or whether any doses were missed. Participants will be asked to bring their log books with them to study visits and the coordinator will photocopy this information.

Quality of life and mood assessment

Questionnaires measuring quality of life (WHO Quality of Life HIV-BEF (WHOQOLHIV-BREF) and the EuroQol-5Dimension (EQ-5D)) and mood (Profile of Mood States (POMS) will be administered at baseline, midway through the study and at the end of the study, as outlined in table 3. WHOQOLHIV-BREF consists of 31 items that measure the following domains: physical health, psychological health, social relationships and environment. It is a shorter version of the original instrument (WHOQOL) and is more convenient for use in clinical trials, taking approximately 10 min to complete. EQ-5D is a descriptive questionnaire examining five dimensions: (1) mobility, (2) self-care, (3) usual activities, (4) pain/discomfort and (5) anxiety/depression. Each dimension has five levels: no problem, slight problems, moderate problems, severe problems and extreme problems. The participant indicates the state of his his or her health by ticking the box most appropriate to the statement in each of the five dimensions. This decision results in a one-digit number that expresses the level selected for that dimension. The digits for the five dimensions can be combined into a five-digit number that describes the participant’s health state. It takes about 10 min to complete. The POMS questionnaire measures the following six factors: (1) tension-anxiety, (2) anger-hostility, (3) fatigue-inertia, (4) depression-dejection, (5) vigour-activity and (6) confusion-bewilderment. It is very sensitive to non-clinical changes in mood states and takes approximately 5 min to complete. These questionnaires will be administered by a trained research coordinator.

Research hypothesis

THC:CBD oils consumed orally—as TN-TC11LM and TN-TC19LM oral capsules—will be safe and well tolerated in PLWHIV. They will also be associated with a reduction in markers of inflammation, reduction in frequency of activated T cells and reduction in HIV reservoir size.

Study outcome measures

The primary objective is to evaluate the safety and tolerability of TN-TC11LM and TN-TC19LM oral capsules in PLWHIV on effective ART. The primary between group comparison is the percentage of participants without any signs of significant toxicity; percentage of participants who are able to complete the study and scores on the WHOQOLHIV-BREF Scale, EQ-D5 and POMS questionnaires from week 0 to week 12 are secondary outcomes that will also be compared between groups. The secondary objective is to determine the effect of TN-TC11LM and TN-TC19LM oral capsules on frequency of activated T-cells and markers of inflammation association with HIV disease progression. Exploratory objectives are to determine the effect of TN-TC11LM and TN-TC19LM oral capsules on: (1) the size of the peripheral HIV reservoir and (2) the composition of the gastrointestinal bacterial and fungal microbiome.

Safety and tolerability

Safety will be assessed at regular intervals (table 3) by vital signs and AEs monitoring, as reported by the participant and actively sought at each study visit by the coordinator or physician. Biological safety will be evaluated by haematology, biochemistry and other clinical, laboratory or other diagnostic tests done on participants during the course of the study. Lab results for all participants for assessed safety variables will be reviewed by the trial investigator. The Data Safety Monitor will also review safety information. Toxicity of TN-TC11LM and TN-TC19LM will be assessed using the WHO toxicity scale. All AEs, regardless of the grade, will be documented, and it will be noted whether these symptoms were already present at baseline. Any AEs that occur during the study will be evaluated by the trial investigators, and grades 3 and 4 AEs will be recorded on the CRFs. If required, blood specimens will be collected for haematology and biochemistry tests. Participants having AEs will be monitored with relevant clinical assessments and laboratory tests, as determined by the trial investigator. The trial investigator will report ongoing AEs at the completion of the clinical study to the primary treating physician at the CVIS who will determine the need for and provide standard medical care. The trial investigator will ensure that the event is satisfactorily resolved or that no additional follow-up is needed. Any participant who discontinues the study for an unresolved clinically significant AE will be followed until satisfactory clinical resolution is achieved and the AE recorded on the case report form (CRF), regardless of severity grading. AEs that may be related to TN-TC11LM and TN-TC19LM will be managed by dose reduction of TN-TC11LM and TN-TC19LM. TN-TC11LM and TN-TC19LM will be discontinued permanently in the event of any life-threatening AEs.

T cell subsets and immune activation

The frequency of different CD4+ and CD8+ T cell subsets will be defined using multiparameter flow cytometry (BD Fortessa X-20) in peripheral blood. The expressions of CD3, CD4 and/or CD8, CD45RA, CCR7 and CD27 will be used to measure the frequency of naïve (CD45RA⁺CCR7⁺CD27⁺), central memory (CD45RA⁻CCR7⁺CD27⁺), transitional memory (CD45RA⁻CCR7⁻CD27⁺), effector memory (CD45RA⁻CCR7⁻CD27⁻) and terminally differentiated (CD45RA⁺CCR7⁻CD27⁻) cells. Regulatory T (Treg) cells will be defined as CD3⁺CD4⁺CD25^highFoxP3^highCD127^low cells. Expression of the CD39 and CD73 ectoenzymes involved in Treg-mediated immunosuppression and HIV disease progression (via the adenosine pathway) will be also assessed.62 63 Various subsets of Th cells (T helper) will be defined as Th1 (CD45RA⁻CCR6⁻CCR4⁻CXCR3⁺), Th2 (CD45RA⁻CCR6⁻CCR4⁺CXCR3⁻), Th17 (CD45RA⁻CCR6⁺CCR4⁺CXCR3⁻) and Th1/Th17 (CD45RA⁻CCR6⁺CCR4⁻CXCR3⁺). Levels of CD8+ and CD4+ T cell immune activation (CD38/HLA-DR coexpression) and senescence (CD28⁻CD57⁺) will be also assessed on all T cell subsets. These markers will be assessed at week 0, 1, 2, 6, 12 and 14.

Inflammatory markers assessments

Plasma levels of various inflammatory markers including interferon-α, IL-1β, IL-6, IL-10, IL-17, TGF-β and IP-10 will be assessed via Luminex (Millipore) and levels of d-dimer, C reactive protein and markers of microbial translocation LPS and sCD14 will be assessed by ELISA in batch from blood drawn at weeks 0, 1, 2, 6, 12 and 14.

Peripheral blood HIV reservoir size

Blood for HIV reservoir assessment will be collected at two time points prior to cannabis initiation (1 week prior to cannabinoid capsule initiation and immediately prior to cannabinoid capsule initiation) to account for normal fluctuations in baseline levels of HIV persistence markers. Subsequently, virological measures will be done at weeks 1, 2, 6, 8, 12 and 14. CD4+ T cells isolated form peripheral blood mononuclear cells (PBMCs) by magnetic negative selection. To capture all infected cells, the frequency of cells harbouring total and integrated HIV DNA will be measured using well-established assays on a total of 500 000 cells (sensitivity of 1 copy/reaction).17 64 As most of the HIV genomes are defective, the recently developed ‘tat/rev induced limiting dilution assay’ (TILDA), which provides a more functional measurement of the HIV reservoir,65 will be employed. To assess if residual levels of viral replication may occur, we will measure 2-LTR circles, which are proposed to be a surrogate marker for ongoing HIV replication during ART.64 66 67 Specifically, this combination of assays will indicate if cannabis has an impact on the size of the total reservoir (DNA), the functional reservoir (TILDA) and ongoing viral replication (2-LTR circles). Measurements will be performed in batch.

Gastrointestinal microbiome composition

A stool sample without preservative will be collected from each participant at the beginning of the study prior to consuming the capsules and during the final week (week 12) of capsule consumption. Specimens will be stored at −80°C until analysed in batch, as previously described.68 Bacterial DNA will be extracted with PCR amplified targeting of the 16S rRNA gene using universal primers that flank the V3–V4 region of the 16S gene modified with the addition of TruSeq Illumina adapters, also as previously outlined.69 Internal transcribed spacer for fungal DNA extraction will be used for fungi. PCR amplification, PCR amplicon quantification and sequencing will be performed as previously described.68 69

Sample size and statistical analyses

In this proof-of-concept study, we are exploring a phenomenon with little in vivo data and with a limited study budget. For this reason, we have chosen a convenience sample of 26 participants, 13 per arm, without formal power calculations. This number of individuals will enable us to assess feasibility (willingness of patients to participate, attend study visits and complete questionnaires and numbers of drop-out participants) as well as safety and tolerability. The data obtained will help to guide future sample size calculations for future studies. Although the small number of participants may result in wide CIs for AEs, this number of participants should give us an idea about the degree of variability for continuous outcomes we are measuring.

For the primary endpoint, the proportions of participants without any signs of significant toxicity (grades 0–2 scores on the WHO toxicity scale), proportions of participants who complete the study and scores on the WHOQOLHIV-BREF, EQ-5D and POMS questionnaires will be examined using descriptive statistics. We will also compare these proportions for group 1 versus group 2 using a Fisher’s exact test. For quality of life and mood measures, we will use analysis of covariance with 12-week score as outcome and baseline score as covariate and treatment as independent variable. With regards to the POMS questionnaire, we will consider only overall scores (and not subscores) due to the small sample size that would make comparisons of the subscale inappropriate.

Immune activation levels for groups 1 versus group 2 at week 12 will be compared using analysis of covariance with adjustment for the week 0 activation levels. The mean change and associated 95% CI will be reported for each of the secondary endpoints described above. Furthermore, the study drug treatment period will be compared with the baseline period with each arm. If the treatment effect is similar within the two arms, then an analysis of the treatment effect will be pooled over the two arms, using analysis of covariance. The change is immune activation levels following discontinuation of study drug (ie, from week 12 to week 14) will be reported as a mean with corresponding 95% CI. A 50% reduction will be considered significant.70

Analogous analyses will be conduction for reservoir assessments other outcome measures listed above as endpoints. A signed rank (non-parametric) test will be used to compare number of copies of total and integrated DNA at baseline 2 versus at week 12. Group differences in the change of HIV reservoir size from baseline to 12 weeks will be assessed by the Mann-Whitney U test. Wilcoxon signed-rank test will be used to compare the HIV reservoir and inflammatory markers in blood samples of the same patient from baseline to 12 weeks. At least a twofold decrease in frequency of infected cells in both groups from baseline to 12 weeks of treatment will be considered significant.71 Microbiome composition will be described with regards to the frequencies of microorganisms families for the groups at baseline and then at 12 weeks of treatment. Due to the exploratory nature of this objective, no formal statistics will be applied.

Patient and public involvement

The CTN Community Advisory Committee (CAC) was involved in the peer-review process of this study proposal, deemed that the research questions addressed were of very high priority to people living with HIV and voted for the funding of this study. The CAC’s critiques of the initial proposal were taken into account in the revised proposal. Two members of the CAC (SM and EM) were involved in finalising the study design, inclusion/exclusion criteria, outcome measures and monitoring plans and are formal study investigators and coauthors. Preliminary and final results of the study will be shared with community members, patient participant and the public at biannual CTN meetings, through the CTN newsletter and on the CTN website in addition to the annual Canadian Association of HIV Research meeting.

Data management

All clinical data and electronic files will be stored in the secure environment of the CVIS of the MUHC. All data published will be anonymised. Only researchers affiliated with the study will have access to participant data. Study progress and safety will be evaluated in an ongoing fashion by the principal investigators and coinvestigators. The study will be monitored for safety and ongoing progress by a standing Data and Safety Monitoring Committee of clinicians and methodologists established by the Canadian HIV Trials Network. The committee meets every 6 months or as needed.

Storage of biological specimens

Within 1 hour of being drawn, blood from the CVIS will be transferred to the laboratory at the Research Institute of the McGill University Health Centre (RI-MUHC) (in the adjacent, connected building), and the plasma will be separated from PBMCs by Ficoll density centrifugation by an experienced laboratory personnel. PBMCs and plasma will be stored in liquid nitrogen tanks at the RI-MUHC laboratory until time for analysis. Patients will be contacted the day before their clinic visit to remind them when a stool specimen is due. They will be instructed to record the time of the provision of the sample on the paper bag containing the sterile container and store it in a refrigerator (−4°C) until brought to the clinic. Once at the CVIS, the stool specimen will be placed in a large fridge designated specifically for the storage of stool specimens.

Ethics and dissemination

Written informed consent will be obtained from all study participants. The study protocol and informed consent are in the process of being reviewed by Health Canada’s Therapeutic Product Directorate. The study will be conducted in accordance with the application Health Canada regulations, International Conference on Harmonization guidelines on current Good Clinical Practice and the Declaration of Helsinki. Patient enrolment for this trial is anticipated to begin August 2018.

Regardless of outcome, trial results will be disseminated through scientific peer-review publication, international and national conferences and the CTN according the Standard Protocol Items: Recommendations for Interventional Trials and Consolidated Standards of Reporting Trials guidelines for transparent reporting of trials.72 73 CTC will be responsible for initially drafting the manuscripts, and professional writers will not be used for any of the publications. Authorship will be determined based on criteria defined by the International Journal of Medical Editors.74 We aim to write the manuscript of the final results within 6 months of completing the study. Participants who have been involved in the trial will be given the option of having a summary of results sent to them.