Methods/Design
Study design
The clinical trial will follow a 2-period double-blind cross-over design. It will take place at the Chronic Disease Innovation Centre (CDIC) at Seven Oaks General Hospital in Winnipeg, Canada. The study protocol flow chart is shown in Figure 1. This trial will be conducted in compliance with Good Clinical Practice (GCP) and all local and national guidelines.
Participants will consent to follow a 26-week study regimen. Each participant will receive 2 sachets per day containing either 15 grams of RPS (MSPrebiotic, resistant potato starch treatment) or 15 grams corn starch (Amioca TF, digestible starch control). The powder in the sachets will be mixed in water and consumed, one sachet in the morning and one before bed. Participants will be instructed to consume the investigational product at least 2 hours prior to or after taking any medication.
For the first two weeks, participants will go through a run-in period, where they will all receive the corn starch control. During weeks 3 to 12 (period 1) participants will receive either RPS or cornstarch. The first treatment received will be determined by a randomization procedure. During weeks 13 to 16, participants will undergo a washout period where they will all consume cornstarch. During weeks 17 and 26 (period 2), participants will receive the treatment they did not previously consume (RPS or cornstarch, respectively).
Participants will complete questionnaires and food records at the beginning and end of each treatment period (Table 1). They will also provide blood, urine, and fecal samples for analysis (Table 1).
Study participants:
Thirty-six adults (18-85 years) with CKD will be recruited to the study via the advanced CKD clinic at Seven Oaks Hospital. Dr. Tangri and Dr. Mollard will oversee recruitment.
Inclusion Criteria: The participant may enter the trial if all of the following apply: The participant is willing and able to give informed consent for participation in the trial and has the ability to speak and read English. Male or female, aged 18 years or above; females of child-bearing potential must agree to use a medically approved method of birth control for the duration of the study. All hormonal birth control must have been in use for a minimum of three months. Acceptable methods of birth control include: hormonal contraceptives including oral contraceptives, hormone birth control patch, vaginal contraceptive ring, injectable contraceptives, or hormone implant, double barrier method, intrauterine devices, non-heterosexual lifestyle or agrees to use contraception if planning on changing to heterosexual partner(s), vasectomy of partner at least 6 months prior to screening. Estimated glomerular filtration rate (eGFR) <15 mL/min/1.73m^2 for the past 3 months. In the investigator’s opinion, participant is able and willing to comply with all trial requirements.
Exclusion Criteria: The participant may not enter the trial if any of the following apply: The participant is cognitively impaired and cannot give consent or participate in the group program. The participant has an existing relationship with the research team, such as supervisory relationship (student, employee) or familial relationship (child, spouse, etc.). Participants who indicate that they cannot consume study treatments. Participants who indicate they are allergic to potatoes or corn. Female participants who are pregnant, lactating or planning pregnancy during the trial. History of renal transplant, ongoing dialysis, use of antibiotics (last 3-months), bowel diseases, cancer, surgically removed bowel, or any gastrointestinal surgery (e.g. intestinal resection, gastric bypass, colorectal surgery). Inability to consume treatment due to swallowing or GI issues. Participating in another interventional trial that could influence the intervention or outcome of this trial. Participants with uncontrolled diabetes with A1C > 10%. Participants who consume probiotic supplements. Participants with abnormal constrictions of the gastrointestinal tract, diseases of the oesophagus and/or the superior opening of the stomach (cardia), potential or existing intestinal blockage, paralysis of the intestine, megacolon, faecal impaction, appendicitis, a sudden change in bowel habits that has persisted for more than 2 weeks, undiagnosed rectal bleeding, or failure to defaecate following the use of another laxative product. Participants with severe anemia (hemoglobin less than 70). Participants taking medications which inhibit peristaltic movement (e.g. opioids, loperamide). Participants able to maintain high fiber/adequate fiber intake through diet or taking other fiber supplements.
Recruitment: A research coordinator will work with someone within the circle of care (such as nurse, a clinical clerk or a physician) of patients attending the Manitoba renal programs interdisciplinary CKD clinic at Seven Oaks hospital to pre-identify potentially eligible participants.
The first point of contact will occur during routine clinical care or through virtual meetings. An individual in the patient’s circle of care will inform the patient that they may be eligible to participate in a research study and ask for their permission for the research coordinator to contact them to discuss the study in detail that day or at a later time, through a virtual or in-person visit. This is meant to be a brief conversation, and the main purpose is to seek permission for the individual to be contacted by the research coordinator, and to do this via someone in the patient’s circle of care. We will ensure that all clinical conversations (e.g. patient’s health, updates, appointment reminders) will be completed first before the study is introduced to them.
The second point of contact will be made by the research coordinator through virtual meeting or in person visit to explain the study in greater detail to the patient. Patients that are interested in the study will be given a letter of information and consent form to review and sign through the Research Electronic Data Capture (REDCap) platform or in paper form. A participant ID log will be kept recording the patients who enrol into the study. It is expected that the recruitment of the 36 participants will occur over a three-month period.
Treatments:
The treatments will consist of 1) 30 grams of RPS (MSPrebiotic) or 2) 30 grams digestible corn starch (Amioca TF) per day. Digestible corn starch was chosen as the comparator because it is visually similar to the RPS but contains no resistant starch. These treatments will be given to participants in 2 sachets per day, which are to be mixed with water and consumed, one in the morning and one in the evening before bed. The dosage of 30 g is based on a previous study of RPS in older adults [17]. The resistant potato starch and corn starch will be pre-packed in labelled packages provided by the manufacturer. The corn starch and resistant potato starch sachets will be stored at room temperature until it is dispensed to participants. Courier will be available as an option to deliver the packages to the study participants.
Compliance:
Diaries with daily checklists for the 2 sachets will be given to the participants for each day during the run-in, washout, and treatment periods. These will be used to monitor their compliance with the study protocol. Additionally, participants will be instructed to return all empty sachets for counting purposes. Compliance for both groups will be defined as an attendance of ≥90% of the scheduled visits with the research team and ≥80% of the required treatments consumed according to diary entries and returned sachets. Participants will be monitored every week by the research team through phone calls or online video. Additionally, there will be a study dispensing log which will be used to track all study treatments given to each of the participants. This log will include participant ID, date of dispensing, randomization code and the returned sachet counts. Concomitant medication will be recorded at the end of run-in and each treatment period.
Assessments:
Baseline Assessments
After screening, eligible participants will receive corn starch sachets for the first two weeks during a run-in phase, however they will be instructed that they could be receiving corn starch or resistant potato starch. Besides corn starch sachets, participants will also receive the Medical Outcomes Study Short Form 36-item (SF-36) and Edmonton Symptom Assessment Scale (ESAS) questionnaires, the link to an Automated Self-Administered 24-hour Canada (ASA24) dietary recall survey, a tape measure, a weight scale and fecal sample collection kits.
At the end of the second week of the run-in period, study coordinators will book a virtual/in person meeting with participants to instruct and measure body weight and waist circumference; go through medication/supplement condition and give a general instruction of how to fill in SF-36, ESAS, and ASA24 questionnaires. Study coordinators will also give a brief introduction about blood, urine, and fecal collection steps and then send a lab requisition to the participants.
Ongoing Assessments
Participants will be able to contact the study team at any time and will be given options to do so (text, voice call, video link, urgent in person appointment). After enrolment in the study, participants will have follow-up virtual/in-person study visits as described in Table 1. Study visits will be acceptable on days within +/- 5 days.
Outcome measures:
Serum concentrations of indoxyl and p-cresol sulphate are considered the primary outcome of this trial. This is to investigate how RPS consumption influences blood uremic toxins. The abundance, diversity, and functionality of the gut microbiome will be investigated as the secondary outcomes. Samples will be sent out for blood, fecal and urine metabolomics analysis, and gut microbiome analysis, following shipping guidelines of the contracted service providers, University of Manitoba policies and all applicable local and national regulations.
Uremic toxin measurement: Serum samples of indoxyl and p-cresol sulphate will be collected by the start and end of each treatment period. The concentrations of free and total p-cresol sulphate and indoxyl sulphate will be measured by high pressure liquid chromatography [21]. Total p-cresol sulphate and indoxyl sulphate will be me measured after deproteinization of serum with ethanol. These analyses will be conducted at McMaster University in Hamilton, ON.
Gut Microbiome: Two fecal samples will be collected from consecutive days for analysis at the end of the run-in and each treatment period. Participants will collect the fecal sample; they will be provided collection kits and an ice pack and instructed to collect a single sample from 3 separate places on the stool using a spoon attached to the cap of the collection tube. Participants will be instructed to store the collected fecal samples in their household –20°C freezer with the provided ice pack, until transport back to the study center. At the study center, samples will be aliquoted and then stored at –80°C. Fecal samples will undergo genomic DNA extraction following the manufacturer’s protocol. Experimental negative controls will be included in extraction protocols to confirm the reliability and consistency of the extracted nucleic acid. The V4 hypervariable region of 16S rRNA gene will be amplified; the sequencing library will be generated and sequenced by Microbiome Insights (www.microbiomeinsights.com) in Vancouver, BC.
Anthropometry: Body weight and waist circumference will be measured at the beginning and end of each period.
Edmonton Symptom Assessment Scale (ESAS): ESAS will be completed in the beginning and end of each period thru paper or online by REDCap. The ESAS is a widely used tool for measuring physical and psychological symptom distress that has been validated in individuals with CKD [22], which consists of nine visual analogue scales (0–10 scale) for: pain, activity, nausea, depression, anxiety, drowsiness, appetite, well-being, and shortness of breath. The scale for each symptom is anchored by the words ‘No’ and ‘Severe’ at 0 and 10, respectively.
Quality of Life: The Medical Outcomes Study Short Form 36-item questionnaire (SF-36) will be used to measure quality of life and has been validated in individuals with CKD [23]. SF-36 will be filled in by the beginning and end of each period. The questionnaire is designed for use across diverse populations and healthcare settings and is composed of eight scales: physical functioning (PF), role functioning/physical (RP), bodily pain (BP), general health (GH), vitality (VT), social functioning (SF), role functioning/emotional (RE), and mental health (MH). These scales are scored from 0 to 100, with higher scores indicating better function.
Dietary intakes and nutritional assessment: Participants will complete three dietary recall surveys (for two weekdays and one weekend) following the run-in, and in the last week of each treatment period, using the Automated Self-Administered 24-hour Canada (ASA24®) dietary assessment tool. The ASA-24 is a web-based tool that enables multiple, automatically coded, self-administered 24-hour recalls. Participants will receive training and have support from study staff in filling out the recalls.
Clinical chemistry: Blood and urine sample for clinical chemistry will be collected at the start and end of each treatment period. In the blood, albumin, BUN, bicarbonate, calcium, chloride, creatinine, eGFR, glucose, phosphorus, potassium, sodium and HbA1c will be measured. In the urine, albumin, albumin/creatinine ratio, glucose, and total protein will be measured. All samples will be collected, and analytes will be measured by Shared Health Diagnostics at Seven Oaks Hospital.
Metabolomics: Serum, fecal, and urine samples will be collected at the end of run-in and treatment periods. The metabolomics analyses and informatics will be conducted by McMaster University in Hamilton, ON. Targeted analysis of water soluble metabolite classes including amino acids, sugars, alcohols, organic acids, amines, TCA cycle intermediates, and short chain fatty acids, using quantitative NMR spectroscopy will be conducted on serum collected at the endpoint of each study period [24]. A targeted quantitative metabolomics approach will be used to analyze urine samples using a direct injection mass spectrometry with a reverse-phase LC-MS/MS assay. Serum and urine samples will be collected in accordance with metabolomic sample collection guidelines, allowing as much as possible that original metabolic profile of the fresh samples is maintained and minimizing potential pre-analysis sample collection or handling issues that could bias the results of the metabolomic analyses [25].
Sample Handling
Blood and urine samples will be collected by the certified phlebotomist in Shared Health Diagnostics at Seven Oaks Hospital. The laboratory will process and run the analysis for the clinical chemistry and store the serum and urine sample in –80°C freezer in the Shared Health lab for uremic toxic measurement and metabolomics analysis. The blood and urine samples will be collected by the start and end of each treatment phases. For stool samples, participants will be provided collection kits and an ice pack and instructed to collect a single sample from 3 separate places on the stool using a spoon attached to the cap of the collection tube. Participants will be instructed to store the collected fecal samples in their household –20°C freezer with the provided ice pack, until transport back to the study centre. At the study centre, samples will be aliquoted and then stored at –80°C in the Shared Health Diagnostics lab. These samples may be moved to storage located at the University of Manitoba if required due to space constraints. Fecal samples will be collected at the beginning and end of treatment period 1 and 2. At least one sample will be obtained during those two consecutive days if possible. Samples will be sent out for blood, fecal and urine metabolomics analysis, and gut microbiome analysis, following shipping guidelines of the contracted service providers, University of Manitoba policies and all applicable local and national regulations.
Qualified Investigator Responsibilities
The Qualified Investigator (QI) will be responsible for determining eligibility of a individuals to participate in the trial. Although some tasks may be delegated to other qualified clinical trials staff members, the QI will ensure the individual or party is qualified to perform those study tasks and is responsible for supervising any individual or party to whom tasks are delegated at the trial site. The QI assumes ultimate responsibility for the overall conduct of a clinical trial and for compliance with all applicable regulations and guidelines. The QI must document the delegation of tasks/duties. The QI will evaluate the general health and review the participants’ medical history, vital sign results, concomitant medications, and all laboratory results to determine eligibility into the trial. The QI will have ongoing responsibilities of monitoring adverse events, serious adverse events, concomitant medications, and any additional laboratory results to ensure participant safety through the entire clinical trial. All study-related medical decisions are made, unless delegated to a qualified and trained sub-investigator by the QI. The QI is ultimately responsible for the welfare and safety of all participants on the trial. The QI agrees that the site will permit, if required, study-related monitoring, audits, REB review, and regulatory inspection(s), providing direct access to source data/documents. It is the responsibility of the QI or designee to maintain adequate clinical study records. Copies of all clinical study material must be archived for a period define by Canadian regulations. It is also the responsibility of the QI to ensure that the study in conducted in accordance with the principles of Good Clinical Practice and according to applicable local laws and regulations concerning studies conducted on human participants which are outside the definition of a medical product or medical device. The QI will have access to all product codes and may break randomization codes if necessary, for handling an adverse event. Lastly, the QI or their designee will review the deviations and determine if the deviation(s) would significantly affect the results, and if deemed necessary, not include such data in statistical analysis. The QI will ensure that all study documents are maintained up to date as specified in Essential Documents for the Conduct of a Clinical Study and as required by the applicable regulatory requirements. The QI will ensure that all serious adverse events are immediately reported to Health Canada, the University of Manitoba REB and any other applicable regulatory authorities.
Safety Monitoring Committee
The interventions in this trial are low risk and they are in addition to standard care that the participants will continue to receive throughout the trial period and after the trial in completed, therefore no data and safety monitoring board (DSMB) will be formed. However, there will be an external review of the participant clinical chemistry and any trial AEs at the trial midpoint when all participants have completed their first treatment period. This review will be conducted by a nephrologist from outside of Manitoba. This review will monitor evidence for treatment harm, looking for trends in the clinical chemistry and/or increases in un/expected events, related to the treatments and take appropriate action. These actions may include proposing protocol changes, they could include early stopping of the trial due to clear harm of a treatment depending on the results of the review.
Statistical Analysis
The bioinformatics and statistical analyses of microbiome data will be performed with the assistance of Microbiome Insights and the Data Science platform at CHI, it will be updated based on recommendations and technology advancements between now and processing of the samples. In general, the default settings of FLASH assembler [22] will be used to merge the overlapping paired-end Illumina fastq files. UPARSE algorithm [23] will be used for a) quality filtering of the reads based on maximum expected error value = 1.0, b) identification of unique sequences, c) abundance sorting and removal of singletons, d) clustering the reads into operational taxonomic units (OTUs) based on 97% identity threshold, e) de novo and reference-based chimera checking (against GOLD database [22]), and f) construction of OTU table. Taxonomic classification will be then carried out using QIIME [21] implementation of UCLUST [24] and will be aligned against the Greengenes database using PyNAST algorithm [25]. Phylogenetic trees were built with FastTree [26] for further comparison between microbial communities. Prior to performing downstream analyses, the resulting OTU table will be filtered to remove all the samples with low sequencing depths. Community richness and diversity indices will be then calculated using QIIME at a given even depth per sample. Phylogenetic- (weighted UniFrac distances) and abundance-based (Bray-Curtis dissimilarity) β-diversity metrics will be calculated following normalization of the final OTU table using the cumulative sum scaling (CSS) transformation [27]. Principal coordinate analysis (PCoA) will be applied on the resulting distance matrices to generate two-dimensional plots using default settings of the PRIMER-6 software (PRIMER-E Ltd, Plymouth). Unsupervised clustering analysis will be performed to relate clustering patterns of samples to the proportion of the core OTUs within each niche (core OTUs defined as those present in at least 75% of samples in each niche). The relative abundances of the selected OTUs will be normalized across samples. Bray–Curtis dissimilarities will be calculated using R "vegan" package [28] and the resulting matrix will be subjected to unsupervised hierarchical clustering using R "dendextend" package [29] and will be visualized over the heatmap of abundance matrix using R "complexheatmap" package [30]. The UNIVARIATE procedure of SAS will be used for testing the normality of residuals for α-diversity measurements. Non-normally distributed data will be either log or Box-Cox power transformed and then subjected to analysis of variance (ANOVA) test using MIXED procedure of SAS. All pairwise comparisons among the groups will be tested using Tukey studentized range adjustment. Permutational multivariate analysis of variance (PERMANOVA; implemented in Primer6 software) will be used to detect significant differences between β-diversity metrics of microbial communities. The relative abundances of selected core OTUs will be tested for statistically significant associations with available metadata using multivariate analysis with linear modeling (MaAsLin) [31] accounting for all potential confounders (covariates) that could be associated with the profile of microbiome (i.e. sex, age, BMI) and participants (treated as a random factor). Significant associations will be considered below a q-value threshold of 0.1. To assess the shifts in functionality of microbiome, correlation network analysis (CoNet, [32]) will be used to explore microbial co-occurrence/mutual-exclusion relationships and identify hub OTUs that show the highest number of positive/negative correlations with other OTUs under treatment conditions.
Effects of treatment on linear outcomes at the end of each period will be analyzed by the SAS MIXED (SAS 9.4) procedure. Sequence and sex will be included in the model as fixed factors and participant will be included as a repeated factor. The normality of the data distribution will be done using Shapiro Wilk test and the non-normal variables will be transformed prior to analysis. Demographic data will be reported as the mean ± standard deviation. The results will be reported as least-squared means ± standard error of the mean (SEM) unless otherwise specified. Statistical significance will be set at P < 0.05 for all analyzes. The Data Science Platform in CHI will provide data management support in addition to biostatistics support for the project.
Randomization, Blinding and Code-breaking
Eligible patients (n=36) will go through assessments at baseline and will be randomly allocated to 2 groups, each consisting of 18 participants. Randomization will be done by an independent researcher in the biostatistics platform at the George and Fay Yee Centre for Healthcare Innovation (CHI) at the University of Manitoba. Randomization will be performed using code written in the R statistical programming language (Version 3.5.3). Treatments will be assigned with a 1:1 ratio. A total of 48 randomization cards will be prepared, one set of 24 for each sex. The randomization schedule will be transferred into sets of opaque sealed envelopes. After a participant’s baseline visit study staff will open a sealed envelope which will contain the participant’s allocation. The order of the interventions will be blinded for both the investigators and the participants. Treatments will be given in sealed sachets; sachet content will be blinded by an outside party, given to the study staff labelled as A or B, and dispensed to participants according to their treatment period. Treatments will not be unblinded until the analyses are complete, unless required due to adverse event during the clinical trial. There will also be a midpoint review of the participants clinical data by an independent Nephrologist who will be unblinded (See Section Qualified Investigator Responsibilities).
Sample Size Calculation
A final samples size of 30 participants in this study will be able to detect a difference between treatments in total p-cresol sulphate of 17.5 μmol/L, or ~15% change, at a power of .88 (alpha =0.05, two sided), given a within person correlation of .79 [23] and an estimated standard deviation of 45 μmol/L [9] for total p-cresol sulphate. A 30% or greater drop in uremic solutes would be considered clinically significant, warranting additional trials investigating this prebiotic intervention in CKD, and we are confident we would be able to detect such a change should it occur. To account for loss of power due to dropouts we will recruit 36 participants.
Inclusion in Analysis
The primary analysis will be conducted using the All Participants (intent to treat) analysis set. The primary analysis will be repeated in the Completers analysis set. Demographics and all other baseline measurements will be analysed in the All Participants set as well as in the Completers set.
Completers Analysis Set: All participants who have completed the trial.
All Participants Analysis Set: All randomized participants.
Discontinuation/Withdrawal of Participants from Trial Treatment
Each participant has the right to withdraw from the trial at any time. Participants may discontinue trial participation at any time and are requested to contact a research team member to inform them of their decision. In addition, the investigators may discontinue a participant from the trial at any time if the investigators considers it necessary for any reason including pregnancy; ineligibility (either arising during the trial or retrospectively having been overlooked at screening); significant protocol deviation; significant non-compliance with the protocol; disease progression which results in inability to continue to comply with the protocol; withdrawal of consent; and loss to follow up.
Withdrawal will not result in exclusion of the data for that participant from analysis. As the primary analysis will be based on an Intention-to-treat, there will also be a completer only analysis performed.
If a participant is withdrawn within the first 4 weeks of the trial they will be replaced. If the replacement participant is withdrawn there will be no subsequent replacement.
The reason for withdrawal will be recorded in the CRF, if provided.
Remuneration
Participants will be remunerated $200 for each completed period, or the prorated value if they withdraw from the trial. Each participant would receive $400 in total if they complete the full trial.
Participant’s name and address will be used for preparing, printing, mailing, and financial record keeping or remuneration cheques. Pre-paid postage and envelope will be provided to the participant with a form requiring their signature upon receipt of remuneration cheque. Participants will be asked to return the form to CDIC. This record will be kept for a maximum of 7 years.