Study to Explore the Effects of Probiotics on Endotoxin Levels in Type 2 Diabetes Mellitus Patients

Design

In this 26-week, single-center, double-blind, randomized, placebo-controlled study, 60 patients with T2DM will be treated with probiotics and 60 will be treated with placebo. Interventions will be done at weeks 0, 4, 8, 12 and 26 from all subjects. Patients allocated to the probiotics group will receive capsules containing four strains of freeze-dried bacteria, namely Lactobacillus acidophilus, Lactobacillus bulgaricus, Bifidobacterium bifidum, and Streptococcus thermophiles (Bio-plus®, Supherb, Israel), at a daily dose of 2 × 1010 CFU. Participants in the placebo group will receive capsules containing wheat-based non-fermentable fibers, as used in another clinical trial [Pereg et al, 2011].

Recruitment of Patients

Recruitment of patients to this study will be made possible by collaboration with primary care centers throughout Riyadh and the Biomarkers Research Program, College of Science, King Saud University.

Inclusion Criteria

• Stable and well controlled T2DM (HbA1c < 7.5% and no change in oral antidiabetic medications during the last 6 months)
• Age 20-75 years
• Provision of written informed consent

Exclusion Criteria

• Chronic gastrointestinal disease (except IBS)
• Systemic antibiotics within 6 weeks before inclusion
• Use of probiotics within 3 months before inclusion (Appendix A lists products currently marketed internationally and available to purchase in Saudi Arabia)
• Regular intake of insulin or insulin analogs, antibiotics or probiotics, antacids, H2-receptor blockers, proton pump inhibiters, loperamide, cholestryramine, ω3 supplements, fibrates, corticosteroids or sex steroids
• Daily alcohol consumption > 30 g
• Significant immunodeficiency
• Known cardiac valvular disease
• Breast-feeding or pregnant
• Non-Arab ethnicity
• Participation in another clinical trial within the last 6 months
• Legal incapability

Allocation to Treatment

After confirmation of eligibility and obtaining written informed consent, the patients will be given a unique subject number by the probiotics company, who also will perform the randomization (stratified for gender), The patients and clinicians at the primary care center will be blinded to the treatment received. The eligible patients will be allocated (1:1) to treatment for 26 weeks with either the probiotics supplement or placebo. Subjects will be instructed to

• Ingest 1 capsule before breakfast and 1 capsule before the evening meal, either directly or mixed in standard yoghurt.
• The capsules will be stored in the refrigerator after the jar has been opened.

Data Handling and Record Keeping

Case report form (CRF) will be used to record data for all participants, and will be completed by the research nurse, who will also enter the data into an electronic database.

Study schedule and location

After inclusion, all further treatments will be managed at the primary care center where the subject was recruited (see appendix C for scheduled visits). A research nurse and a research dietician will be responsible for all contact with patients. Doctors associated with the research team will be available if problems arise.

Acquisition of Clinical Data and Assessment of Compliance

• Medical history, including the presence of chronic diseases, regular medication, smoking and alcohol habits, will be recorded before inclusion.
• Nutritional habits will be assessed using a standardized 14-day recall questionnaire, which will be discussed with a dietician
• Changes in medication during the study period will be recorded
• Anthropometric measurements will be made using standardized methods. Height will be measured at the start of treatment. Weight (in light clothing without shoes or items in pockets), waist circumference (measured as a horizontal plane at the level of the umbilicus) and hip circumference (measured as a horizontal plane at the level of the trochanter major) will be measured at the start and end of the treatment.
• Liver ultrasonography will be performed either at screening or at the start of treatment to determine the presence or absence of a bright liver indicative of steatosis. Patients with fatty liver and liver transaminases > 1.5 × the upper limit of normal will be offered referral to a hepatologist.
• Compliance will be monitored at weeks 4, 8, 12 and 26. Patients will bring their previously received capsules to the primary care center to calculate the mean number of capsules ingested per day.

Acquisition of Routine Biochemical Data and Biological Samples

• Blood samples obtained will be analyzed at the Biomarkers Research Program to measure glucose, HbA1c, insulin, C-peptide, total cholesterol, HDL-cholesterol, LDL-cholesterol, triglycerides, alanine aminotransferase and aspartate aminotransferase levels.
• Fasting blood samples will be collected at week 0, 8 and 26. Peripheral venous blood will be drawn into pyrogen-free tubes without any anticoagulant. The tubes will be immediately placed in ice, allowed to coagulate, and centrifuged (2500 ×g for 10 min at 4°C) within 2 hours of collection. Serum samples will be stored at -80°C until use. At least two 2-mL serum samples will be collected at each time-point.
• Stool samples will be taken by asking patients to store the sample in a plastic sealable bag in their freezer until transfer to the clinic. (samples taken at the same points as they are requested for blood samples. Once at the clinic, the samples will be divided, flash frozen, and stored at -80°C. Changes in gut microbiota will be profiled using signature-tagged sequencing (pyrosequencing) of amplified 16S rRNA genes followed by automated bioinformatics analysis. Targeted RT-PCR will also be used to examine bacterial species such as Enterobacteria and Bifdobacteria relative to the universal bacterial primer set. Pyrosequencing and RT-PCR will be performed in a sub-cohort (n=15) of patients considering the final systemic findings. Samples will be retained for subsequent studies.

Subject Withdrawal

Subjects may withdraw from the trial at any time at their own request, or they may be withdrawn at any time at the discretion of the investigator for safety, behavioral or administrative reasons. The subjects will also be withdrawn from the study in case of:

• Treatment with systemic antibiotics for > 1 weeks during week 0-22
• Any use of systemic antibiotics during week 22-26

Patient Safety

Adverse Events

Adverse events (AEs) are undesirable signs or symptoms that occur during the study and may or may not be causally related to the treatment. All AEs considered possibly, probably or definitely related to the test product will be recorded on CRFs.

Serious Adverse Events

Serious adverse events (SAEs) are defined as events that are fatal, life-threatening, disabling, incapacitating or result in hospitalization or prolong hospital stay, or result in malformation. All SAEs will be recorded in the CRF, whether they are related to the test product or not. According to previous studies (9), probiotics are very safe, and any SAE that might be possibly, probably or definitely related to the test product will be regarded as unexpected. All unexpected SAEs will be reported immediately to the Medicines and Healthcare products Regulatory Agency (MHRA). Any SAE that might be related to the test will immediately lead to discontinuation of the test product.

Ethical and Regulatory Aspects

The study will be performed in accordance with the protocol and the ethical principles that have their origin in the Declaration of Helsinki as well as with the International Council for Harmonization Guidance on Good Clinical Practice. These documents state that the informed consent of subjects is an essential precondition for participation in the clinical study. The study will commence only after obtaining approval from the local ethical review board of the College of Medicine Research Center, King Saud University.

Plan for Data Analyses

As this trial is designed to assess the physiological effects rather than support clinical indications for treatment with probiotics in T2DM, we plan to perform per protocol analyses. Only subjects treated with at least 80% of the planned doses for at least 80% of the time will be considered as treated per protocol.

Raw data will be entered in a statistical software (Statistical Package for the Social Sciences), SPSS version 16.5 (Chicago, IL, USA). Preparation of data set will be done prior to analyses. Descriptive statistics will be done; frequencies will be presented in percentage (%). Continuous variables will be presented as mean ± standard deviation. Variables exhibiting non-Gaussian variables will be transformed prior to analyses. Repeated measures analysis of co-variance (ANCOVA) will be used to compare groups, which will represent a combination of analysis of variance (ANOVA) and linear regression. Confounding and fixed variables such as age and gender, as well as BMI will be taken if proper matching has not been achieved prior to repeated measures. Post-hoc Banjamini-Hohberg corrections will be done for multiple comparisons. For comparison of 2 groups, independent T-test will be done for continuous variables and Mann-Whitney for non-continuous variables. Analysis of adverse events (if any) will be done using Chi-Square test and Fisher's exact test following assumptions of randomness, independence and size. Significance will be set at p < 0.05.