Study to Explore the Effects of Probiotics on Endotoxin Levels in Type 2 Diabetes Mellitus Patients
Condition: Diabetes Mellitus Type 2
MeSH terms: Diabetes Mellitus; Diabetes Mellitus, Type 2
Study type: Interventional

Summary: 
      Probiotics, which are believed to be health promoting live microorganisms, have been reported
      to influence circulating endotoxin levels. Ingestion of the live cultures may alter gut
      mircobiota in a beneficial manner to reduce inflammation; although their mechanism and
      influence to reduce inflammation in T2DM is not established for this disease state.
      Therefore, the aim of this study is to (1) characterize the beneficial effects of probiotics
      on circulating endotoxin levels and other biomarkers related to systemic low-grade
      inflammation in patients with T2DM; (2) Compare circulating endotoxin levels and inflammatory
      cytokine levels between patients treated with probiotics or placebo to examine the beneficial
      effects of probiotics on reducing the inflammatory status, through assessment of systemic
      markers (adipokines, endotoxin, cytokines); (3) to examine the effects of probiotics on gut
      microflora in order to understand the mechanism for such change in inflammatory status. To
      achieve this, 120 consenting adult Saudis, naïve or newly diagnosed T2DM patients without
      co-morbidities, will be enrolled in this clinical trial and randomized to receive twice-daily
      placebo or probiotics for 26 weeks in a double-blind manner. Glycemic inflammatory markers
      will be measured and fecal samples analysed, interventions will be done at baseline, 4, 8, 12
      and 26 weeks. It is envisaged that probiotics will induce beneficial changes in gut
      mircobiota, reduce the systemic inflammatory state through altering systemic endotoxin levels
      and, as such, reduce the systemic inflammatory response observed in T2DM subjects. This will
      have a fundamental impact on how we should treat the inflammatory component of T2DM,
      particularly once the results are verified in a larger cohort of patients, as this could have
      very dramatic effects on how we treat patients with T2DM. Reducing the pathogenesis of T2DM
      by dampening the inflammatory response, which may also impact on insulin resistance status
      and health of the individual, will have clear benefits. This could have profound effects on
      preventative T2DM management, as well as current T2DM care without excessive cost for the
      wider Saudi health economy.
    

Detailed description: 
      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.
    

Intervention name(s): Probiotics; Placebo
Intervention type(s): Dietary Supplement; Dietary Supplement
Intervention description(s): Administration of probiotics daily for 26 weeks and compared to placebo; Administration of placebo daily for 26 weeks and compared to probiotics group
Other intervention name(s): Ecologic Metabolic
Allocation: Randomized
Intervention model: Single Group Assignment
Masking: Triple (Participant, Care Provider, Investigator)
Primary purpose: Supportive Care
Keywords: Diabetes Mellitus