Probiotics in Metformin Intolerant Patients With Type 2 Diabetes
Probiotics in Metformin Intolerant Patients With Type 2 Diabetes
Metformin, the first-line drug in the treatment of type 2 diabetes (T2DM), may cause dose
 dependent undesirable side-effects like diarrhea, abdominal pain, nausea or bloating which
 may affect up to 20 % of patients treated with this drug. The mechanism of the
 gastrointestinal intolerance in patients treated with metformin is poorly understood. The
 number of studies on this topic increases and data are mounting that metformin treatment is
 associated with changes in gut bacterial composition. Among other drugs, metformin also leads
 to enrichment of short chain fatty acids (SCFAs) producing microbiota which exert positive
 influence on the human metabolic state.

 It has been shown that the therapeutic effect of metformin depends on the microbiota and
 metformin's main site of action in humans is the intestine. It is also known that patients
 with T2DM, in general, show evidence of gut dysbiosis followed by alterations of an
 intestinal barrier leading to an increase in intestinal permeability and elevated
 inflammatory state.

 Therefore, it has been speculated that metformin's versatile effect mediated through the gut
 microbiota is responsible not only for its therapeutic effect but also for its undesirable
 digestive symptoms.

 Probiotics, defined as "live microorganisms, that when administered in adequate amounts,
 confer a health benefit on the host", may have the potential to modulate the gut bacterial
 composition. This is why the investigators hypothesize that it may also reduce the intensity
 of adverse effects associated with metformin use.

 The investigators have chosen Sanprobi Barrier multi-strain formula probiotic because it is
 identical, in relation to bacterial strains and number, to Ecologic® BARRIER which has been
 proven in in vitro studies to improve the function of epithelial barrier of the intestine. It
 was also shown that 12-week administration of strains included in Ecologic® BARRIER in obese
 postmenopausal women improved intestinal barrier permeability marker (lipopolysaccharide) and
 cardiometabolic risk factors (waist, fat mass, subcutaneous fat, uric acid, total
 cholesterol, triglycerides, low-density lipoprotein cholesterol, glucose, insulin, and
 homeostatic model assessment - insulin resistance (HOMA-IR).
The optimal daily dose of metformin is thought to be 2000 mg, however patients with metformin
 intolerance cannot reach this target dose. Participate in this study are metformin
 intolerant. Metformin intolerant patients have been defined as those not able to be treated
 with the metformin daily dose exceeding 1500 mg due to gastrointestinal upset. Patient's
 metformin intolerance assessment will be questionnaire based (questionnaire adapted from
 Laura J. Mc Creight et al.). Patients will fill out questionnaires regarding the
 gastrointestinal symptoms associated with metformin at a dose they did not tolerate and at a
 time of reduced daily dose of metformin they do tolerate (patients are accepted to have
 gastrointestinal symptoms which they accept). Sanprobi Barrier probiotic/placebo will be
 administrated and the gastrointestinal symptoms will be repeatedly assessed with the use of
 above mentioned questionnaire at certain time points during the study. Patients will be
 advice to increase the daily dose of metformin as described below. At certain visits patients
 will undergo the tests measuring the intestinal permeability (blood and stool zonulin
 immunoenzymatic tests). Inflammatory state will be assessed by measurement of blood
 C-reactive protein (CRP) as well as blood and stool calprotectin. Zonulin is an endogenous
 protease, which concentration provides information about the condition of tight junctions
 between intestinal cells and has been used a a marker of intestinal permeability.
 Calprotectin, constitutes up to 60% of soluble cytosolic proteins in human neutrophils. In
 addition, it occurs in monocytes, macrophages and epithelial cells. Therefore, fecal and
 serum calprotectin content may be proportional to the number of neutrophils migrating through
 the gastric and intestinal mucosa and may be associated with inflammatory diseases of the
 gastrointestinal tract. Calprotectin has been widely used in contemporary clinical practice
 to monitor inflammation of the gut mucosa. CRP blood concentration is a marker of
 inflammation. Additionally, fecal samples will be used for microbial analysis (16S ribosomal
 ribonucleic acid (rRNA) sequencing) and blood samples for oxidative stress parameters, HbA1c,
 lipogram and alanine aminotransferase activity will be collected. Oxidative stress being a
 disturbance in oxidative balance, has been associated with type 2 diabetes and linked to
 adverse health effects, including alterations within the intestinal microbiota and vascular
 endothelium. Probiotics have already been shown to modify the intestinal microbiota and their
 usage may exert a beneficial effect on the oxidative stress parameters. This will be a 32 -
 weeks, prospective, single center, randomized, double-blind, cross-over study consisting of
 10 site visits and 4 telephone contacts.

 Visit 1. Written informed consent for participation in the study and medical history
 collection.

 Visit 2 - month 0. Randomization visit. (within 3 ± 1 days of the visit 1) Fasting state.
 Blood pressure, heart rate, body mass index (BMI) measurements, waist-hip ratio (WHR).

 Blood collection for zonulin and immunoglobulins against zonulin, calprotectin, CRP, HbA1c,
 hemoglobin (HGB), red blood cells (RBC), white blood cells (WBC), platelet count (PLT),
 creatinine, lipogram, alanine aminotransferase (ALT) activity and oxidative stress parameters
 (antioxidant enzymes: superoxide dismutase (SOD), glutathione peroxidase (GPx); catalase
 (CAT); glutathione reductase (GR), radical damage indicators of free of lipids and proteins:
 total oxidation capacity (TOC); concentration of lipid hydroperoxides (LHP); concentration of
 lipofuscin (LPS) - serum and lysate of erythrocytes; concentration of sulphydryl protein
 (PSH); malondialdehyde (MDA) concentration - serum and erythrocyte lysate; concentration of
 reduced glutathione (GSH); non-enzymatic antioxidant system: total antioxidant capacity (TAC)
 of plasma (total antioxidant status (TAS)) .

 Stool collection for microbial analysis, short chain fatty acids, zonulin and calprotectin
 concentration.

 Gastrointestinal symptoms questionnaire administration. Randomization to probiotic/placebo
 group with the permutation method. Patient will be advised to consume 2 capsules in the
 morning and 2 capsules in the evening. There will be two groups of probiotic/placebo products
 namely "A" and "B". Patients who will be randomized to probiotic/ placebo "B" at visit 2 will
 be switched to probiotic/ placebo "A" at visit 6 and patients who will be randomized to group
 probiotic/ placebo "A" at visit 2 will be switched to probiotic/ placebo "B" at visit 6. The
 patient will receive two probiotic/ placebo packs containing 120 capsules each.

 Visit 3 - month 1 (4 weeks ± 3 days from the visit 2) Gastrointestinal symptoms questionnaire
 administration. Return of the empty packages or unused probiotic/placebo issued at the visit
 2. The patient will receive two probiotic/ placebo packs containing 120 capsules each.

 The current dose of metformin will be increased by 500 mg per day if possible.

 Visit 3 A - telephone contact (1 week ± 3 days from the visit 3) Gastrointestinal symptoms
 will be assessed after increasing the dose of metformin. Decision about the possibility of
 continuing the increased dose of the drug will be made.

 Visit 4 - month 2 (4 weeks ± 3 days from the visit 3) Gastrointestinal symptoms questionnaire
 administration. Return the empty packages or unused probiotic/placebo issued at the visit 3.
 The patient will receive two probiotic/ placebo packs containing 120 capsules each. Depending
 on the patient's clinical symptoms and tolerability of previously increased dose of
 metformin, increasing the dose of metformin (additional 500 mg/day) will be advised in order
 to achieve metformin dose of at least 2000 mg. In the case of side-effects from the
 gastrointestinal system, the dose will be reduced to the dose where there were no symptoms or
 there were symptoms that patients accepts.

 Visit 4 A - Telephone contact (1 week ± 3 days from visit 4) Gastrointestinal symptoms will
 be assessed after increasing the dose of metformin. Decision about the possibility of
 continuing the increased dose of the drug will be made.

 Visit 5 - month 3 (4 weeks ± 3 days from the the visit 4) Fasting state. Blood pressure,
 heart rate, BMI measurements, WHR. Blood collection for zonulin and immunoglobulins against
 zonulin, calprotectin, CRP, HbA1c, HGB, RBC, WBC, PLT, creatinine, lipogram, ALT activity and
 oxidative stress parameters (SOD, GPx,CAT, GR, TOC, LPS, PSH, MDA, GSH, TAS (TAC).) .

 Stool collection for microbial analysis, SCFAs, zonulin and calprotectin concentration.

 Gastrointestinal symptoms questionnaire administration. Return the empty packages or unused
 probiotic/placebo issued at the visit 4. Probiotic / placebo will be discontinued.

 Visit 6 - month 4. Cross-over visit. (4 weeks ± 3 days from the visit 5).

 Fasting state. Blood pressure, heart rate, BMI measurements, WHR. Blood collection for
 zonulin and immunoglobulins against zonulin, calprotectin, CRP, HbA1c, HGB, RBC, WBC, PLT,
 creatinine, lipogram, ALT activity and oxidative stress parameters (SOD, GPx,CAT, GR, TOC,
 LPS, PSH, MDA, GSH, TAS (TAC).) .

 Stool collection for microbial analysis, SCFAs, zonulin and calprotectin concentration.

 Gastrointestinal symptoms questionnaire administration. Patient will be cross-over to the
 different group of probiotic/placebo ("A" or "B" as described on the visit 2) and will be
 advised to consume 2 capsules in the morning and 2 capsules in the evening.

 The patient will receive two probiotic/ placebo packs containing 120 capsules each.

 Visit 7 - month 5 (4 weeks ± 3 days from the visit 6) Gastrointestinal symptoms questionnaire
 administration. Return the empty packages or unused probiotic/placebo issued at the visit 6.
 The patient will receive two probiotic/ placebo packs containing 120 capsules each.

 Depending on the patient's clinical symptoms and tolerability of previously increased dose of
 metformin, increasing the dose of metformin (additional 500 mg/day) will be advised in order
 to achieve the metformin dose of at least 2000 mg. In the case of side-effects from the
 gastrointestinal system, the dose will be reduced to the dose where there were no symptoms or
 there were symptoms that patients accepts.

 Visit 7 A - telephone contact (1 week ± 3 days form the visit 7) Gastrointestinal symptoms
 will be assessed after increasing the dose of metformin. Decision about the possibility of
 continuing the increased dose of the drug will be made.

 Visit 8 - month 6 (4 weeks ± 3 days from the visit 7) Gastrointestinal symptoms questionnaire
 administration. Return the empty packages or unused probiotic/placebo issued at the visit 7.
 The patient will receive two probiotic/ placebo packs containing 120 capsules each.

 Depending on the patient's clinical symptoms and tolerability of previously increased dose of
 metformin, increasing the dose of metformin (additional 500 mg/day) will be advised in order
 to achieve the metformin dose of at least 2000 mg. In the case of side-effects from the
 gastrointestinal system, the dose will be reduced to the dose where there were no symptoms or
 there were symptoms that patients accepts.

 Visit 8 A - telephone contact (1 week ± 3 days form the visit 7) Gastrointestinal symptoms
 will be assessed after increasing the dose of metformin. Decision about the possibility of
 continuing the increased dose of the drug will be made.

 Visit 9 - month 7 (4 weeks ± 3 days from the visit 8) Fasting state. Blood pressure, heart
 rate, BMI measurements, WHR. Blood collection for zonulin and immunoglobulins against
 zonulin, calprotectin, CRP, HbA1c, HGB, RBC, WBC, PLT, creatinine, lipogram, ALT activity and
 oxidative stress parameters (SOD, GPx,CAT, GR, TOC, LPS, PSH, MDA, GSH, TAS (TAC).) .

 Stool collection for microbial analysis, SCFAs, zonulin and calprotectin concentration.

 Gastrointestinal symptoms questionnaire administration. Return the empty packages or unused
 probiotic/placebo issued at the visit 8 Probiotic / placebo will be discontinued.

 Visit 10 - month 8 (4 weeks ± 3 days from the visit 9) Fasting state. Blood pressure, heart
 rate, BMI measurements, WHR. Blood collection for zonulin and immunoglobulins against
 zonulin, calprotectin, CRP, HbA1c, HGB, RBC, WBC, PLT, creatinine, lipogram, ALT activity and
 oxidative stress parameters (SOD, GPx,CAT, GR, TOC, LPS, PSH, MDA, GSH, TAS (TAC).) .

 Stool collection for microbial analysis, SCFAs, zonulin and calprotectin concentration.

 Gastrointestinal symptoms questionnaire administration. The patient will finish participation
 in the study.
Inclusion Criteria:

 1. Written informed consent for participation in the clinical trial

 2. Age 18-75 years

 3. Type 2 diabetes mellitus diagnosed at minimum 6 months prior to the study

 4. Metformin intolerance defined as gastrointestinal adverse effects occurrence at the
 daily metformin dose higher than 1500 mg assessed by the Questionnaire adapted from
 Laura J. McCreight et al., which disappeared or decreased to the accepted tolerable
 level after dose reduction to 1500 mg per day.

 5. Metformin treatment in the daily dose not higher than 1500 mg

 6. Stable metformin dose in the last 3 months before inclusion to the study

 Exclusion Criteria:

 1. Estimated Glomerular Filtration Rate (eGFR) < 60 ml /min/ 1.73m2

 2. Elevation of ALT and aspartate aminotransferase (AST) activity in the blood serum,
 three times above the reference value

 3. Chronic bowel disease

 4. Any other acute or chronic disease that may cause gastrointestinal symptoms

 5. Acute or chronic pancreatitis

 6. Chronic alcohol consumption >30 g/day for men and > 20 g/day for women

 7. Antibiotic therapy in the last 6 months prior to the study

 8. Probiotics use in the last 3 months before the study

 9. Chronic use of steroid drugs or other immunomodulators

 10. Heart failure (New York Heart Association (NYHA) III and IV)

 11. Pregnancy or breast feeding