BIFI-OBESE: Clinical Trial in Paediatric Obesity

Obesity is a major, public health concern that affects at least 400 million individuals and is associated with severe disorders including diabetes and cancers. Worldwide, the prevalence of overweight and obesity combined in children, adolescents and youth, between 1980 and 2013, increased to 47.1%, with alarming data also in developing countries. Obesity is often caused by imbalance between excessive caloric intake and reduced physical activity.

Recently, microbial changes in the human gut was proposed to be another possible cause of obesity and it was found that the gut microbes from fecal samples contained 3.3 million non-redundant microbial genes. However, it is still poorly understood how the dynamics and composition of the intestinal microbiota are affected by diet or other lifestyle factors. Moreover it has been difficult to characterize the composition of the human gut microbiota due to large variations between individuals.

The role of the digestive microbiota in the human body is still largely unknown, but the bacteria of the gut flora do contribute enzymes that are absent in humans for food digestion. Moreover, the link between obesity and the microbiota is likely to be more sophisticated than the simple phylum-level Bacteroidetes: Firmicutes ratio that was initially identified, and it is likely to involve a microbiota-diet interaction.

Obese and lean subjects presented increased levels of different bacterial populations. It is hypothesized that the obese microbiome is set up to extract more calories from the daily intake when compared to the microbiome of lean counterparts. In addition, a caloric diet restriction impacted the composition of the gut microbiota in obese/overweight individuals and weight loss.

In lean subjects there are Coriobacteriaceae, Lactobacillus, Enterococcus, Faecalibacterium prausnitzii, Prevotella, Clostridium Eubacterium, E. coli and Staphilococcus. By contrast, Bifidobacterium, Methanobrevibacter, Xylanibacter, Bacteroides characterize the composition of lean gut microbiota.

For this reason, in a cohort of obese paediatric subjects with visceral adiposity, the aim of the study is to assess the efficacy of a supplementation with probiotic bifidobacteria with respect to a conventional treatment on weight loss and improvement of cardio-metabolic risk factors.

Study design: A single-center pilot open-label randomized control trial. Population: The study will comprise a total of 100 subjects of both sexes, between 6 and 18 years of age, obese, according to the IOTF criteria and with visceral adiposity, as waist circumference ≥ 90th percentile, pubertal stage ≥ 2 according to the Tanner stage, HOMA-IR \> 2,5 or insulin \> 15 µU/ml, diet naïve or with failure of weight loss (defined as -1 kg/m2 BMI in 1 year).

Inclusion/ Exclusion criteria (see Eligibility Criteria). Intervention: In the first part of the study (Study 1, V0-V1) patients will be randomized in a open-label, into two groups homogeneous for number and sex of the subjects. One group will receive a supplementation of probiotic containing Bifidobacterium breve B632 and Bifidobacterium breve BR03, 15 gtt/die (3x108 CFU/die) and one group will receive a placebo for a total of 2 months of treatment. Both group receives a Standard Diet according to routine care and practice. For patients who wants to continue the study there will be a cross-over study (study 2, V2-V3) after one month of wash-out.

Dietary restriction: The standard diet will be distributed with 55-60% of carbohydrates (45-50% complex and no more than 10% refined and processed sugars), 25-30% lipids and 15% proteins, and will be performed in accordance with the calories of an isocaloric balanced diet calculated throughout the Italian LARN Guidelines for age and gender.

Physical activity: all subjects will receive general recommendations about performing physical activity. Exercise will be conducted daily and will consist of 30 minutes of aerobic physical activity.

Randomization: Participants will be randomly assigned in a 1:1 to probiotic intervention group or placebo group.

Timing: Patients will be evaluated firstly at time of enrollment (V0) and, at the end of the first part of study (Study 1, V1), biochemical evaluations will be completed. Next there will be one month of wash-out when the patients don't take any probiotic or placebo. In the second part of the study 2, patients will be evaluated at V2 and, after 2 months of treatment (Study 2, V3). The following anthropometric measures, biochemical and ultrasound evaluations and questionnaires will be obtained:

1. Anthropometric measures:

   * height (V0, V1, V2, V3);
   * weight (V0, V1, V2, V3);
   * body mass index (BMI; Kg/m2) (V0, V1, V2, V3);
   * waist and hip circumferences (V0, V1, V2, V3) for the calculation of the following ratios: waist/hip, waist/height;
   * Tanner stage (V0, V1, V2, V3);
   * blood pressure and heart rate (V0, V1, V2, V3). Biochemical evaluations (after a 12-h overnight fast): CBC with formula, serum insulin-like growth factor 1 (IGF1, ng/mL), 25-hydroxy (OH) vitamin D (ng/mL), uric acid (mg/dL), alkaline phosphatase (U/L), ACTH (pg/mL), cortisol (microg/dL), TSH (uuI/mL), fT4 (ng/dL) (V0, V1, V2, V3); aspartate aminotransferase (AST, IU/L), alanine aminotransferase (ALT, IU/L); AST-to-ALT ratio will be calculated as the ratio of AST (IU/L) and ALT(IU/L) (V0, V1, V2, V3); serum creatinine concentration (mg/dL) will be measured with the enzymatic method; according to the NKF-K/DOQI Guidelines for CKD in children and adolescents, the eGFR will be calculated using updated Schwartz's formula: eGFR (mL/min/1.73 m2) = \[0.413 x patient's height (cm)\] / serum creatinine (mg/dL) (V0, V1, V2, V3); glucose (mg/dL), insulin (μUI/mL); insulin-resistance (IR) will be calculated using the formula of Homeostasis Model Assessment (HOMA)-IR: (insulin \[mU/L\] x glucose \[mmol/lL) / 22.5) (V0, V1, V2, V3); lipid profile: total cholesterol (mg/dL), High-Density Lipoprotein (HDL)-cholesterol (mg/dL), triglycerides (mg/dL); Low-Density Lipoprotein (LDL)-cholesterol will be calculated by the Friedwald formula and non-HDL (nHDL)-cholesterol will be also calculated(V0, V1, V2, V3); oral glucose tolerance test (OGTT: 1.75 g of glucose solution per kg, maximum 75 g) and samples willbe collected for the determination of glucose and insulin every 30 min. The area under the curve (AUC) for parameters after OGTT will be calculated according to the trapezoidal rule. Insulin sensitivity at fasting and during OGTT will be calculated as the formula of the Quantitative Insulin-Sensitivity Check Index (QUICKI) and Matsuda index (ISI). The stimulus for insulin secretion in the increment in plasma glucose as insulinogenic index will be calculated as the ratio of the changes in insulin and glucose concentration from 0 to 30 min (InsI). Βeta-cell compensatory capacity will be evaluated by the disposition index defined as the product of the ISI and InsI (DI) (V0, V1, V2, V3); a collection at rest of first-morning urine sample. Physical and chemical urinalysis; urine albumin (mg/L) will be determined by an advanced immunoturbidimetric assay and urine creatinine (mg/dL) will be measured using the enzymatic method. Urine albumin to creatinine ratio (u-ACR - mg/g), will be calculated using the following formula: \[urine albumin (mg/dL) / urine creatinine (mg/dL)\] x 1000. For these calculations both albumin and creatinine will be in the same unit. The subjects whose urine will be found positive, they will undergo a collection of two more samples and will be considered the u-ACR mean value of these (V0, V1, V2, V3). A sample of feces will be taken for microbic count (V0, V1, V2, V3). LPS (V0, V1, V2, V3). LPS will be measured with commercial kits (Limulus amoebocyte lysate assay) with standard procedures. Citokines IL1, IL1β, IL6, IL10, TNFα will be evaluated (V0, V1, V2, V3) (ELISA kit).
2. A health diary will be taken during the 2 months of treatment: each patient will complete the diary with collateral effects or antibiotic treatment ecc.
3. NGS (Next Generation Sequencing) will be analized for fecal analysis (V0, V1, V2, V3)
4. Metabolomic analysis will be taken with mass spectrometry on fecal samples (V0, V1, V2, V3)
5. SCFA analysis on fecal samples (V0, V1, V2, V3).

Outcomes (see Outcome Measures). Information retrieval: A case report form (CRF) will be completed for each subject included in the study. The source documents will be the hospital's or the physician's chart.

Statistical e sample size: A sample of 16 individuals has been estimated to be sufficient to demonstrate a difference of 10 mg/dl in the basal glucose concentration with 90% power and a significance level of 95% and a drop-out rate of 10% at the 8th weeks of treatment. A sample of 34 individuals in each group has been estimated to be sufficient to demonstrate a difference of 1,4 point in the HOMA-IR index with 90% power and a significance level of 95% and a drop-out rate of 10% at the 8th weeks of treatment. Statistical significance will be assumed at P\< 0.05. The statistical analysis will be performed with SPSS for Windows version 17.0 (SPSS Inc., Chicago, IL, USA).

Organization characteristics: The study will be conducted at the Pediatric Endocrine Service of Division of Pediatrics.

All blood samples will be measured evaluated using standardized methods in the Hospital's Chemistry Laboratory, in Maggiore della Carità hospital, in Novara, previously described. Fecal analysis will be measured in the Department of Sciences and Technologies, University of Bologna, in Bologna.

Good Clinical Practice: The protocol will be conducted in accordance with the declaration of Helsinki. Informed consent will be obtained from all parents prior to the evaluations after careful explanations to each patient.

Eligibility Criteria:
Inclusion Criteria:

1. both sexes
2. between 6 and 18 years of age
3. obese, according to the IOTF criteria (Cole TJ et al., 2000)
4. pubertal stage ≥ 2 according to the Tanner stage (Tanner et al., 1961)
5. HOMA-IR \> 2,5 or insulin \> 15 µU/ml

Exclusion Criteria:

1. Adverse reactions to the product or component of the product (allergies...)
2. Genetic obesity (Prader Willi syndrome, Down syndrome), Metabolic obesity (Laurence-biedl syndrome...), endocrinological obesity (Cushinch syndrome, hypotiroidism)
3. Chronic diseases, hepatic or gastroenterological diseases
4. Medical treatment for chronic diseases
5. Probiotic or prebiotic therapies and antibiotic treatment