Tablet formulation

The active product B. coagulans MTCC 5856, 2 billion spores per tablet (2 × 109 spore/tablet), was supplied by Sabinsa Corporation, Utah, USA. Tablets were packed in 70 mL HDPE container. Each active had 2 billion spores per tablet i.e., 333.33 mg of B. coagulans MTCC 5856, 222.67 mg of microcrystalline cellulose, 10 mg of starch, 30 mg of sodium starch glycolate and 4.0 mg of magnesium stearate. Viable spore count of B. coagulans MTCC 5856 was determined as per the method described previously [13] Briefly, 1.0 g of B. coagulans MTCC 5856 was mixed in sterile saline (0.9 % NaCl, w/v) and then incubated in a water bath for 30 min at 75 °C, followed by immediate cooling to below 45 °C. The suspension was further serially diluted in sterile saline and the viable count was enumerated by plating on glucose yeast extract agar (HiMedia, Mumbai, India) by pour plate method. The plates were incubated at 37 °C for 48–72 h. Analysis was performed twice in triplicate. Average means of spore viable counts was expressed in cfu/g. For the placebo tablet, maltodextrin of equivalent weight was used and formulated in similar shape and size as that of the active, and packed in HDPE containers.

Ethics and informed consent

The trial (Clinical Trial Registry India, # CTRI/2014/03/004502) was conducted in three clinical sites i) Mysore Medical College and K R Hospital, Mysore, India ii) Sapthagiri Institute of Medical Sciences and Research Center, Bangalore, India and iii) Kempegowda Institute of Medical Sciences, Bangalore, India. The institutional ethics committees of the aforesaid clinical sites provided a written favorable opinion for the conduct of this study in respective clinical sites. No further changes or amendments were made to the approved protocol and the study was executed in its complete form. This trial was conducted in accordance with the principles enunciated in the Declaration of Helsinki (Edinburgh, 2000) [14] and the ICH-harmonized tripartite guideline regarding good clinical practice (GCP). Written and oral information about the study was provided to all the subjects in a language understandable by the subject. Every subject was informed by the investigator, prior to the screening evaluation, of the purpose of this clinical trial, including possible risks and benefits and documented the informed consent process in the subject’s chart. Sufficient time was provided for each subject to decide whether to participate in the study and all the questions and clarifications regarding the study were clarified by the investigator.

Study design and selection of study subjects

This randomized, double blind, parallel group, placebo controlled, multi-centered study had a total of 5 visits to the clinical site by the study subjects, besides screening visit. Subjects were included in the study if indicated “Yes” to all of the inclusion criteria and “No” to any of the exclusion criteria. Inclusion Criteria: 1) Male or female subjects ranging in age from 18 to 55 years (both inclusive) diagnosed as having gastro intestinal disorders and based on the medical history record were included in the study by the investigator. 2) Fulfilling Rome III diagnostic criteria for functional IBS [15]. Criterion fulfilled for the last 3 months with symptom onset at least 6 months prior to diagnosis. a) Recurrent abdominal pain or discomfort (uncomfortable sensation not described as pain) at least 3 days/month in the last 3 months associated with two or more of the following: (i). Improvement with defecation (ii). Onset associated with a change in frequency of stool (iii). Onset associated with a change in form (appearance) of stool. b) Recurrent feeling of bloating or visible distension at least 3 days/month in the last 3 months. c) Loose (mushy) or watery stools without pain occurring in at least 75 % of stools. 3) Willingness to follow the protocol requirement as evidenced by written, informed consent. 4) Willingness to complete subject diaries and respond to study questionnaires. 5) Except standard treatment of care, agree not to use any other (including vitamins and minerals) medication during the course of the study. 6) Agree not to use any yogurt during the course of this study. 7) Subjects whose blood chemistries are within a normal range or not considered clinically significant if outside the normal range. 8) Subject’s assurance that they have not taken antibiotics or other products whose primary site of action is in the gastro intestinal tract (GIT) for a period up to 1 month prior to the start of the study. Exclusion Criteria: 1) Sufficient criteria for a diagnosis of functional dyspepsia or other functional GI disorder. 2) Any clinically significant medical history, medical finding or an ongoing medical or psychiatric condition exists which in the opinion of the Investigator could jeopardize the safety of the subject, impact validity of the study results or interfere with the completion of study according to the protocol. 3) Significant abnormal findings as determined by baseline history, physical examination, vital signs (blood pressure, pulse rate, respiration rate) hematology, serum chemistry, urinalysis. 4) History or presence of significant alcoholism or product abuse in the past one year. 5) Participation in a clinical study during the preceding 90 days. 6) History of malignancy or other serious disease. 7) Any contraindication to blood sampling. 8) Smoking or consumption of tobacco products. 9) Blood or blood products donated in past 30 days prior to study ingredient administration. 10) Female subjects on pregnancy and lactating women.

Sample size calculations, randomization and treatment allocation and procedures

A sample size of 30 subjects was calculated based on a 40 % reduction in severity of clinical symptoms of IBS (bloating, vomiting, diarrhea, abdominal pain, stool frequency) between the two treatment groups with a power of 80 % at the 5 % level of statistical significance. With an expected dropout rate of 20 %, the sample size was increased to 36 (18 subjects per group). Eligible subjects were randomized in a 1:1 ratio (placebo: active) in a randomly-permuted order by computer into 3 blocks of 16 each, along with overages. Each participant was assigned a 6-digit randomization code and the respective site personnel dispensed the investigational product as per the randomization code list generated by an independent statistician. Clinical site staff and participants remained blinded to the treatment received throughout the course of the study. Double blinding was accomplished by independent blinding of the dosing kits. Newly diagnosed or untreated patients who were not on any other treatment in the past 3 months with mild to moderate IBS in severity were enrolled into the study. Two drugs, one a combination of Domperidone 30 mg and Esomeprazole 40 mg, and the other drug Metronidazole 400 mg, once a day was considered as standard treatment of care for diarrhea predominant IBS for the study subjects for both active and placebo groups, by the investigators of the three clinical sites. In addition to the aforesaid standard treatment, subjects were asked to self administer one tablet per day, either placebo or active, at least 30 min before a meal, preferably in the morning as a dietary supplement for a period of 90 days. This was subject to a gap of at least 4 h between the study product (placebo/active) and standard care of treatment was maintained. Subjects used this product on an outpatient basis and were advised to return for clinical evaluation at day 30, day 60, day 90 and day 105. The dosing period was for 90 days.

Compliance with study supplement was reviewed at each visit by examination of the returned supplements. All accountability records were incorporated into the investigator’s study file. The patients were instructed against the use of any kind of yoghurt during the study duration. The daily food intake of the patients was recorded in the patient diaries provided to them at visit 1. The same was verified at subsequent visits by the investigators. Respective hospital laboratories were used for all assessments pertaining to this study. Clinical trial monitors who were independent of the study staff monitored the progress of all clinical investigations that were conducted and ensured that the protocol is adhered in all aspects. Data collection during this clinical study and statistical analysis were performed by separate functional groups and a certified, independent statistician respectively. No changes or amendments were made to the approved protocol after the trial commenced and no interim analysis was done during the study period. The screening and enrollment of study subjects is seen in Fig. 1.

Fig. 1 Flow chart of study procedures. Legend: From eligible thirty six subjects who were fit into the inclusion and exclusion criteria, 31 completed the study. At every follow up visit, study evaluations and assessments were made in both the study groups Full size image

Safety and efficacy outcomes

The safety outcomes were measured by: 1) Physical examination and vitals, 2) Assessment of reported adverse events (AEs), if any. The primary efficacy outcomes were measured by 1) modified gastro intestinal (GI) discomfort questionnaire for bloating, vomiting and diarrhea [16] 2) Stool frequency and consistency by subjective evaluation using Bristol stool form score [17]. 3) Self assessment of abdominal pain, measured on a 10 cm visual analog scale –VAS [18], 2) The secondary efficacy outcomes were measured by 1) Physician’s global assessment for disease severity [19], 2) A 34 item IBS quality of life (QOL) questionnaire [20].

Statistical analysis

The baseline values of VAS, GI discomfort questionnaire, Bristol stool form score, Physician’s global assessment and IBS QOL questionnaire were compared to that of end of study visit for both treatment groups by appropriate statistical tools. Statistical analysis software (SAS) version 9.2 software was used for data analysis. Paired ‘t’ test, analysis of covariance (ANCOVA) and Wilcoxon signed rank sum test were used for appropriate data set variables to reach the best possible statistical conclusion between the active and placebo receiving groups. A ‘p’ value <0.05 was considered as statistically significant. The baseline descriptors were summarized as mean and standard deviation for continuous variables and as frequencies and percentages for categorical variables. Last observation carry forward (LOCF), the intent to treat method was followed for efficacy evaluations of subjects.