Study design
This was an observational study involving patients with chronic constipation. Patients were recruited from gastroenterology outpatients departments at four institution (Yokohama City University Hospital, Namiki Koiso Clinic, Iwasaki Naika Clinic, and Kanagawa Dental University Yokohama Clinic) from February 6, 2021, to June 30, 2022.
Ethical considerations and registration
The study protocol followed the Declaration of Helsinki [5] and the Ethics Guidelines for Clinical Research published by the Ministry of Health, Labour and Welfare, Japan. This study was approved by the Ethics Committee of Yokohama City University Hospital on December 25,2020 (approval number: B201100034). This trial was registered in the University Hospital Medical Information Network (UMIN) Clinical Trials Registry as UMIN000043262 on February 5, 2021. Written informed consent for participation in the study was obtained from all participants.
Patients
The inclusion criteria for the study were as follows:1) 20-79 years of age as of the date of informed consent, 2) patients diagnosed or previously diagnosed with chronic constipation according to the Rome IV criteria [6], and 3) willingness to provide written informed consent. The exclusion criteria were as follows: 1) patients who could not collect specimens due to defecation disorder; and 2) patients who were judged by the physician to be unsuitable for the study.
Study protocol
Patients were observed for a baseline of two weeks, during which time information about their histories, blood tests, and bowel habits was gathered. Patients were also assessed for disease-specific QOL, using the Japanese version of the PAC-QOL (Japanese Patient Assessment of Constipation Composite Index JPAC-QOL) [7,8]. The enrolled patients received two tablets of BBG9-1 three times per day for 8 weeks. Participants were permitted to continue taking any medication for constipation they were previously taking during the study period without changing the dosage. During the study period, no new drugs for the treatment of constipation were permitted save the study product. The participants were also told to continue taking their normal diets and nutritional supplements during the research time. In the absence of defecation for several days, participants were allowed to take 48 mg of sennoside. During the 8-week study period, participants recorded their daily bowel movements during the screening period. Records containing data for more than five days per week were required for the data to be considered valid. Fecal samples were collected before the start of treatment and 8 weeks after BBG9-1 administration for SCFA analysis. All patients were instructed to visit the study site 8 weeks after the initiation of treatment. At the visit, patients completed the JPAC-QOL and were interviewed about any side effects, their defecation diaries, and numbers of residual study products were checked. The schematic of this study is shown in Fig1.
Study product
The study product was BBG9-1 tablets (Biofermin® tablets, Biofermin Pharmaceutical Co., Ltd., Kobe, Japan). One tablet contained 12 mg (viable cell count: 1×106–1×109 CFU/g) of Bifidobacterium bifidum. The doctors in charge of the study prescribed the study product at the start of the study at the study site. The participants were instructed to take two tablets of BBG9-1 daily after each meal. Compliance was monitored by counting the number of remaining BBG9-1 tablets at the end of the 8-week study period.
Outcome measures
The primary endpoint of this study was the quantity of SCFAs in feces, such as butyric acid, acetic acid, and others. The secondary endpoints were changed in the Japanese version of the Japanese Patient Assessment of Constipation Composite Index (JPAC-QOL), frequency of bowel movements, stool form, sense of incomplete evacuation, straining, number of rescue use, SBM (defecation without rescue attempts), and CSBM (defecation without rescue attempts and no residual stool sensation).
Before and after BBG9-1 administration for 8 weeks, the variation in JPAC-QOL scores was compared. Additionally, the JPAC-QOL scores before, 4 weeks after, and 8 weeks after therapy were looked at. A reliable and targeted self-administered questionnaire called the PAC-QOL was created and validated to evaluate quality of life impairments in people with chronic constipation. [7]. It consists of 28 questions, each on a 5-point Likert scale (0, not at all, 1, slightly, 2, moderately, 3, quite a bit, 4, extremely or a great deal). Physical discomfort, psychological discomfort, worry/concern, and satisfaction are the four subscales of the JPAC-QOL scale. The average score for each item is used to express the total subscale scores [8].
Stool form was scored using the BSFS, which is a visual scale for stool type graded from type 1 to type 7 (type 1, hard lumps, type 2, sausage shaped but lumpy, type 3, like a sausage but with cracks on the surface , type 4, like a sausage or snake, smooth and soft, type 5, soft blobs with clear-cut edges, type 6, fluffy pieces with ragged edges, a mushy stool, type 7, watery, no solid pieces, entirely liquid)[9]. Patients recorded their own stool consistency according to the BSFS scale in their recording diary. Incomplete sensation of evacuation was assessed on a binary scale (0, absent; 1, present), and the degree of straining was assessed on a 5-point ordinate scale (1, none, 2, mild, 3, moderate, 4, strong, 5, extremely strong). These were also evaluated using patient self-assessments. The mean scores for the baseline period, the latter two weeks of 4 and 8 weeks of BBG9-1 administration, and the washout period were analyzed. Patients who experienced an incomplete sense of evacuation during more than half of their defecation events were defined as experiencing an incomplete sense of defecation. Frequency of bowel movements was defined as the number of days with at least one bowel movement.
Analysis of SCFA
LC-MS/MS chromatography-mass spectrometry of fecal samples were collected and was immediately stored at -80℃ until analysis. Pretreatment was performed according to the Shimazu Manual. SCFAs were analyzed according to a previously described method[10].
Statistical analyses
Data are expressed as mean ± standard deviation, unless indicated otherwise. We analyzed the data using JMP 15.0 (SAS Institute Inc, Cary, North Carolina, USA). The contentious variables were analyzed using Student`s paired t-test without adjustment for multiple testing. All t-tests were two-sided, with a significance level of 5% (p = 0.05).