Subjects
The study of 32 healthy volunteers was conducted prospectively from April to August 2010 at Osaka Medical College Hospital. Subjects eligible for inclusion were healthy adults who 1) were aged between 20 and 79 years of age at the time of obtaining consent, 2) had freely given their fully informed consent based on their full understanding, and 3) had taken no medication during the one-month period before the start of the study. The exclusion criteria were 1) a history of peptic ulcer or gastrointestinal bleeding, 2) significant hepatic, renal, heart, or respiratory disease, 3) a history of gastrointestinal surgery other than appendectomy, 4) oral use or planned oral use of a drug other than an antiulcer drug, 5) alcohol or chemical dependency, 6) a history of intestinal obstruction or suspected gastrointestinal obstruction on other tests, 7) a lack of consent to the surgery required if the capsule endoscope was retained in the body, and 8) a determination by the investigator, at his discretion, that a subject was ineligible for participation in the study for any other reason. All subjects received oral and written explanation of the study prior to participation and gave written informed consent. The study was conducted in accordance with the Declaration of Helsinki (1995) after the protocol had been approved by the Ethics Review Committee of Osaka Medical College.
Protocol
This was a prospective, randomized, study. Every day for two weeks, the irsogladine group (Group I) received diclofenac sodium 75 mg plus irsogladine maleate 4 mg, and the omeprazole group (Group O) received diclofenac sodium 75 mg plus omeprazole 10 mg. The dose of diclofenac sodium was determined based on the dose approved by the Japanese Ministry of Health and Welfare and the doses used in other clinical trials [8–10]. Generally, the dosage of a PPI used for the prevention of NSAID-induced gastric ulcers is half the dosage used for the treatment of gastric ulcers in Japan. On this basis, we determined that the appropriate dosage of omeprazole should be 10 mg/day.
The subjects were assigned to either Group I or Group O prior to the study. Bowel preparation, capsule endoscopy with a PillCam™SB video capsule (Given Imaging, Yoqneam, Israel) and image evaluation were conducted as previously reported [11]. We conducted a preliminary analysis of the results of these baseline capsule endoscopy examinations to determine subject eligibility for the remainder of the study. Images were analyzed with the software program Rapid Reader 4 (Given Imaging). Lesions were evaluated according to the Los Angeles classification or the Lanza score [12] by esophagogastroduodenoscopy, and the number of small-intestinal mucosal lesions was assessed by capsule endoscopy, serum biochemistry, fecal occult blood, and fecal calprotectin before and after two weeks of treatment. A diagnosis of Helicobacter pylori (H. pylori) infection was confirmed by a blood antibody test at the beginning of the trial.
Esophagogastroduodenoscopy
To standardize the reporting criteria for the endoscopic findings, the two endoscopists (T. K. and E.U.) attended each other’s endoscopic sessions before and regularly during the trial.
Capsule endoscopy
Mucosal breaks in the small intestine were defined as lesions with slough surrounded by erythema, corresponding to the grade 2 category of Goldstein et al. [4]. Typical examples of the bleeding, mucosal breaks and reddish lesions found in this study are shown in Figure 1A–C. Reddish lesions, such as reddened folds, denuded areas, and petechiae, were grouped in a single classification: reddened lesions. Mucosal breaks, reddened lesions and bleeding were identified and evaluated by independent blinded reviewers as described below. The number of mucosal breaks, reddened lesions and sites of bleeding in the small intestine found at baseline and post-treatment by capsule endoscopy was calculated for each subject and compared between Groups I and O. The percentage of subjects with at least one mucosal break in each treatment group was also calculated.
Investigators who were to evaluate the results of capsule endoscopy of the small intestine were required to attend a standardized training session on the use of the Given Diagnostic System. These two investigators (T.K. and E.U.) independently assessed the capsule endoscopic images under blinded conditions. Positive findings were classified as either mucosal bleeding or mucosal injury. If the two observers recorded different findings, they discussed the case until they reached agreement.
Noninvasive tests of intestinal damage
Subjects collected a stool sample for determination of fecal calprotectin as a measure of intestinal inflammation at baseline and the final visit. Stools were frozen within 12 h of receipt and stored at −20°C for subsequent analysis with an enzyme-linked immunosorbent assay kit (Immundiagnostik, Bensheim, Germany) as previously described [13]. Results are expressed as micrograms of calprotectin per gram of stool, and a cutoff value of 50 μg/g stool was used, as recommended by the manufacturer [14]. The fecal calprotectin value suffers the problems of variation, so we determined to use the fold increase after treatment when the calprotectin concentration before treatment was set to 1. Before and after the study, fecal occult blood was assessed with the tetramethylbenzidine and guaiac tests by using occult fecal blood slide kits from Shionogi Pharma (Osaka, Japan). In both tests, the color intensity of the oxidation product was assigned to one of three categories, +, ± or −, and on this basis differences between before and after treatment were denoted “exacerbation”, “invariable” or “improvement”. The hemoglobin and transferrin antibody tests for occult fecal blood were performed with an OC-Micro analyzer (Eiken, Tokyo, Japan). Generally, fecal occult blood is influenced by the intake of meat, fish, bright red, green or yellow vegetables, and so on. Therefore, we explained to our subjects how these foods affect the results of the occult blood tests(the tetramethylbenzidine test and the guaiac test), and suggested that they pay attention to their food intake during the period 4 days prior to the examination date.
Sample size
The sample size was based on our estimation of the proportion of subjects that would be expected to exhibit mucosal breaks at post-treatment by capsule endoscopy. We estimated that the incidence of mucosal injuries would be approximately 20% in the irsogladine group, on the basis of a preliminary study by Niwa et al. [8] showing that the incidence of NSAID-induced small-intestinal lesions was lower in subjects on daily rebamipide (20%) than in subjects on placebo (80%). In rats, irsogladine suppresses indomethacin-induced small-intestinal lesions as effectively as rebamipide [7]. In addition, we estimated that the incidence of mucosal injuries would be approximately 70% in the control group, because a recent study found small-intestinal lesions in 55–68% of subjects taking NSAIDs [3, 4]. Thus, 15 subjects would need to be recruited to each group (30 subjects in total) for a chi-square test, a significance level of 5% (two-sided), a power of 80%, and equal allocation. On the assumption that two subjects would not be able to complete the study, a minimum of 32 subjects was required.
Randomization
A coordinator performed a simple fixed-allocation randomization by using a block-randomization scheme. Random numbers were generated by SAS (SAS Institute, Cary, NC, USA).
Statistics
For continuous or categorical variables, the statistical significance of differences between groups was determined with the t test or Wilcoxon rank-sum test, and the statistical significance of differences within a group was determined with the Wilcoxon signed-rank test. For binary variables, the statistical significance of differences between groups was determined with the chi-square test. All reported p values are two-sided, and values of less than 0.05 were considered to indicate statistically significant differences. All statistical values were calculated with SAS Ver. 9.2 (SPSS, Chicago, IL, USA), Windows Edition.