Effects of a Protease Inhibitor Camostat Mesilate on Gut Microbial Function in Patients with Irritable Bowel Syndrome: A Pilot Randomized Placebo-Controlled Study

Irritable bowel syndrome (IBS) is one of common disorders of gut-brain interaction (DGBI) of multifactorial origin [1, 2]. The pathophysiology of IBS has not been well understood so far although several functional alternations on brain-gut axis have been described. Recently, studies suggest a role for altered gut microbiota and bacterial metabolites in IBS [3].

The coordinated interactions of host (e.g., mast cells) and bacterial-derived proteases maintain homeostasis but become relevant to impaired intestinal immune and barrier function if deregulated [4]. Increased fecal protease activity (PA) has been observed in patients with IBS [5, 6]. In a rodent model, fecal supernatants of diarrhea-predominant IBS patients with elevated serine protease contents induced visceral hypersensitivity and increased paracellular permeability via the protease-activated receptor (PAR-)2 [5]. In addition, IBS subset with high fecal PA has decreased fecal microbial diversity and different community structure compared with the low PA subset [6]. From these points of view, serine protease is considered to play an important role on pathophysiology of IBS.

The stress response of the hypothalamic-pituitary-adrenal axis is attenuated via negative feedback though binding of cortisol to glucocorticoid receptors in the hypothalamus and pituitary [7]. In animal models, augmentation of the hypothalamic-pituitary-adrenal axis has been linked to increased visceral sensitivity, a cardinal feature of IBS [8]. FK506-binding protein (FKBP), which shows peptidylprolyl cis-trans isomerase (PPIase) activity, plays a role in immune regulation [9] and interacts with glucocorticoid receptors [10]. Serine proteases can modulate PPIase activity. Thus, FKBP-type PPIase activity might contribute to visceral sensitivity and/or mucosal barrier function in the gut.

It has been investigated that camostat mesilate (CM), one of the serine protease inhibitors, dose-dependently inhibited stress-induced colonic hypersensitivity and decreased fecal PA in an animal model [11]. The other kinds of serine protease inhibitors including nafamostat mesilate also reversed colorectal pain sensitivity in rats with post-inflammatory visceral hypersensitivity [12]. Furthermore, nafamostat mesilate reduced enhanced mucosal permeability of rectal biopsy tissues from patients with diarrhea-predominant IBS in vitro [13]. These preclinical findings indicate that serine protease inhibitors might ameliorate GI symptoms as a new potential therapeutic strategy for IBS.

It has not been confirmed so far whether protease inhibitors modify gut microbial function in patients with IBS. The aim of the present study was to investigate whether CM modifies fecal bacterial function related to serine protease and/or FKBP-type PPIases using a pilot randomized, double-blind, placebo-controlled trial in patients with IBS.

Thirty-four patients with IBS meeting Rome III criteria [14] were recruited by advertisement at Tohoku University Campus in Sendai, Japan. Subtyping of predominant bowel habit classified patients as IBS with diarrhea (IBS-D), IBS with constipation (IBS-C), mixed IBS (IBS-M), or unsubtyped IBS (IBS-U) [14]. Of 34 patients, 32 were finally included in this study (online suppl. Fig. S1; for all online suppl. material, see https://doi.org/10.1159/000542758). In addition, 21 healthy subjects without any significant or recurring GI symptoms participated as healthy controls (HC). No subjects had visited at any healthcare providers. Any other medication had not been prescribed in all subjects at the time of the study.

Each participant carefully underwent a medical interview and physical and laboratory examinations so that subjects with organic disease could be excluded. The subjects were excluded if they had undergone abdominal surgery other than appendectomy, had suffered from any serious physical or psychiatric complication, and they were not pregnant at the time of study. Participants were also excluded if they had suffered from infectious gastroenteritis and/or had been treated with probiotics or antibiotics within 6 months prior to enrollment in the study.

Symptoms and quality of life (QOL) were assessed by the validated questionnaires on day 0. A binary adequate relief (AR) of global IBS symptoms [15] and GI symptom severity were asked just after treatment (on day 14, for patients alone). GI symptom severity was evaluated by the IBS Symptom Severity Scale (IBS-SSS) [16, 17], psychological tendencies were evaluated by the Self-rating Depression Scale (SDS) [18, 19] and State-Trait Anxiety Inventory (STAI) [20, 21], and disease-specific QOL was examined by the IBS-QOL [22, 23].

Patients were requested to fast for at least 12 h prior to sensory and motility testing. The barostat is a computer-controlled pump (Distender II model; G&J Electronics, Willodale, ON, Canada) used for testing perception thresholds and smooth muscle tone in the lumen of the bowel [24]. At approximately 9:00 a.m., the barostat catheter was placed in the rectosigmoid colon prior to testing. All sensory and motility testing was performed with the subject lying in a left-lateral position according to the previous studies [24‒26].

After sample distentions were performed by inflating the barostat bag, the individual operating pressure (IOP) during measurement of smooth muscle tone was determined as the minimum distending pressure required to overcome mechanical forces plus 2 mm Hg [25, 26]. Perception thresholds in the rectosigmoid colon were assessed using the ascending method of limits [24]. Phasic distentions were 30 s in duration and were separated by 30-s rest intervals, starting at 2 mm Hg and increasing by 2-mm Hg steps until either the subject requested the protocol be stopped or a pressure of 40 mm Hg was reached. Subjects were instructed to report their moderate pain and urgency to defecate experienced at the end of each distention using a six-point scale (0 = no sensation; 1 = weak; 2 = mild; 3 = moderate; 4 = strong; 5 = intense). The moderate pain or urge threshold was defined as the pressure at which the subject first reported moderate pain or urge to defecate [25]. The pain or urge threshold was confirmed by applying pressure around the thresholds randomly in 2-mm Hg steps every 30 s.

After patients rested for at least 10 min after the sensory testing, rectosigmoid smooth muscle tone and contractility at IOP were evaluated during 20 min at the resting period. Average bag volume at IOP was recorded as a measure of smooth muscle tone. Phasic volume events (PVEs) served as a measure of phasic contractions [24]. To control for occasional, minor changes in muscle tone, the bag volume had to differ more than 10% from the baseline tone occurring at a frequency of 1–4 per minute to be characterized as a change.

Additional motility response was observed after intravenous administration of corticotropin-releasing hormone (CRH, 2 μg kg⁻¹) just after the resting baseline on day 14 alone. Average smooth muscle tone and number of PVEs at IOP were evaluated for 60 min to observe the motility response to peripheral CRH administration in the same way of our previous study [26].

We determined a target sample size of 17 participants in each group, assuming a 15% dropout rate, estimated based on previous GI physiological studies using a barostat test for IBS as a preliminary study. All participants were instructed to keep their usual diet at least 14 days before and during the study. In all subjects, fecal and blood samples were collected, subjective symptoms were assessed, and colonic sensitivity and smooth muscle tone were measured with the barostat test at the beginning of the study (on day 0).

This study was conducted as randomized, double-blind, placebo-controlled clinical trial for patients with IBS alone. After the pretreatment period, eligible patients were randomly assigned regarding age, gender, and IBS subtype to receive either placebo or CM at a dose of 200 mg po tid for 14 days ending with fecal and blood sampling, the self-reported questionnaires on AR and the IBS-SSS, and the visceral sensory and motility testing again on day 14. Randomization into two groups (equal ratios) was performed by an independent collaborator (K.I.) using a Web-based randomization system. Patients and clinical investigators were unaware of the study-group assignments.

Stool samples were collected and frozen by patients, brought to the hospital, and deep-frozen at −80°C before the sensory and motility testing (i.e., on day 0 and day 14). The collected samples were sent to the laboratory of Miyarisan Pharmaceutical Co., Ltd., and stored at −80°C. To conduct 16S ribosomal RNA gene sequencing, DNA was extracted from the fecal samples using a glass bead extraction method and purified, according to a previously reported method [27]. The amount of DNA was determined using a QuantiFluor dsDNA System and Quantus Fluorometer (Promega, Madison, WI, USA). Genomic DNA from 20 Strain Even Mix Genomic Material (ATCC MSA-1002) was used in the study to evaluate data analysis procedures.

The V3-V4 region of the 16S rRNA gene was amplified from stool using a TaKaRa Ex Taq Hot Start PCR mixture (TAKARA Bio., Shiga, Japan). The primers used for PCR amplification were 341F and 785R, which contained Illumina index and sequencing adapter overhangs, according to a previously reported method [28]. The PCR products were purified using SPRIselect (Beckman Coulter, Brea, CA, USA). DNA concentrations were quantified with a QuantiFluor dsDNA System and Quantus Fluorometer; then equal amounts of purified PCR products were pooled for subsequent Illumina MiSeq sequencing. Sequencing was carried out with a MiSeq Regent Kit V3 (600 cycles) (Illumina, San Diego, CA, USA), according to the manufacturerʼs instructions. Sequence processing and quality assessment were performed using the Quantitative Insights Into Microbial Ecology (QIIME) package (version 1.9.1) (https://qiime2.org/) [29]. Paired-end reads were merged using the fastq-join script in ea-utils with the parameters m = 6 and p = 20, then quality filtered using QIIMEʼs script split_libraries_fastq.py (r = 3, p = 0.75, q = 20, n = 0). De novo and reference-based chimera detection and removal were performed using USEARCH v6.1 [30] with the Greengenes v13.8 database. Operational taxonomic units (OTUs) were chosen using an open reference OTU-picking pipeline against the 97% identity of the pre-clustered Greengenes v13.8 database using UCLUST [28]. Data quality was evaluated using sequence data from ATCC MSA-1002. A representative sequence for each OTU was aligned with PyNAST [31]. Bacterial taxonomy was assigned using UCLUST. Diversity analyses were performed with QIIME script core_diversity_analyses.py using high-quality 5,000–7,000 reads/sample. Principal coordinate analysis was performed by weighted unifrac distance. Statistical significance of sample groupings was assessed using permutational multivariate ANOVA (PERMANOVA) (QIIME script compare_categories.py). To determine bacterial taxonomy that explained differences between conditions, linear discriminant analysis (LDA) effect size (LEfSe) method was used [32]. LDA values >2 were considered significant.

Predicted metagenomics analyses were performed as follows. The metagenomic content of samples was inferred from 16S rRNA gene sequence data using Phylogenetic Investigation of Communities by Reconstruction of Unobserved States and the Kyoto Encyclopedia of Genes and Genomes database. PICRUSt analysis [33] was performed by using the online galaxy version 1.0.0 (http://huttenhower.sph.harvard.edu/galaxy/).

Numerical values are expressed as the mean ± standard deviation (SD). After checking the normal distribution, Student’s t test was used to compare between groups. For non-normal distributions, the Mann-Whitney U test was used. Two-way analysis of variance or generalized estimating equations were used to compare changes before and after treatment in the symptom severity scores, the concentrations of serum cytokines or hormones, the sensory and motility data, and the relative abundances of 16S rRNA gene sequence data with >1.0% genus level between CM and placebo groups. The Spearman correlation coefficient was used to assess the relationships among these parameters. The relative abundances of the predicted 16S rRNA genes and metagenome function were also compared between patients who relieved IBS symptoms after treatment (i.e., responders) and those who did not considering a potential confounder. For all analyses, a p value of 0.05 defined statistical significance.

Characteristics of the participants are shown in Table 1. Overall, patients showed significantly higher scores of IBS symptom severity and psychological symptoms (with the exception of the trait anxiety score on the STAI) and lower IBS-QOL scores compared with HC. There was no significant difference in age, gender ratio, the proportion of subtypes, or the scores on the symptoms or QOL between CM and placebo groups at the pretreatment period on day 0 (online suppl. Table S1).

The urge thresholds at baseline in IBS patients were significantly lower than those in HC (p < 0.05, respectively, Table 2). There was no significant difference in the pain threshold, IOP, the average smooth muscle tone, or the number of PVEs at baseline between IBS and HC. There was no significant difference in any sensory or motility parameter between CM and placebo groups at the pretreatment period (Table 3).

In the fecal samples at baseline, LDA effect size (LEfSe) revealed that the relative abundances with >1.0% genus level of Faecalibacterium and Coprococcus were significantly higher and that of Dialister was significantly lower in patients with IBS compared with HC (Fig. 1a–d). Among IBS, there was no difference in the relative abundance of OTU between CM and placebo groups at the pretreatment period.

With respect to fecal metagenome function, there was no significant difference in relative abundance of any FKBP-type PPIase between IBS and HC at baseline (online suppl. Table S2). The relative abundance of serine protease showed a trend toward a lower level in IBS compared to HC (p = 0.06). Among IBS, there was no significant difference in the relative abundance of serine protease or any FKBP-type PPIase between CM and placebo groups at the pretreatment period. Also, there was no difference in the metagenomic function among the subtypes of IBS (data were not shown).

After the treatment on day 14, there was no significant difference in the proportion of AR (43% vs. 57%) between CM and placebo groups. There was no significant difference in the score of IBS-SSS except abdominal bloating (43.4 vs. 19.4, p = 0.02) between the treatment groups (online suppl. Table S3). There was no difference in the responder rate or the symptom severity among the subtypes of IBS (data were not shown). No adverse event has been observed during the study.

In the fecal sample at the posttreatment period on day 14, the permutational multivariate ANOVA (PERMANOVA) detected significant differences in fecal microbiota comparisons between CM and placebo groups (p < 0.05, online suppl. Fig. S2). LEfSe revealed that the relative abundance with >1.0% genus level of Streptococcus was significantly higher in CM group than placebo group (Fig. 2a, b). Generalized estimation equation analyses revealed that the relative abundance of Streptococcus was significantly increased in CM group compared with placebo group (p < 0.05, interaction effect, Fig. 2c). LEfSe also showed that the relative abundances with less than 1.0% genus level of the class bacilli, the family Porphyromonadaceae, and the genus Parabacteroides were significantly higher in CM group than placebo group (Fig. 2a).

The relative abundances of serine protease (K14645; p < 0.05, interaction effect, Fig. 3a) and FKBP-type PPIase FkpA (K03772; p = 0.05, Fig. 3b), FklB (K03773; p < 0.05, Fig. 3c), and SlyD (K03775; p < 0.05, Fig. 3e) were significantly increased in CM group compared with placebo group but those of SlpA (K03774; p > 0.1, Fig. 3d) were not. There was no statistically difference in effects of CM for FKBP-typePPIase activity and serine proteases among subtypes of IBS (data were not shown). The relative abundance of serine protease was positively correlated with FKBP-type PPIase FkpA, FklB, and SlyD at the baseline and posttreatment periods, respectively (online suppl. Table S4).

No significant difference was demonstrated regarding changes in the relative abundance of Streptococcus or any predicted metagenome function between responders who reported AR and nonresponders after either treatment (online suppl. Table S5). These findings were still maintained after adjusting the types of treatment. In other words, CM modulated the fecal microbiome composition and functional potentials that are related to FKBP-type PPIase activity regardless of the self-reported general symptom relief.

There was no significant difference in any perception threshold between the treatment groups at the posttreatment period. Lower number of PVEs was found in CM group compared to placebo at the resting baseline and at the first 20 min after CRH administration (p = 0.02 and p = 0.06, Table 3). The number of PVEs was significantly increased after CRH administration in both CM and placebo groups (p < 0.01, treatment effect), although a significant group or interaction effect failed to be shown (see Table 3).

This preclinical study aimed to confirm whether oral administration of CM modifies fecal bacterial metagenome function of serine protease and FKBP-type PPIases, which may regulate immune function and glucocorticoid receptors. We also investigated whether CM ameliorates not only IBS symptoms but also visceral sensitivity and motility. The main findings of the present study were 14-day administration of CM (200 mg tid, equivalent to usual clinical dosage for patients with chronic pancreatitis), significantly altered gut microbiota composition, and metagenome function associated with serine protease and FKBP in the fecal samples of IBS. CM was not superior to placebo in improvement of the self-reported IBS symptoms or visceral pain sensitivity although colonic motor activity partially changed.

This is the first randomized placebo-controlled double-blind trial to assess the efficacy and safety of CM in patients with IBS. It has been demonstrated that psychological stress induces gut barrier dysfunction [34] and that increased mucosal permeability is associated with symptom severity of IBS [35]. It has also been reported that tryptase activity is associated with increased mucosal permeability of rectal biopsy tissues from patients with diarrhea-predominant IBS in vitro and the elevated permeability could be repressed by addition of a tryptase inhibitor, nafamostat [13]. Furthermore, a mast cell stabilizer and a histamine 1 (H1) receptor antagonist, ketotifen, ameliorated visceral hypersensitivity and decreased abdominal pain and other bowel symptoms after 8 weeks of treatment in patients with IBS, whereas this agent did not affect rectal mucosal mast cell counts or the spontaneous release of tryptase or histamine [36]. Therefore, mast cells and their mediators in the intestinal mucosa may play important roles in the pathophysiology of IBS [37].

Despite our expectation, 2-week oral administration of CM (200 mg tid) did not significantly improve IBS symptoms or modify colonic sensitivity or CRH-stimulated motor activity compared with placebo in the present study. The clinical dosage of CM for remission of acute symptoms of chronic pancreatitis is up to 600 mg daily (200 mg tid) in Japan. It has been reported that improvement of epigastric pain severity and no serious adverse events were observed after administration of 200 mg tid of CM at 2 and 4 weeks in patients with functional dyspepsia [38]. Thus, we chose the dosage and duration in patients with IBS as a pilot study for safety reasons. Previously, it has been demonstrated that much higher doses of CM (i.e., single administration of 30–300 mg/kg) dose-dependently inhibited stress-induced colonic hypersensitivity and paracellular permeability in an animal model [11]. Thus, administration of higher dose of CM might be required to ameliorate abdominal pain and abnormal bowel habit in a similar fashion. On the other hand, a higher placebo response rate was observed in the present study. It was investigated that pooled placebo response rate in pharmacological treatment for IBS was 37.5% and was the highest especially in trials using shorter duration (1–4 weeks) at 46.0% [39]. Further sophisticated clinical trials for IBS patients using different (especially higher) doses of CM should be warranted to confirm the hypothesis in consideration to safety.

In the present study, the relative abundances of Faecalibacterium and Coprococcus were significantly more increased and that of Dialister was significantly more decreased in the baseline fecal samples of patients with IBS compared with those of HC. It has been confirmed that Faecalibacterium and Coprococcus are butyrate producers [40]. Some studies demonstrated a significant increase in the proportion of total fecal short chain fatty acids (SCFA) in patients with IBS [41, 42]. Dialister species are known to generate acetate, lactate, and propionate but not butyrate [43]. It has been reported that Dialister abundance was negatively correlated with IBS symptom severity, anxiety, and depression level [44]. Butyrate promoted the secretion of nerve growth factor, which may be involved in butyrate-induced noninflammatory visceral hypersensitivity from enteric glial cells in the colon in an animal model [45]. However, several studies that have compared gut microbiota composition in patients with IBS to healthy subjects have failed to provide consistent findings [46]. The variability in findings among studies is possibly due to the heterogeneity of disease pathophysiology, differences in study design, and different study samples from different cultural populations. Thus, multicenter studies using a common protocol from different counties should be needed to ensure generalizability and reproducibility as a next step.

We found that the relative abundance of Streptococcus was significantly increased in the fecal samples of CM group compared with those of placebo group after treatment regardless of symptom improvement. Interestingly, our fecal metagenome function analyses revealed that CM significantly leaded to increased relative abundances of serine protease and FKBP-type PPIases that may regulate immune function [9] and stress response [10]. In this study, the relative abundance of serine protease was positively correlated with those of FKBP-type PPIases (FkpA, FklB, and SlyD). Streptococcaceae family encompasses protease producers [47]. The PPIase activity is required for maturation of the secreted streptococcal protease as a trigger factor [48]. Carroll et al. [49] confirmed that Streptococcaceae family was positively associated with fecal PA in humans. LEfSe also showed that the class bacilli, the family Porphyromonadaceae, and the genus Parabacteroides were significantly higher in CM group than placebo group. Bacillus spp. can produce extracellular enzymes such as glycoenzyme, protease, and lipase and also has antibacterial and immune regulation effects [50]. Porphyromonadaceae plays a role as adiposity modulators through the production of SCFA, acetate, and propionate [51]. Parabacteroides is associated with regulating immunity, relieving inflammation, carbon metabolism, secreting SCFA, and antibiotic resistance [52]. These bacterial functions may apply a variety of survival strategies as residents of the gut, some of which may be beneficial to humans.

Proteolytic activity (PA) is often tightly regulated at different levels (i.e., host-derived serine proteases and gut microbial proteases) under physiological conditions [53]. Both host and gut microbial serine peptidases are assumed as potential actors of this crosstalk. Proteolytic imbalance and its dysregulation may be associated with inflammation and/or pain sensitivity in the gut. Although few reports have previously revealed significant contributions of bacterial proteases to the proteolysis in the human large intestine, maintaining the GI health has been reported to involve a sophisticated interplay between serine proteases, their inhibitors, and specific receptors known to be localized on various cells of the human intestinal epithelium under physiological conditions [47]. Accordingly, inhibition of PA caused by CM treatment might promote compensatory increase in bacterial composition of protease producers (e.g., Streptococcaceae family) to maintain the GI health.

Our study has limitations. First, fecal PA was not measured before and after CM treatment. Edogawa et al. [6] demonstrated that high PA patients with IBS have greater symptom severity as well as higher colonic permeability. It has not been investigated whether oral administration of CM could modify luminal PA so far. Second, intestinal mucosal biopsies were not performed. It has been confirmed that a number or distribution of mucosal mast cells, which potentially release serine proteases, was different between IBS and healthy subjects [54]. However, it has been demonstrated that mucosal microbiota was significantly associated with fecal microbiota using a large cohort samples from both IBS patients and healthy subjects [55]. Third, each meal for the participants was not strictly controlled throughout the study. None of them had received any specific dietary treatment before and during the intervention.

In conclusion, our preliminary findings showed that a protease inhibitor, CM, significantly modulated not only the fecal microbiome composition but also the metagenome function of serine protease and FKBP-type PPIases despite the fact that it failed to improve their self-reported symptoms in patients with IBS. Further large clinical trials using different doses of CM should be warranted to develop new strategies for management of IBS.

The study protocol was designed in accordance with the Declaration of Helsinki and was approved by Tohoku University Ethics Committee (Study Number: 2014-1-38), and written informed consent to participate was obtained from all participants. This clinical trial for IBS patients was conducted from 2009 to 2018 and registered with the University Hospital Medical Information Network clinical trials registry (UMIN000001673).

K.M., K.O., and M.T. are employees at Miyarisan Pharmaceutical Co., Ltd. Mo.K., Mi.K., K.I., N.M., and S.F. declare no conflict of interest on this study.

This study was supported by Grant-in-Aids for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology, Japan, and by Research Grant from Miyarisan Pharmaceutical Co., Ltd., Tokyo, Japan.

Study concept and design: S.F., Mo.K., and N.M. Data collection and sample processing: Mo.K., Mi.K., and K.I. Analysis and interpretation of the data: Mo.K., K.M., K.O., M.T., and S.F. Drafting the manuscript: Mo.K., K.M., and S.F. All authors contributed to the critical revision of the manuscript for important intellectual content and approved of the final manuscript.

The data that support the findings of this study are not publicly available due to their containing information that could compromise the privacy of research participants but are available from the corresponding author upon reasonable request.