Efficacy and mode of action of mesalazine in the treatment of diarrhoea-predominant irritable bowel syndrome (IBS-D): a multicentre, parallel-group, randomised placebo-controlled trial

Article history

The research reported in this issue of the journal was funded by the EME programme as project number 09/20/16. The contractual start date was in October 2010. The final report began editorial review in May 2014 and was accepted for publication in December 2014. The authors have been wholly responsible for all data collection, analysis and interpretation, and for writing up their work. The EME editors and production house have tried to ensure the accuracy of the authors’ report and would like to thank the reviewers for their constructive comments on the final report document. However, they do not accept liability for damages or losses arising from material published in this report.

Declared competing interests of authors

none

Copyright statement

© Queen’s Printer and Controller of HMSO 2015. This work was produced by Lam et al. under the terms of a commissioning contract issued by the Secretary of State for Health. This issue may be freely reproduced for the purposes of private research and study and extracts (or indeed, the full report) may be included in professional journals provided that suitable acknowledgement is made and the reproduction is not associated with any form of advertising. Applications for commercial reproduction should be addressed to: NIHR Journals Library, National Institute for Health Research, Evaluation, Trials and Studies Coordinating Centre, Alpha House, University of Southampton Science Park, Southampton SO16 7NS, UK.

Existing research

Irritable bowel syndrome (IBS) is one of the commonest conditions seen by gastroenterologists, experienced by 1 in 10 of the population at some time in their lives and accounting for up to 40% of new referrals to gastroenterology outpatients departments. The condition is characteristically heterogeneous but all patients have abdominal discomfort and disturbed bowel habit. 1 The other feature found in at least half of patients with IBS is a history of anxiety or depression, and the presence of multiple unexplained physical symptoms, otherwise known as ‘physical symptom disorder’ or ‘somatisation’, is present in two-thirds of patients. Patients often believe that stress aggravates their symptoms but the effect does not appear to be immediate, as there is a poor correlation between stress and symptoms on a day-to-day basis. 2 The effect appears to be more long term and patients with chronic stressors rarely recover until these are relieved. 3 In animals, chronic stress causes diarrhoea, an effect that appears to be mediated through the release of corticotropin-releasing factor within the brain, where it activates descending autonomic pathways, delaying gastric emptying and accelerating colonic transit. Corticotropin-releasing factor is also found locally in the mucosa, where its release may activate mast cells. In addition to these effects of acute stress, chronic stress in experimental animals, generated by repetitive water avoidance or lifelong stress following maternal deprivation in the neonatal period, increases the number of mast cells within the mucosa. This leads to increased gut permeability and increased translocation of bacteria with associated low-grade immune activation within the mucosa. 4

More recently, evidence has been accumulating that similar activation of mast cells can occur in stressed humans. Acute stress induced by immersion of the hand into ice-cold water has been shown to stimulate jejunal water secretion and the release of mast cell products, including tryptase and histamine, in healthy volunteers (HVs). 5 In patients with diarrhoea-predominant irritable bowel syndrome (IBS-D), mast cell numbers have been shown to be increased in jejunal biopsies, along with intraepithelial CD3⁺ T lymphocytes (CD3). 6 This study also showed higher tryptase concentration in aspirated jejunal fluid, suggesting that the mast cells were activated,6 and more recent studies show that the ensuing increase in gut permeability is confined to females, suggesting an important gender difference in susceptibility to stress-induced gut changes, which accords with the known female predominance of IBS. We have recently confirmed increased numbers of mast cells and intraepithelial lymphocytes in duodenal biopsies from patients with IBS-D in Nottingham, who also show increased tryptase release into the supernatants of incubated duodenal biopsies. 7

Importance of mast cells in generating irritable bowel syndrome symptoms

There are numerous reports now of increased mast cell numbers in patients with IBS, in the terminal ileum,8,9 caecum10 and rectum. 11–13 Mast cells contain many mediators, including histamine, serotonin (5-HT) and tryptase. 14 Recent interest has focused on the tryptase content because it has been shown to activate protease-activated receptor 2 (PAR2) receptors, which are found on afferent nerves, and their activation increases sensitivity of the bowel to distension. Supernatants from IBS mucosal biopsies have been shown to activate afferent nerves in an isolated mouse jejunal segment,15 and, more recently, in human colonic submucosal nerves. 14 We propose that anxiety and chronic stressors act in humans to increase the number of activated mast cells throughout the gut in patients with IBS, thereby inducing the characteristic visceral hypersensitivity and abdominal pain. We hypothesised that mesalazine slow-release granule formulation (2 g; PENTASA^®, Ferring Pharmaceuticals Ltd) treatment, through its anti-inflammatory effects, would reduce the number of mast cells and thereby reduce abdominal pain, stool looseness and stool frequency.

Previous studies of mast cell stabilisers and anti-inflammatory agents in irritable bowel syndrome

Although there were some poorly designed trials two decades ago, claiming to show that sodium cromoglycate was effective for IBS,16–18 these studies remain unconfirmed and the treatment is not widely used. There have been other smaller studies targeting mast cells with antihistamines, such as ketotifen;19 although this reduced visceral hypersensitivity, it had no effect on mast cell numbers or release of mast cell mediators. Our own trial of prednisolone in postinfective IBS (PI-IBS) was of limited duration – just 3 weeks – and, although this showed a halving of CD3, patient symptoms were already subsiding and we were not able to show any difference from the control subjects. In that study, the fall in mast cell numbers on prednisolone was twice that on placebo but this difference was not statistically significant. We felt that 3 weeks was too short a time period to make an impact on mucosal histology. A strategy that reduced mast cell numbers over the long term might well be more effective than specific inhibitors of mast cell activation, or indeed any specific mast cell products, as these are numerous [histamine, tumor necrosis factor (TNF), prostaglandin, substance P, mast cell tryptase and nerve growth factor (NGF)], all with quite variable modes of action.

Previous studies of mesalazine treatment for irritable bowel syndrome

The first anecdotal open-label trial of 12 patients with resistant IBS-D who responded to mesalazine20 showed a benefit that took about 2–3 months to become apparent. There have since been two further reports of open-label treatment21,22 and two small randomised controlled trials. 23,24 All but the Corinaldesi et al. 23 trial used patients with IBS-D. The Bafutto et al. trial22 used mesalazine 800 mg three times a day for 30 days in 61 patients with IBS-D – 18 of whom had PI-IBS and showed benefit with a reduction in stool frequency, stool consistency and abdominal pain – but was uncontrolled, with no placebo arm. The Andrews et al. study21 involved just six patients but this showed that mesalazine decreased biopsy proteolytic activity. Both of the randomised controlled trials23,24 are rather too small for their significance to be sure, with n = 20 and n = 17, respectively. One study23 showed a significant reduction of mast cell numbers and an overall reduction in inflammatory cells.

Risk and benefits

Mesalazine has been widely used for > 45 years and there are extensive data on its side effects. In general, the drug is well tolerated. Nephrotoxicity is seen at a rate of about 1 per 100,000 prescriptions;25 commoner, but less serious, side effects include diarrhoea, nausea, vomiting and abdominal pain, together with headaches, pancreatitis and blood disorders, which are all rare. Balancing this, patients with IBS suffer a marked decrease in quality of life, similar to that of other chronic diseases such as diabetes and heart failure. They also spend significant amounts of time off work and when they are at work they work less efficiently. A simple safe and effective treatment would be of undoubted benefit to what is a substantial subgroup of the population, given that IBS-D affects around 3% of the general population.

Rationale for the current study

Studies in Nottingham over the last decade have identified the importance of inflammation in various subgroups of IBS. We have focused on the group of patients with IBS who develop symptoms following acute bacterial gastroenteritis, the so-called postinfectious IBS. In this group, we have been able to show that the acute inflammatory insult associated with acute Campylobacter jejuni enteritis is followed by a more prolonged indolent phase with increased chronic inflammatory cells long after the infecting organism has left the body. In this subgroup of IBS we have demonstrated activated circulating peripheral blood mononuclear cells with increased cytokine production and an associated increase in inflammatory gene expression. 26 We have also demonstrated the importance of anxiety and depression,27 which, along with adverse life events, increase the risk of PI-IBS. 28 The changes observed in PI-IBS are similar to those in IBS-D, the predominant bowel disturbance being diarrhoea with a similar prognosis. 29 This work has been supported by others who have shown inflammatory changes in patients with IBS-D who do not have a background of previous infection. 30,31 Such studies have also shown increased inflammatory cells and increased expression of inflammatory cytokines, including interleukin-1 beta (IL-1β). 32 Increased gut permeability has also been shown in IBS-D,33 making a trial of an anti-inflammatory treatment a logical choice. Safety is of pre-eminent importance with IBS drugs, as can be seen by the recent withdrawal of tegaserod34 (Zelnorm^®, Novartis) and the previous withdrawal of alosetron (Lotronex^®, Prometheus). 35 Both drugs, which were therapeutically effective, had to be withdrawn as a result of rare side effects (incidence < 1 per 700 patients treated). This leaves such patients bereft of effective treatments, a gap that mesalazine might well fill. Our hypothesis was that mesalazine, by virtue of its anti-inflammatory actions, would alter the inflammatory mediators leading, over a number of weeks, to a reduction in the number of mast cells and a reduction in the release of inflammatory mediators, and hence to a reduction in symptoms. Previous studies have shown that 5-aminosalicylic acid (5-ASA) inhibits the release of inflammatory mediators, including histamine and prostaglandin D₂. 36 It also inhibits activation of the transcription factor ‘nuclear factor kappa-light-chain-enhancer’ of activated B cells, which is a major link in the inflammatory cascade. 37 More recently, it has been recognised that 5-ASA exerts an anti-inflammatory effect that is mediated via peroxisome proliferator-activated receptor gamma (PPARγ receptors). 38 Whether directly or indirectly, 5-ASA has also been reported to inhibit inducible nitric oxide synthetase production and also prostaglandin production via its cyclooxygenase-2 inhibitory effects. 39 Mesalazine, therefore, both by virtue of inhibiting other inflammatory pathways and by directly inhibiting mast cell pathways, may reduce mucosal immune activation. We plan to investigate the effect of long-term mesalazine on mast cell numbers, the chronic inflammatory cells and the mucosal production of inflammatory cytokines, IL-1β and tumour necrosis factor alpha (TNF-α) as well as mast cell-specific tryptase. We also examined its effect on stool calprotectin, a marker of colonic inflammation, which is widely used to exclude inflammatory bowel disease and is also recognised to be modestly elevated in around 25% of most IBS series.

Purpose

The purpose of the trial was to define the clinical benefit and possible mediators of the benefit of mesalazine in IBS-D.

We therefore evaluated symptoms (primarily bowel frequency) and objective biomarkers reflecting mast cell activation and small bowel tone.

Primary objective

Effect of mesalazine on stool frequency in weeks 11 and 12.

Secondary objectives

Effect of mesalazine on the following:

1. overall IBS symptoms

2. mast cell numbers, mucosal lymphocytes and faecal tryptases

3. small bowel tone by measurement of fasting small bowel water content through magnetic resonance imaging (MRI).

Our overall aim was to assess the ability of the biomarkers listed above to predict treatment response.

Trial/study configuration

This was a multicentre, two-arm, parallel-group, double-blind randomised placebo-controlled trial comparing mesalazine with placebo in patients with IBS-D. The design of the study was modified after consultation with a selection of interested patients from the Nottingham Digestive Diseases Biomedical Research Unit patient advisory group, who provided a lay member for the Trial Steering Committee (TSC).

Randomisation and blinding

This was a double-blind, parallel-group study. The participant, supervising doctor and study nurse were not aware of the treatment allocation.

The randomisation was based on a computer-generated pseudo-random code using random permuted blocks of randomly varying size, created by the Nottingham Clinical Trials Unit (CTU) in accordance with their standard operating procedure (SOP), and held on a secure server. The randomisation was stratified by the recruiting centre. The supervising doctor or study nurse obtained a randomisation reference number for each participant by means of a remote, internet-based randomisation system that was developed and maintained by the Nottingham CTU.

The sequence and decode of treatment allocations were concealed until all interventions were assigned, recruitment, data collection and all other trial-related assessments were complete, and data files were locked.

Participants

Clinical outcomes

Mechanistic outcomes

Assessment of efficacy

We used descriptive statistics to compare the randomised groups at baseline. The primary outcome was assessed using ITT without imputation. We used a generalised linear mixed model to compare the mesalazine group and placebo group for the primary outcome, with adjustment for the baseline value of the outcome, and study centre as a random effect. Additionally, we adjusted for any variables showing imbalance at baseline in secondary models. We compared the characteristics of participants who did and did not adhere with the study medication before estimating the treatment effect if the medication was actually taken using Complier Average Causal Effect (CACE) analysis. We investigated the effect of missing primary outcome data using multiple imputation. The secondary outcomes were assessed using similar models as for primary outcome, or logistic or Poisson regression, as appropriate, dependent on outcome type.

We undertook subgroup analyses by including appropriate interaction terms in the linear mixed model for primary outcome according to baseline daily mean stool frequency, baseline mean abdominal pain score and baseline mean HADS anxiety score.

Secondary outcomes were treated similarly, after transformation if appropriate, whereas binary and count outcomes were handled by multiple logistic or Poisson regression, as appropriate. Given the large number of potential comparisons, p-values for mechanistic variables will not be presented. All analyses were performed using Stata version 13 (StataCorp LP, College Station, TX, USA) adopting the ITT principle without imputation for missing data (with a sensitivity analysis using multiple imputation for the primary outcome).

We planned to conduct a number of prespecified subgroup analyses.

For each of the three outcomes listed below, we investigated whether or not there were any differences in between-group effects according to the following baseline variables: anxiety, stool frequency, abdominal pain and mast cell activation:*

1. stool frequency during weeks 11–12

2. number of days with any stool consistency scoring 6 or 7 during weeks 11–12

3. mean score of worst pain for each day, averaged over weeks 11 and 12.

*Mast cell activation will be defined as elevation of any of the inflammatory mediator components, such as mast cell tryptase, IL-1β, TNF-α, histamine and 5-HT in biopsy supernatant.

These subgroup analyses were conducted by including appropriate interaction terms in the regression models and, as the study has not been powered to detect any such subgroup effects, were considered as exploratory and would require confirmation in future research.

The primary mechanism hypothesis to be investigated was that treatment with mesalazine reduces inflammation, which, in turn, reduces clinical symptoms. The aim of this type of analysis is to estimate how much of any observed treatment effect can be attributed to a variable that is thought to be an intermediate on the causal pathway, or mediator.

After summarising inflammatory markers at baseline and 11–12 weeks’ follow-up by trial arm using appropriate descriptive statistics, we will examine change in these markers (stool calprotectin, mast cell tryptase, mast cell percentage area stained) and change in stool frequency using a scatter plot.

Definition of populations analysed

Safety set All randomised participants who receive at least one dose of the study drug. All AEs in this set are reported.

ITT set All randomised participants for whom at least one post-baseline assessment of the primary end point is available.

Stool samples and sigmoid colon biopsies

These were collected at week 0 and the end of trial (EOT) from patients recruited in Nottingham. Stools collected were analysed for calprotectin. A commercially available calprotectin enzyme-linked immunosorbent assay (ELISA) kit (Buhlmann, Schönenbuch, Switzerland) was used for extraction and quantification of stool calprotectin. Samples were analysed for faecal tryptase based on a method published recently by our group. 42 Faecal protease activity is expressed in trypsin units per mg of protein.

Sigmoid biopsies were collected for immunohistochemistry for mast cell tryptase, CD3, CD68 (a marker of macrophage) and 5-HT, and tissues were processed and stained in the histopathology laboratory in Nottingham University Hospitals NHS Trust, UK. A further set of biopsies was maintained in culture and supernatants collected were assayed for (1) mast cell tryptase, chymase, carboxypeptidase A3 (CPA3) and histamine, and (2) IL-1β and TNF-α levels. The biopsy tissues were incubated immediately in Hanks’ medium for 30 minutes before storing at –80 °C until assays for (1) mast cell mediators were performed by the Immunopharmacology Group at the University of Southampton; and (2) IL-1β and TNF-α levels were performed by RI, Research Fellow in Centre of Biomolecular Science, University of Nottingham. Levels of IL-1β and TNF-α was analysed by using a commercial kit V-PLEX immunoassay (Meso Scale Discovery, Rockville, MD, USA).

Histological methods (see Appendix 2)

Mast cell numbers were assessed as the mean of area percentage stained per region of interest (m²).

Faecal tryptase

Stool samples were collected at baseline (randomisation day) and at the EOT (end of week 12).

Demographics

A total of eight sites participated in this study. Table 2 and Appendix 2 (see Table 16) show a summary of recruitment by site and by treatment arm.

Characteristics of enrolled patients in both groups were similar at baseline (Table 3).

Primary and secondary outcome data were collected for 115 (85%) and 116 (85%) participants, respectively, at 11–12 weeks of follow-up.

Clinical primary outcome

The primary ITT comparison showed no evidence of any clinically significant difference between mesalazine and placebo for the primary outcome (Table 4). Additional adjustments for variables (age, abdominal pain score, number of days with urgency and PHQ-15 score) displaying imbalance at baseline did not materially change the results (Table 5a) and nor did multiple imputation analysis or CACE analysis (Table 5b–c).

Subgroup analyses (Table 6a) of the primary outcome by baseline daily mean stool frequency suggest that mesalazine may be more effective among patients with greater baseline stool frequency and is associated with larger treatment effect, but this could be a chance finding and would require confirmation in further studies. There was no evidence that treatment effect differed according to baseline pain or HADS (Table 6b and c). Our sensitivity analysis, using multiple imputation of missing data for the primary outcome, showed no effect on primary outcome (see Table 6b).

Clinical secondary outcomes

No differences were apparent for any of the secondary outcomes, with the exception of number of days with urgency (Table 7), which were increased by about 20% on mesalazine treatment compared with placebo.

Compliance

Compliance was defined a priori as taking ≥ 75% of the medication throughout the 12 weeks. Each patient was given two boxes of medication during the 12-week study, each box containing 100 sachets. The amount of medication taken was calculated by 200 minus the number of medication sachets returned at EOT. Compliance with medication (Table 8) and baseline characteristics of compliers (defined as taking ≥ 75% of the medication throughout the 12 weeks) were similar in both groups. Analysis of the primary outcome using the CACE approach showed no difference between the two treatment arms (mean difference 0.2, 95% CI –0.6 to 0.9).

Adverse events

The most frequently occurring side effect was exacerbation of IBS symptoms, which could be worsening abdominal pain or diarrhoea. Two patients (3%) from the mesalazine group and three (5%) from the placebo group complained of this and were withdrawn from the study. Other less frequent side effects are listed in Appendix 2 (see Table 15). One patient was pregnant in the middle of the trial period, although she had a negative result in a pregnancy test at the start of the trial. She was withdrawn from study with no adverse consequence to her or her newborn. 43 One patient from the mesalazine group was found to have breast cancer and she was withdrawn from the study, as her IBS symptoms and stool diary would have been very difficult to interpret. All participants who developed these AEs were withdrawn from the study and their symptoms settled on follow-up – see Appendix 2, Table 15.

Mechanistic primary outcome

Mast cells

The mast cell percentage area stained was not elevated in patients with IBS-D compared with our normal range that was established previously in our laboratory. The baseline mast cell percentage area stained for patients with IBS-D was median 2.25% [interquartile range (IQR) 1.86–2.73%], compared with median 2.42% (IQR 2.09–3.39%) in the healthy control subjects (Figure 2). There was no reduction in mast cell percentage area stained following treatment with mesalazine (Figure 3 and Table 9).

FIGURE 2.

Mast cell count assessed from percentage area stained comparing healthy control subjects and patients with IBS-D (median, IQR).

FIGURE 3.

Effect of mesalazine vs. placebo on mast cell percentage area stained in patients with IBS-D (median, IQR).

TABLE 9 - Effect of mesalazine vs. placebo on mast cell percentage area stained/m2 in patients with IBS-D [median (IQR)]

Mesalazine		Placebo
Baseline	After treatment	Baseline	After treatment
2.11 (1.06–2.11)	2.29 (1.86–2.75)	2.33 (1.88–2.74)	2.25 (1.5–2.59)

Mechanistic secondary outcome

Mast cell tryptase and other mediator release during biopsy incubation

Baseline supernatant levels were compared between patients with IBS-D and our normal range in healthy volunteers. There was no significant increase in the baseline mediator levels except CPA3 (Figure 4 and Table 10).

FIGURE 4.

Baseline CPA3 levels in patients with IBS-D. Shaded area indicates normal range in HVs (median, IQR).

TABLE 10 - Baseline supernatant levels (ng/ml) between HVs and patients with IBS-D [median (IQR)]

Mediator	HVs, n = 21	IBS-D patients, n = 45	p-value
Tryptase	6.7 (3.8–11.4)	4.3 (1.8–8.9)	0.07
Chymase	0	0 (0–0.9)	0.14
CPA3	0.34 (0.28–0.52)	0 (0–0.9)	0.0496
Histamine	1.6 (0.7–3.8)	0.7 (0–1.3)	< 0.01

Following treatment with either mesalazine or placebo in the patients with IBS-D, there was no change in the supernatant levels of the mediators (Table 11 and Figures 5–9).

TABLE 11 - Supernatant mediator levels (ng/ml) following treatment with mesalazine or placebo [median (IQR)]

Supernatant mediators	Mesalazine baseline (n = 21)	Placebo baseline (n = 23)	Mesalazine after treatment	Placebo after treatment
Tryptase	4.3 (1.5–8.6)	4.6 (2.5–9.1)	4.9 (1.8–8.2)	5.8 (2.1–10.3)
Chymase	0 (0–0.3)	0 (0–0.8)	0 (0–1.7)	0 (0–0.4)
CPA3	0 (0–0.3)	0 (0–1.0)	0 (0–0.8)	0 (0–0.5)
Histamine	0.9 (0.3–1.4)	0.7 (0–1.4)	0.8 (0–1.2)	0.7 (0.2–1.0)
5-HT	9.4 (6.1–15.1)	6.3 (2.7–13.7)	10.7 (5.4–14.0)	9.3 (3.4–14.7)

FIGURE 5.

Tryptase levels before and after treatment with mesalazine or placebo (median, IQR).

FIGURE 6.

Chymase levels before and after treatment with mesalazine or placebo (median, IQR).

FIGURE 7.

Carboxypeptidase A3 levels before and after treatment with mesalazine or placebo (median, IQR).

FIGURE 8.

Histamine levels before and after treatment with mesalazine or placebo (median, IQR).

FIGURE 9.

Serotonin levels before and after treatment with mesalazine or placebo (median, IQR).

Small bowel tone assessed by volume of fasting small bowel water

Fasting small bowel water showed wide variability, ranging from 5 to 220 ml. This did not alter significantly after treatment with either mesalazine or placebo (Table 12, Figures 10 and 11).

TABLE 12 - Fasting small bowel water content (ml) following treatment with either mesalazine or placebo

Fasting small bowel water content (ml), median (IQR)
Mesalazine baseline (n = 17)	Placebo baseline (n = 20)	Mesalazine after treatment	Placebo after treatment
58 (27–122)	58 (28–106)	48 (36–79)	58 (21–96)

FIGURE 10.

Fasting small bowel water before and after treatment with mesalazine or placebo (median, IQR).

FIGURE 11.

Changes in fasting small bowel water before and after treatment with either mesalazine or placebo.

Faecal tryptases

Totals of 27 and 30 pairs of stool samples were collected in the mesalazine and placebo groups, respectively. The baseline faecal tryptase level was in the range of between 6.8 and 577.8 trypsin units/mg of protein, which was highly variable. There was a significant increase in faecal tryptase after treatment with mesalazine (Table 13 and Figure 12). There was no correlation between baseline faecal tryptase level and baseline supernatant tryptase level: Spearman’s r = 0.13, p = 0.41. There was no significant correlation between baseline faecal tryptase level and anxiety, depression or bowel symptoms.

TABLE 13 - Faecal tryptase levels (trypsin units/mg protein) following treatment with mesalazine or placebo

Baseline, median (IQR)		After treatment, median (IQR)
Mesalazine (n = 30)	Placebo (n = 27)	Mesalazine	Placebo
61.2 (37.6–101.4)	66.5 (44.8–126.5)	82.7 (40.5–194.8)	70.9 (36.0–191)

FIGURE 12.

Change in faecal tryptase following treatment with mesalazine compared with placebo (median, IQR).

Post hoc analysis

Other immune cells, for example CD3⁺ T lymphocyte, CD68- and serotonin-containing enterochromaffin cells

There was no effect on either treatment with mesalazine or placebo on CD68-staining macrophages or on 5-HT-containing enterochromaffin cells. There was a paradoxical increase in CD3 count following treatment with mesalazine for reasons which are unclear (Figure 13).

FIGURE 13.

CD3⁺ T lymphocyte count before and after treatment with mesalazine or placebo (median, IQR).

Stool calprotectin

Samples were obtained from 53 patients (30 placebo group, 23 mesalazine group). Baseline stool calprotectin levels varied widely, ranging from undetectable to as high as 420 µg/g. There was a negative correlation between calprotectin levels and baseline total HADS score but this did not reach significance (r = 0.25, p = 0.07) (Figure 14).

FIGURE 14.

Correlation between baseline calprotectin levels (µg/g) and baseline total HADS score.

When patients were divided into two groups based on stool calprotectin levels of ≤ 100 µg/g (group B) and ≥ 101 µg/g (group A), the group with higher calprotectin levels (≥ 101 µg/g, group A) at baseline showed a significantly lower total HADS score than the normal calprotectin level group (group B) (Figure 15). Otherwise, comparing these two groups, there were no differences with their baseline clinical characteristics such as abdominal pain severity, average daily stool frequency and stool consistency.

FIGURE 15.

Baseline stool calprotectin levels when divided into two groups (median, IQR).

Postinfectious irritable bowel syndrome

Thirteen participants met the previously published criteria for PI-IBS. 11 They had to meet Rome III criteria1 for IBS following an episode of infectious gastroenteritis characterised by ≥ 2 of the following symptoms: fever, vomiting, diarrhoea and positive stool culture. 11 Eight participants were randomised into the mesalazine group and five were allocated to the placebo group. There was significant improvement in clinical symptoms – such as abdominal pain, urgency and stool consistency – following treatment with mesalazine but not with placebo (Figures 16–18).

FIGURE 16.

Abdominal pain severity before and after treatment with mesalazine or placebo.

FIGURE 17.

Urgency symptoms before and after treatment with mesalazine or placebo.

FIGURE 18.

Stool consistency before and after treatment with mesalazine or placebo.

Limitations

Despite strict entry criteria, our population was still heterogeneous. In retrospect, it would have been better if we had stratified by postinfectious onset. We did consider this but felt that this would make it very difficult to recruit to the trial. We could overcome this in future studies by having a great many more recruitment sites and around five times as many participants, given that PI-IBS accounts for only around 20% of all cases of IBS-D, but this would require more resources than those that we had available to us. It is worth noting that there is an appreciable loss to follow-up (15.5%) but this is not out of line with other similar studies. Dropouts are mostly likely due to failure of treatment and so are unlikely to account for our negative result.

Research recommendations

1. Our data suggest that it is unlikely that future trials of mesalazine in unselected IBS would be fruitful.

2. If there is a subgroup that may benefit it is likely to be those with postinfective IBS, particularly those postinfective patients with more severe diarrhoea.

3. The link between mast cells and clinical symptoms is weak and, again, future work on the role of mast cells needs to better characterise the patients, as the majority of unselected patients with IBS do not have elevated mast cell numbers. It may be that, as others have reported, it is the number of activated mast cells that are important53 and better markers of activation would be useful rather than the current gold standard of electron microscopy, which is expensive and time-consuming.

4. Finally, the release of mediators from biopsies does not link well to symptoms or mast cell numbers. The dominant factor for release is likely to be crushing and tissue injury by the biopsy process, which is not well standardised and may overwhelm other factors that would be of more interest. We need a better way of assessing in vivo activity of the mucosal cells.

Contributions of authors

Ching Lam (clinical research fellow/trial manager): managed, overall, the running of the whole trial, managed the clinical trial site in Nottingham, the analysis of data and the writing of reports/abstracts.

Wei Tan (statistician): data analysis for the clinical primary outcome data.

Matthew Leighton (research manager): trial manager for initial set-up of the study.

Margaret Hastings (research nurse): study supervision and recruitment of participants into study.

Melanie Lingaya (technician): analysis and technical support.

Yirga Falcone (technician): analysis and technical support.

Xiaoying Zhou (technician): analysis and technical support.

Luting Xu (research fellow): analysis and technical support.

Peter Whorwell (principal investigator): study concept and design.

Andrew F Walls (reader in immunopharmacology): analysis and technical support.

Abed Zaitoun (consultant histopathologist): analysis and technical support.

Alan Montgomery (senior statistician): statistical analysis and interpretation of data.

Robin C Spiller (chief investigator and senior author): obtained funding, study concept and design, interpretation of data, study supervision, recruitment.

We would like to thank:

1. The Clinical Research Network (CLRN) in assisting with the set-up of study in other centres and providing research nurses to assist in this study.

2. The PCRN who assisted with identifying potential patients with IBS in primary care.

3. Ferring Pharmaceuticals for sponsoring mesalazine and the placebo medication.

4. The CTU, University of Nottingham, for its information technology/electronic database support.

5. Nurses and technicians from the NIHR Nottingham Digestive Diseases Biomedical Research Unit, Nottingham University Hospitals NHS Trust, for assisting and supporting this study.

6. Dr Andrew F Walls, Xiaoying Zhou and Laurie Lau from the Immunopharmacology Group, University of Southampton, who helped in processing the biopsy supernatants for tryptase, histamine, CPA3 and chymase.

7. Dr Abed Zaitoun and the histopathology/immunohistochemistry laboratory in the Nottingham University Hospitals NHS Trust, who helped with preparation of biopsy samples for histology/immunochemistry stains.

8. All participating principal investigators and CLRN nurses who have assisted in the recruitment and running of this study in other sites:

   1. Dr Jessica Williams, Gastroenterology, Royal Derby Hospitals NHS Foundation Trust

   2. Dr Stephen Foley, Gastroenterology, King’s Mill Hospital

   3. Dr Anurag Agrawal, Gastroenterology, Doncaster Royal Infirmary

   4. Dr Sandip Sen, Gastroenterology, United Hospitals North Staffordshire

   5. Dr Matthew Rutter, Gastroenterology, University Hospital of North Tees

   6. Dr Arvind Ramadas, Gastroenterology, James Cook University Hospital

   7. Professor Peter Whorwell, Neurogastroenterology Unit, University Hospital of South Manchester NHS Foundation Trust.

Publication

Leighton MP, Lam C, Mehta S, Spiller RC. Efficacy and mode of action of mesalazine in the treatment of diarrhoea-predominant irritable bowel syndrome (IBS-D): study protocol for a randomised controlled trial. Trials 2013;14:10.

Notes

This study was accepted as an oral presentation in the Digestive Disease Week 2014 in Chicago, IL, USA. See Appendix 3 for abstract.

Disclaimers

This report presents independent research. The views and opinions expressed by authors in this publication are those of the authors and do not necessarily reflect those of the NHS, the NIHR, MRC, NETSCC, the EME programme or the Department of Health. If there are verbatim quotations included in this publication the views and opinions expressed by the interviewees are those of the interviewees and do not necessarily reflect those of the authors, those of the NHS, the NIHR, NETSCC, the EME programme or the Department of Health.

Excluded medication

Dose-controlled medication

Methodology for immunohistochemical staining of cells for mast cell tryptase, CD3⁺ T lymphocytes, CD68 and serotonin

Biopsies were formalin fixed and embedded in wax prior to standard sectioning for staining.

The slides prepared were scanned into the computer using the nanozoomer and was magnified ×40 for ease of portability. Cell counting was performed by a single person (LTX, fellow from the FRAME Laboratory, University of Nottingham) who was blinded to the study. Detection of each stained cell type was checked for reproducibility (> 95%) before cell counting began. At least 5–10 areas around lamina propria were drawn, and CD68 cells were counted, giving an average cell number per mm². CD3, which is a marker of lymphocytes, was assessed by counting the number of stained cells at the superficial epithelium per area drawn (mm²) and an average obtained. The 5-HT cells were counted at the deep lamina propria and an average of number of cells/mm² were obtained. Mast cell tryptase expression was detected in the lamina propria using automatic software (i-Tem Desktop, Olympus Soft Imaging Solutions GMBH, Münster, Germany), as some mast cells may be in a degranulated state, thus making cell counting difficult. Results were presented as the percentage area stained for mast cell tryptase.

Mast cell proteases (tryptase, chymase and CPA3) were measured by sandwich ELISA assays developed by the Immunopharmacology Group, University of Southampton, as described previously. 58–60 Briefly, antibodies specific for tryptase (EAR), chymase (CC2) and CPA3 (CA2) were coated on Costar™ 96-well EIA/RIA Plates (Fisher Scientific, Loughborough, UK) for 16 hours at +4 °C. Plates were washed three times and blocked with 2% bovine serum albumin (BSA) for 1 hour at room temperature and samples or protein standards of tryptase, chymase or CPA3 were added for 90 minutes. After a further washing stage, detecting antibodies that were specific for tryptase (AA5), chymase (CC2) or CPA3 (CA5) were added, the plates again washed, avidin–horseradish peroxidase added and cleavage of TMB substrate (3,3′,5,5′-tetramethylbenzidine, Sigma-Aldrich, Gillingham, UK), measured colorimetrically at 450 nm. Prior to assays being performed, the ELISA were validated for use with cell supernatants, by measuring recovery of each of the proteases spiked into samples prior. Histamine (Life Science Format) was measured using a commercially available enzyme immunoassay kit (ELISA, Neogen, Lexington, KY, USA). All assays were performed blind.

Adverse events

Recruitment

Recruitment was slow initially owing to other staff seeing patients and not being aware of the study. The multiple visits were a deterrent and many patients said that they could not take time off work. We advertised widely. Advertising on the local buses produced no volunteers but advertising in local free papers produced a better response.

FIGURE 19.

Total recruitment by date.

FIGURE 20.

Recruitment by site and date.

Reprinted from Gastroenterology volume 146 issue 5, Lam C, Tan W, Leighton M, Williams J, Agrawal A, Sen S, et al. 712 A Multi-Centre, Parallel Group, Randomised Placebo Controlled Trial of Mesalazine for Treatment of Diarrhoea-Predominant Irritable Bowel Syndrome (IBS-D). S23–S24, 2014 with permission from Elsevier.

Stool diary