We hypothesised an imbalance of the endocannabinoid system in IBS, and that endocannabinoid‐like dietary compounds may improve IBS symptoms, principally abdominal pain. In particular, palmithoylethanolamide, the saturated fatty acid amide of palmitic acid, is a dietary component commonly found in egg yolk and peanuts, consistently reported to exert anti‐inflammatory and analgesic activities both in vitro and in vivo . 12 , 13 Polydatin, a resveratrol glucoside, is a common dietary component derived from grapes which may act synergistically with palmithoylethanolamide in reducing mast cell activation and local oxidative stress. 14 Thus, we designed a pilot study evaluating the efficacy and safety of dietary compounds palmithoylethanolamide/polydatin in patients with IBS.

The endocannabinoid system consists of cannabinoid 1 and 2 receptors, located both in central and enteric nervous system (particularly cannabinoid 1 receptor), and in the immune system, such as mast cells (particularly cannabinoid 2 receptor); their endogenous ligands, belonging to the group of lipid mediators; and enzymes involved in ligand biosynthesis and degradation, such as fatty acid amide hydrolase. 5 , 6 The involvement of the endocannabinoid system, including its receptors and metabolic pathways, in IBS pathophysiology was suggested by basic science showing that the endocannabinoid system may regulate all the mechanisms proposed in IBS pathophysiology, 5 genetic studies indicating that cannabinoid 1 and fatty acid amide hydrolase polymorphisms 7 , 8 are associated with IBS, and clinical trials. These studies showed that the nonselective cannabinoid receptor agonist dronabinol reduces fasting colonic motility in nonconstipated IBS, 9 the cannabinoid 1 receptor antagonist rimonabant induces dose‐dependent diarrhoea as an adverse effect, 10 and compounds inactivating fatty acid amide hydrolase improve diarrhoea and pain in a mouse model. 11 Analgesic lipid mediators, produced by neural and non‐neural cells, such as inflammatory cells, include, among others: (i) endocannabinoids; (ii) and long‐chain fatty acid ethanolamide agonists of peroxisome proliferator‐activated receptor‐α (PPAR‐α). 6 The former mediators, anandamide and 2‐arachidonoyl‐glycerol, suppress sensitisation and neurogenic inflammation by activating cannabinoid 1 and 2 receptors. 6 The latter mediators, such as oleoylethanolamide and palmitoyl‐ethanolamide, are chemically related to anandamide but exhibit low affinity for cannabinoid receptors (endocannabinoid‐like compounds), and participate in the control of inflammation and nociception, which, in the case of palmithoylethanolamide, occurs mainly via down‐regulation of mast cell activity. 6 Interestingly, palmithoylethanolamide may act as mast cell modulator, as recognised by the Nobel laureate Rita Levi‐Montalcini; 12 as a possible agonist for cannabinoid 2‐like receptors; and as agonist for PPAR‐α, transient receptor potential vanilloid type 1 (TRPV1), and ‘orphan’ G protein‐coupled receptor 55; whereas oleoylethanolamide is a more potent agonist at PPAR‐α and TRPV1 and also activates the other ‘orphan’ G protein‐coupled receptor 119. 13 For these reasons, palmithoylethanolamide and oleoylethanolamide have emerged as potential regulators of nociception. 6

Irritable bowel syndrome (IBS) is one of the most common functional gastrointestinal disorders in which abdominal pain and/or discomfort are associated with changes in bowel habit. 1 IBS affects 10–20% of the population causing a marked reduction of quality of life in affected individuals. 1 Nevertheless, its pathophysiology is not completely elucidated and specific biomarkers are not yet available. Therefore, current treatments generally have a poor efficacy. 2 To date, it is believed that IBS is the consequence of dysregulation of the brain–gut axis with both central and peripheral mechanisms involved. 3 , 4 Compelling evidence suggests that IBS results from interactions among environment, host and genetic factors. Different triggers (including diet, microbiota, bile acids, etc.) in genetically pre‐disposed individuals may contribute to the loss of intestinal barrier function with passage of antigens through the mucosal layer. 3 , 4 This may elicit enteroendocrine and mucosal immune responses (including mast cell recruitment and activation), which induce neuroplastic changes and affect afferent and intrinsic nerves, leading to the symptoms and pathophysiological features of IBS. 3 , 4

During the study, the following rescue therapies were allowed: butylscopolamine (no more than two tablets/day according to need, in case of severe abdominal pain); a single lukewarm water enema (if needed in case of severe constipation); loperamide (no more than one tablet once a day according to need, in case of severe diarrhoea).

Explorative analyses were also performed. Patients were classified as responders if they reported a reduction of at least 1 point on a 5‐point Likert scale of their digestive symptoms at the end of treatment as compared with baseline. The difference between the percentages was tested by means of a chi‐square test. This analysis was post‐hoc and performed with the intention to know the proportion of patients who experienced improvement or relief of their symptoms in each arm.

The model adopted to assess drug efficacy included: gender, age, centre, treatment (palmithoylethanolamide/polydatin or placebo), bowel habit, time (baseline, 4 weeks, 8 weeks, 12 weeks or end of treatment), use of rescue medications and the interaction between time and treatment. Therefore, the variable interaction between time and treatment measures the different trend of means among times between treatment groups, and it is the main variable to assess drug efficacy.

All the data were analysed by repeated measure analysis of variance (anova) with unstructured variance/covariance matrix (a generalised, linear‐mixed model), and corrected for multiple comparisons using the Tukey's method when appropriate. Repeated measure analysis allows a better handling of missing data (mediators under the detection limits, protein amounts not sufficient to perform all the Western blot analysis, samples not available). Estimating the variance/covariance matrix, it improves anova effectiveness otherwise conditioned by the homogeneity of variance assumption.

All subjects who have received at least one dose of study treatments were included in the safety population. All randomised patients who took at least one dose of the study drug and with at least one evaluation of the primary endpoint were included in the intention‐to‐treat (ITT) population. All the analyses were performed on the ITT population.

Based on previous studies, 16 , 17 between patients and HC, we considered a 50% decrease in mast cell count from mean 9.0% (standard deviation [s.d.] 2.5%), which requires 12 subjects in each group to show such a decrease with a power of 90% at the 5% α level (primary outcome measure). Furthermore, based on previous work showing a 36% decrease in mast cell count following active treatment in patients with IBS, 20 we computed that 16 patients in each group were needed to show such a decrease with a power of 90% at the 5% α level (secondary endpoint). Finally, to compensate for potential dropouts we invited a total of 48 patients and 12 controls to participate in the study.

The immune complex were visualised using enhanced chemiluminescence's detection reagents (ECL‐kit, Amersham, Italy) according to the manufacturer's instructions and analysed by automatic Image Quant 400 apparatus (GE Healthcare, Amersham, UK). The results were expressed as optical density of the relative protein bands. Beta‐actin was used to normalise all Western blot experiments [anti‐beta Actin antibody (AC‐15:sc 69879 Santa Cruz Biotechnology Inc., Dallas, TX, USA, dil. 1:1000 v:v]. Results were reported as the ratio between the expression of specific protein and beta actin.

Protein expression was assessed by western blot. Briefly, the day of experiments, biopsies stored at −80 °C, were placed in a mortar, finely chopped and homogenised using liquid nitrogen. The obtained homogenised powder was reconstituted with 300 μL of the following lysis buffer: Tris‐HCl pH 7.5 (50 mM), NaCl (150 mM), sodium ortovanadate (1 mM), glycerophosphate (20 mM), EDTA (2 mM), DTT (1 mM), PMSF (1 mM), leupeptin (5 μg/mL), aprotinin (5 μg/mL), pepstatin (5 μg/mL) and then collected in 1.5 mL eppendorf tubes. Therefore, samples were frozen and thawed three times in liquid nitrogen and then placed under rotation for 30 min at 4 °C in order to optimise the homogenisation process. After centrifugation at 850, to remove cell debris, the supernatants were collected and the protein concentration of each sample was determined by using the BSA for protein determination following the manufacturer's instructions (detection limits: 2000–31.25 μg/mL). Protein samples (35 mg/mL) were mixed with gel loading buffer in a ratio 1:1, boiled for 5 min and centrifuged at 850for 10 min. Protein samples were separated under reducing conditions in 12% SDS‐polyacrylamide gel using a Biorad apparatus for mini‐gel (Bio‐Rad Laboratories, Hercules, CA, USA). The proteins were transferred onto nitrocellulose membrane on a semi‐dry Bio‐Rad blotting apparatus according to the manufacturer's instruction. Membranes were incubated in 5% milk buffer at room temperature under shaking for 2 h, to block nonspecific protein sites. Therefore, membranes were incubated at 4 °C overnight with the following primary antibodies diluted in milk buffer:

The levels of the main endocannabinoids, palmitoyl‐ethanolamide and oleoylethanolamide were measured as previously reported. 18 Briefly, tissue samples were homogenised in chloroform/methanol/Tris‐HCl 50 mM pH 7.4 (2:1:1, v/v) containing 10 pmol of d5‐2‐arachidonoyl‐glycerol and 5 pmol of d8‐anandamide, d4‐palmitoyl‐ethanolamide and d2‐oleoylethanolamide. 19 The lipid‐containing organic phase was purified by open‐bed chromatography on silica gel and fractions eluted with chloroform/methanol 9:1 by vol. (containing anandamide, 2‐arachidonoyl‐glycerol, palmitoyl‐ethanolamide, oleoylethanolamide) were analysed by isotope dilution‐liquid chromatography/atmospheric pressure chemical ionisation/mass spectrometry (LC‐APCI‐MS). MS detection was carried out in the selected ion‐monitoring (SIM) mode using m/z values of 356 and 348 (molecular ions +1 for deuterated and undeuterated anandamide), 384.35 and 379.35 (molecular ions +1 for deuterated and undeuterated 2‐arachidonoyl‐glycerol), 304 and 300 (molecular ions +1 for deuterated and undeuterated palmitoyl‐ethanolamide), 328 and 326 (molecular ions +1 for deuterated and undeuterated oleoylethanolamide). The amounts of endocannabinoids, palmitoyl‐ethanolamide and oleoylethanolamide were calculated on the basis of their area ratio with the internal deuterated standard signal area. Amounts in pmols were normalised both for mg of wet tissue and for mg of total extracted lipids.

Histamine detection was performed by the Enzyme Immuno Assay EIA kit IM2562, Immunotech SAS (Marseille, France). The experiments have been performed on the neat supernatants according to manufacture instruction. The sensitivity of the assay was 1.0 nM–100 nM of histamine. In several samples the amount of histamine was under the detection limit of the kit assay (1 nM), and for this reason it was impossible to normalise the samples for the total protein content. Nerve growth factor (NGF) detection was performed by the NGF Emax ImmunoAssay System (Cat. N. G7630), Promega Corporation (Madison, WI, USA). The experiments were performed on the neat supernatants according to manufacture instruction. The assay detects a minimum of 7.8 pg/mL of NGF. Serotonin levels were detected by a serotonin enzyme‐linked immunosorbent assay (ELISA) kit (Cat. N. KA1894), Abnova (Heidelberg, Germany). The experiments were performed on the neat supernatants according to manufacture instruction. The assay detects a minimum of 5.0 ng/mL of serotonin.

Spontaneous release of mucosal mediators from colonic biopsies was obtained as previously described. 16 , 18 Briefly, upon removal, biopsies were rapidly immersed in hard plastic tubes containing 1200 μL of sterile filtered Dulbecco Modified Eagle's Medium buffer, continuously oxygenated (95% O 2 /5% CO 2 ) at 37 °C. 18 After a 25‐min incubation period, samples were centrifuged at 850 g for 5 min, and 200 μL of supernatant aliquoted and stored at −20 °C until the assay, while biopsies were collected and stored at −80 °C. 17

Biopsies were fixed in buffered 10% formalin and processed for either H&E histology or immunohistochemistry using previously validated protocols. 16 , 17 Histological sections were evaluated for the exclusion of microscopic colitis or overt mucosal inflammation by a pathologist who was unaware of the diagnosis. For immunohistochemistry, paraffin‐embedded biopsies were cut and processed as following. Mast cells were detected as previously described, 16 using mouse monoclonal antibody directed against tryptase (1:3000; Millipore, Billerica, MA, USA). For the staining after 2 h of incubation at room temperature with the primary antibody, slides were washed with phosphate‐buffered saline and then incubated with secondary biotinylated anti‐mouse antibody followed by streptavidin – horseradish peroxidase conjugate (Millipore). Mast cells were quantified on immunohistochemically stained sections with a Leitz Dialux microscope in blind fashion using a previously validated computer‐assisted analysis system. 16 , 17 All the samples were centralised in Bologna, and analysed in blind by two experts (CC and MRB). Samples were analysed blindly and codes were opened only at the end of the study. Results are expressed as percentage of cells over lamina propria area (%). Quantification of cells using this method will be also simply defined as ‘mast cell count’ in the manuscript.

The questionnaires collected data regarding digestive symptoms and bowel habit at baseline and every 4 weeks during treatment period, using previously validated 5‐point Likert scales. 16 , 17 For each symptom, severity of influence on usual activities (mild – not influencing usual activity; moderate – diverting from, but not urging modifications of usual activities; severe – markedly influencing usual activity so as to urge modifications; extremely severe – bed rest) and frequency during the week (rare – once a week; occasional – 2–3 time/week; frequent – 4–6 time/week; extremely frequent – 7 time/week) were graded 0–4 and analysed. 16 , 17 The following symptoms of IBS were assessed: abdominal pain/discomfort and bloating. Flatulence, relief of abdominal pain/discomfort by defecation, onset of symptoms associated with a change in frequency or in form (appearance) of stool were assessed as present/absent. Number of bowel movements per day and/or week and bowel habit characteristics, assessed by the Bristol stool scale, were also recorded. Digestive symptoms other than those of IBS were also investigated by previously validated questionnaires. 17 Specifically, each patient completed a symptom questionnaire that assessed the following symptoms of dyspepsia: epigastric pain/burning, post‐prandial fullness, early satiety, epigastric bloating, nausea and vomiting. As for IBS symptoms, both severity and frequency were graded 0–4, as above described. Finally, for gastro‐oesophageal reflux disease, the following symptoms were assessed: heartburn, acid regurgitation, chest pain and dysphagia. General well‐being was monitored using a 10‐point visual analogue scale (0 = none, 10 = worst).

Eligible patients with symptoms meeting Rome III criteria for diagnosis of IBS 1 and controls were recruited from five European study centres, including Bologna, Nantes, Barcelona, Tuzla and Zagreb. Controls were recruited by public advertisement and included in the study after thorough exclusion of gastrointestinal complaints. For the patients, inclusion criteria comprised a positive diagnosis of all IBS subtypes [IBS with constipation (IBS‐C), with diarrhoea (IBS‐D) or mixed (IBS‐M)] 1 , at least 18 and no more of 70 years of age, negative colonoscopy or barium enema examination within the previous 5 years, negative relevant additional screening or consultation whenever appropriate. Patients and controls were excluded if they were pregnant, breast‐feeding or not using reliable methods of contraception. Exclusion criteria also included the current use of nonsteroidal anti‐inflammatory drugs, corticosteroids and mast cell stabilisers, the use of topical or systemic antibiotics in the last month, or the continuous use of stimulant laxatives, major abdominal surgery, a history of untreated food intolerance (in contrast, we included subjects remaining symptomatic despite the withdrawal of the suspected food), inflammatory bowel disease, infectious diarrhoea or diverticular disease, coeliac disease (by detection of anti‐transglutaminase and anti‐endomysial antibodies), allergic diseases, including asthma (excluded by family and personal history and specific anti‐IgE antibodies), and other organic or psychiatric disorders as assessed by medical history, appropriate consultations and laboratory tests.

The protocol was designed by the coordinating centre (GB, CC and VS). Data were monitored by the Sponsor (CM&D Pharma Limited, Padova, Italy, under license of Epitech group Srl, Saccolongo, Padova, Italy) with the supervision of Medical Trials Analysis company (MTA Srl, Ferarra, Italy; an academic‐driven contract research organisation). Medical Trials Analysis personnel, in collaboration with the coordinating centre, analysed the trial data. A statistical analysis plan was released and approved by the Sponsor prior to the database lock and unblinding of the treatment. The protocol was approved by an independent ethics committee at each centre and carried out according to the Declaration of Helsinki and the principles of good clinical practice. All patients gave written informed consent. All authors have access to the study data and have reviewed and approved the final manuscript. The trial was registered in a public registry (ClinicalTrial.gov No. NCT01370720).

Twelve healthy controls (HC) were enrolled to compare their biological data with that of IBS patients at baseline. All subjects underwent a formal clinical assessment and were further phenotyped using validated questionnaires (see below). Within 2 weeks of the screening visit, all subjects underwent left colonoscopy after the cleansing of the distal colon with two 500‐mL water enemas performed the evening before and on the morning of the procedure. In all cases, eight mucosal biopsies were obtained from the proximal descending colon at baseline, while only in patients with IBS a second set of eight biopsies was obtained at the end of treatment period. Two biopsies were used for routine H&E histology and immunohistochemistry, four biopsies for mucosal mediators release experiments, one biopsy for biochemical/biomolecular analysis and the last one for endocannabinoid system and endocannabinoid‐like mediators’ assays.

This is a pilot, phase IIb, randomised, double‐blind, placebo‐controlled, parallel‐arm, multicentre trial, designed to study the efficacy and safety of co‐micronised palmithoylethanolamide/polydatin in adult patients with IBS (as assessed according to the Rome III diagnostic criteria for IBS 1 ). The study included a 2‐week screening period, and a 12‐week placebo‐controlled treatment period (Figure 1 ). There was no follow‐up period at the end of treatment period. After the screening phase, eligible patients were randomly assigned to either co‐micronised form palmithoylethanolamide/polydatin 200 mg/20 mg (EP1844784, patented by Epitech group Srl, Saccolongo, Padova, Italy), or the equivalent placebo (without the active treatment, replaced by equal amount of microcrystalline cellulose), b.d., in a 1:1 ratio, for 12 weeks. Palmithoylethanolamide is approved by the Italian Ministry of Health as ‘Food for special medical purposes’ for visceral pain. Dosing of the active treatment in the present study was based on a previous dose‐finding study (although not performed in IBS). 15 Study visits were conducted every 4 weeks during the treatment period. All the subjects were blindly allocated by means of scratch cards to one of the two treatment groups according to a computer‐generated randomisation list provided by the sponsor. A validated SAS program was used by an independent statistician to generate a randomisation list with blocks, block size = 4, pre‐allocated to centres. Patients, study investigators, and sponsor staff were blinded to the randomisation codes. The codes were kept confidential until the end of the study when the randomisation code was broken after the database lock.

Rescue medications were taken 39 times (17 in palmithoylethanolamide/polydatin group, vs. 22 in placebo group) by 15 patients (seven in palmithoylethanolamide/polydatin group, vs. eight in placebo group). In the placebo group, the average frequency of rescue medication was higher than that of the palmithoylethanolamide/polydatin group in most of the weeks of observation, but the differences were not statistically significant.

Effect of active treatment or placebo on abdominal pain/discomfort severity. Single data are reported for each subject, with data points joined for before, during and at the end of the treatment. Palmitoylethanolamide/polydatin (PEA/PD) treatment was effective in reducing the severity of abdominal pain/discomfort, as demonstrated by the significant interaction between treatment and time ( P < 0.050). P value was computed using a generalised linear mixed model.

Figure 4 shows the effect of active treatment or placebo on abdominal pain/discomfort severity, showing single data for each subject, with the data points joined for before, during and the end of treatment. The severity of abdominal pain/discomfort decreased significantly over the time ( P = 0.001), with a significant effect of treatment ( P = 0.002), that was related to palmithoylethanolamide/polydatin, as demonstrated by the significant interaction between treatment and time ( P = 0.049) (Table 2 ). In contrast, the frequency of abdominal pain/discomfort reduced significantly over the time ( P = 0.001), with a significant effect of treatment ( P = 0.001), but this interaction was not significant ( P = 0.895) (Table 2 ). Interestingly, for the severity of abdominal pain/discomfort we showed that responder patients were 18/29 (62.1%) in the palmithoylethanolamide/polydatin group vs. 10/25 (40.0%) in the placebo group, with a delta difference of 22.1% ( P = 0.115).

As shown in Table S2 (published online), oleoylethanolamide levels were significantly reduced ( P = 0.002), while cannabinoid 2 receptor expression was significantly increased ( P = 0.012) in patients with IBS vs. HC. There was no association between anandamide, 2‐arachidonoyl‐glycerol, palmitoyl‐ethanolamide or oleoylethanolamide and bowel habit (data not shown). Interestingly, the expression of the cannabinoid 1 receptor and fatty acid amide hydrolase was significantly higher in patients with IBS‐C vs. IBS‐D and IBS‐M (cannabinoid 1 receptor: 2.1 ± 3.6 in IBS‐C, 0.7 ± 0.4 in IBS‐D and 1.0 ± 0.5 in IBS‐M, P = 0.011; fatty acid amide hydrolase: 1.4 ± 0.4 in IBS‐C, 0.9 ± 0.4 in IBS‐D and 0.8 ± 0.2 in IBS‐M, P = 0.008). As shown in Table S2 (published online), there was no significant effect of active treatment or placebo on the endocannabinoid system and endocannabinoid‐like mediators over time.

Representative photomicrographs showing tryptase positive mast cells in the colonic mucosa of a healthy control (HC), a patient with IBS at baseline (IBS‐T0) and at the end of the treatment (IBS‐T12) (a). Mast cells were identified by immunohistochemistry for tryptase (bar = 20 μm). Compared with HC, the mean area of lamina propria occupied by tryptase‐positive mast cells (mast cell count, %) was significantly increased in patients with IBS ( P = 0.013) (b). However, there was no significant effect of active treatment or placebo on mast cell count over time, from baseline (T0) to the end of treatment (T12) ( P = 0.501) (c).

As shown in Figure 3 , the area of lamina propria occupied by mast cells (mast cell count, %) was significantly greater in patients with IBS vs. HC ( P = 0.013). There was no effect of gender ( P = 0.702), age ( P = 0.898), centre ( P = 0.335) or bowel habit ( P = 0.689). There was no significant effect of active treatment or placebo on mast cell count over time ( P = 0.501). No effect was observed for gender ( P = 0.839), age ( P = 0.422), centre (0.422), bowel presentation ( P = 0.927), treatment (0.660) and time (0.747).

The flow chart of the enrolment and randomisation of the study was reported in Figure 2 . The study was conducted from June 2010 to December 2012. A total of 157 patients were screened for this study, of whom 103 were excluded because they did not meet inclusion criteria ( n = 37) or declined to participate ( n = 66). Fifty‐four patients were randomised, 29 allocated to palmithoylethanolamide/polydatin and 25 to placebo treatment groups. The primary efficacy outcome was available in all the subjects, and all the subjects received at least one dose of the study drug and were included in the ITT population. Among the 54 randomised patients, 10 patients prematurely discontinued the study being lost during follow‐up (five patients in both the groups refused second sigmoidoscopy). Demographic and baseline characteristics of the subjects included in the study were similar between groups and reported in Table 1 .

Discussion

The main finding of the present study was that the palmitoylethanolamide/polydatin treatment was markedly effective in reducing the severity of abdominal pain/discomfort in IBS. In order to understand the potential mechanisms of action of palmitoylethanolamide/polydatin in IBS symptoms, we studied mast cell infiltration/activation and the peripheral endocannabinoid system. Our data confirmed that compared with controls, unselected patients with IBS have an increased infiltration of mast cells in the colonic mucosa. Furthermore, we showed that the anti‐inflammatory fatty acid amide oleoylethanolamide was significantly reduced, while the peripheral cannabinoid 2 receptor was significantly increased in patients with IBS as compared with controls, suggesting an alteration of the endocannabinoid system and endocannabinoid‐like mediators in IBS. Nonetheless, we found no effect of palmitoylethanolamide/polydatin treatment on mast cell count and activation, endocannabinoid and endocannabinoid‐like levels, cannabinoid 1 and 2 receptors and fatty acid amide hydrolase, suggesting that palmitoylethanolamide/polydatin treatment exerts its effect through different pathways.

Abdominal pain is a common symptom in patients with functional bowel disorders, and IBS in particular. Pain is a key element of symptom severity, is inversely correlated with patient's health‐related quality of life and represents a driver of healthcare utilisation.21 For these reasons, abdominal pain in association with abnormal defecation is now recommended as a major outcome measure by the Food and Drug Administration (FDA) and European Medicines Agency (EMA) in IBS clinical trials.22 Our results showed that palmitoylethanolamide/polydatin was statistically effective on the severity, but not on the frequency of abdominal pain. Although this can be seen as a limitation, previous studies suggest that pain severity may be more relevant than pain frequency and, as a consequence, included as the primary end‐point in IBS trials.22

Although dietary approaches are becoming popular in IBS, their efficacy on abdominal pain has been less strictly assessed. In addition, most studies have been focused on dietary elimination [e.g. low‐fermentable oligosaccharides, disaccharides, monosaccharides and polyols (FODMAPs), gluten avoidance, hypoallergenic diets] rather than supplementation. As a result, patients may be uncertain about foods and supplements allowed in their diets.23 Hence, we believe that our data on a dietary supplementation for the management of pain can be viewed with interest as a potential alternative to strict pharmacological approaches.

Previous findings support the role of mast cells in the pathophysiology of IBS. The major physiological functions of gastrointestinal mast cells (regulation of epithelial functions, contribution to the host defence and crosstalk with the enteric nervous system) are crucial in gut homoeostasis.24 Mast cell hyperplasia and/or activation (assessed by degranulation or activation rate, mucosal content and/or spontaneous release of histamine, protease or tryptase) may be considered a common feature of IBS patients (for review, see25), although inconsistent results are sometimes reported.26, 27 Mucosal mediators, particularly of mast cell origin, may contribute to sensory‐motor dysfunction seen in IBS, as demonstrated by translational approaches in animal models,18, 28 and a relationship exists between mast cell count and/or activation and clinical features of IBS patients.16, 17, 29, 30. Finally, mast cells stabilisers (chromones)31 and H1 histamine receptor antagonists, such as ketotifen26 and ebastine,32 improve symptoms and quality of life of patients with IBS. In our study performed in five different countries, we confirmed previous results by our and other groups16-18, 25, 29, 30 showing a significant increase in colonic mast cell count in IBS. Samples were centralised in Bologna, assessors were blinded and codes were opened only at the end of the study. This study did not confirm previous data showing increased mast cell activation, although a trend towards an increase was shown for several of the immune factor parameters we studied. It is possible that better markers of activation, such as the current gold standard of electron microscopy,16 would be more useful than those utilised in the present study. Although it is possible that there is no effect on these parameters, a type 2 error cannot be discounted as the study, given its pilot nature, was likely underpowered to detect these differences. Finally, contrasting results are not unusual in a heterogeneous condition such as IBS, and are probably related to genetic, regional, dietary, environmental and experimental differences among studies.

This is the first study assessing in depth the endocannabinoid and endocannabinoid‐like systems in IBS. We showed that levels of oleoylethanolamide were significantly reduced in the colonic mucosa of patients with IBS. Interestingly, also the levels of anandamide, 2‐arachidonoyl‐glycerol, and palmitoylethanolamide were reduced, although not in a statistically significant manner, while the expression of cannabinoid 2 receptor was higher in patients with IBS. Altogether these results suggest an impairment of the endocannabinoid system in IBS, supporting the old unproven hypothesis of ‘clinical endocannabinoid deficiency’ in IBS.33 Although endocannabinoids are potentially involved in the regulation of many factors implicated in IBS pathophysiology, including visceral hypersensitivity, pain, inflammation, secretion, motility and also microbiota, which may modulate the expression of cannabinoid 2 receptors,5, 34 the state of this system is virtually unknown in IBS. Based on the well‐known analgesic effect of cannabinoids in various animal models of acute and chronic pain and visceral hypersensitivity,5 it is plausible to suggest the deficiency of the endocannabinoid system in a condition, such as IBS, in which pain or discomfort are the key symptoms.33 To the best of our knowledge, only a recent pilot study assessed plasma levels of endocannabinoid system in 14 female patients with IBS and 7 controls.35 This study showed higher levels of 2‐arachidonoyl‐glycerol and lower levels of oleoylethanolamide and palmitoyl‐ethanolamide in patients with IBS‐D, while the levels of oleoylethanolamide were increased in patients with IBS‐C. In our work we did not observe any difference in the endocannabinoid levels related to bowel habit. Interestingly, we opted to analyse mucosal as opposed to systemic endocannabinoid and related mediator levels to obtain a direct measurement of the fraction possibly involved in changes of gut sensory and motor function. In our work, the most relevant impairment was observed for oleoylethanolamide. Oleoylethanolamide regulates gastric emptying and intestinal motility, but the exact mechanism of action is not yet defined.5, 6 Oleoylethanolamide, like palmitoylethanolamide, exerts its effect principally through activation of PPARα.5, 6 However, in animal models in which oleoylethanolamide inhibited upper gastrointestinal transit, this alteration was still present in PPARα, cannabinoid 1 and 2 knockout mice,36 suggesting that PPARα and cannabinoid receptors are not involved in the suppression of motility by oleoylethanolamide. Whether or not the reduced levels of oleoylethanolamide as observed in our study may impact on gastrointestinal transit or visceral pain is virtually unknown at present. Our results showing increased expression of cannabinoid 2 receptor are in line with the concept that the cannabinoid 2 receptor, although also expressed in colonic epithelial cell, is an established constituent of immune system cells, including mast cells, and is up‐regulated during inflammation.5, 6 The increased expression of cannabinoid 2 receptor is not surprising in a condition characterised by mast cell hyperplasia such as IBS.25

Despite the effect of palmitoylethanolamide/polydatin on abdominal pain demonstrated by the present study, the underlying mechanism of action remains to be elucidated. It is possible that mechanisms more complex than that explored in this pilot study may explain the observed clinical benefit. For example, we have previously demonstrated that only mast cells in close proximity to colonic nerves correlated with abdominal pain in IBS,16 while the mast cell number alone or the activation of mast cells as assessed by electron microscopy or histamine and tryptase release were not associated with IBS symptoms. This is in line with the view that IBS is a complex and multifactorial disease and a single mechanism unlikely explains this complexity.16 Interestingly, other mechanistic studies showing a benefit of active treatment in the management of IBS, failed to clarify the exact mechanism of action of the active compound, confirming the complexity of these type of studies.26, 27, 32 Whether the palmitoylethanolamide/polydatin effect is centrally related, secondary to mast cell stabilisation or to modulation of the endocannabinoid system remains to be further investigated.37-39

We acknowledge the limitations of the present study. Due to its pilot and mechanistic nature, the sample size was limited and thus our study was not powered for the clinical endpoints. We preferred to analyse the severity and frequency of digestive symptoms with a previously validated 5‐point Likert scale,16, 17 and for this reason we did not use the new FDA and EMA recommended end points.22 Thus, it is difficult to compare the clinical benefits observed in this study with the results of studies fulfilling the new end points.22 The therapeutic effect of palmitoylethanolamide/polydatin should be confirmed in larger clinical trials. Our population was heterogeneous because participants were recruited from five different European countries and included all IBS stool pattern subtypes. This decision was based on previous studies showing a similar degree of immune cell activation irrespective of bowel habit.16, 17, 20 Thus, it is likely that we have included patients with different underlying pathophysiological mechanisms, and this may explain why the mechanism of action of palmitoylethanolamide/polydatin remains to be elucidated. Finally, for all these reasons the generalisability of the trial findings needs caution and requires further confirmation.

In conclusion, the results of the present study indicate an alteration of the endocannabinoid and related signalling systems in IBS and suggest that IBS patients may obtain benefits from palmitoylethanolamide/polydatin therapy. These results pave the way to further studies aimed at targeting the endocannabinoid system in IBS.