Field of the Invention

The invention relates to methods for determining the efficacy of treatments for treatment of irritable bowel syndrome and related disorders.

The invention further relates to the detection of histamine metabolites.

BACKGROUND OF THE I NVENTI ON

The irritable bowel syndrome (IBS) is a very common disorder, affecting up to 20% of the western population. IBS is characterized by abdominal discomfort and/or pain associated with altered defecation pattern in the absence of a known organic cause [Ohman L, S Simren M. (2010) Nature rev. Gastroent. hepatol.7, 163-73]. Although the predominant defecation pattern may vary from diarrhoea to constipation, abdominal pain or discomfort is present in all patients and represents the most disturbing and therapy-resistant symptom in IBS. It is linked to abnormal perception of visceral stimuli such as intestinal distension or nutrient infusion, also referred to as visceral hypersensitivity [Ohman L, & Simren M. (2010) Nature rev. Gastroent. hepatol.7, 163-73; Kuiken SD, et al. (2005) Aliment. Pharmacol, ther.22, 157-64; Cam illeri M et al. (2008) Clin. Gastroent. Hepatol.6, 772-81]. Clinical management of IBS largely consists of symptomatic treatment aimed at restoring the dominant defecation pattern, but often leaves visceral pain unaffected. Drugs targeting visceral hypersensitivity may thus represent an important new pharmacological approach to treat IBS. Dysfunction of peripheral nociceptive neurons is demonstrated in both somatic and visceral pain models [Brierley SM & Linden DR. (2014) Nat Rev Gastroenterol Hepatol 11 , 611-27]. In particular, the upregulation and sensitization of receptors located on the peripheral nerve terminals of nociceptors is now recognized as an important mechanism of visceral hypersensitivity [Sousa-Valente J et al. (2014) Br J Pharmacol 171, 2508-27]. TRPV1 , the receptor responsive to capsaicin, heat, acidosis and endovanilloids, is one of the nociceptors involved in this process. Sensitivity to colorectal distension is decreased in TRPV1 knock-out mice [Jones RC, 3 ^rd et al. (2005) J Neurosci 25, 10981-9] and reduced by TRPV1 antagonists [van den Wijngaard RM, et al. (2009) Neurogastroent motil , 21, 1107-e94; Winston J, et al. (2007) Gastroenterology 132, 615-27], while expression of TRPV1 is increased in preclinical models of visceral hypersensitivity [ Miranda A, et al. (2007) Neurosci 148 , 1021-32]. Previously, we showed increased pain responses to rectal application of the TRPV1 agonist capsaicin in IBS patients, but we failed to detect upregulation of TRPV1 mRNA or protein expression [van Wanrooij SJ, et al. (2014) Am J. Gastroent 109, 99-109]. These data suggest that sensitization rather than upregulation of TRPV1 occurs in IBS. To date, no human data are available supporting this hypothesis.

Sensitisation of TRPV1 is well documented in models of neuropathic pain and in the skin [Hirth M, et al. (2013) Pain 154, 2500-11; Morales- Lazaro SL, et al. (2013) J Physiol 591 , 3109-21]. Although microscopic inflammation is reported in IBS, two recent clinical studies failed to demonstrate improvement of IBS symptoms following treatment with the anti-inflammatory agent mesalazine, arguing against inflammation as a potential mechanism underlying sensitization of TRPV1 [Barbara G, et al. (2014) Gut 63, 1530-1532. Lam C, et al. (2016) Gut 65, 91-99]. Alternatively, mast cell mediators such as histamine and serotonin, known to induce visceral hypersensitivity in mice, could be involved [Cenac N, et al. (2010) Gut 59, 481-8] . Mast cell activation and release of histamine and tryptase is indeed increased in IBS [Cenac N, et al. (2007) J clin invest 117, 636-47; Barbara G, et al. (2004) Gastroenterology 126, 693-702] . Moreover, we recently showed improvement of IBS symptoms and visceral pain in a small proof-of-concept study with ketotifen, a mast cell stabilizer and histamine 1 receptor (H1-R) antagonist [Klooker TK, et al. (2010) Gut 59, 1213-21.].

Summary of the invention

One aspect of the present invention relates to methods for determining whether treatment with an H1 -receptor antagonist is likely to cure abdominal pain in a irritable bowel syndrome (IBS) patient functional dyspepsia patient, a Crohn's disease patient or a patient with diverticulosis, comprising determining whether a body sample of said patient has increased levels of a histamine metabolite relative to levels in a normal tissue sample of a healthy individual, wherein an increase of a histamine metabolite is indicative for the effectivity of a treatment to be performed with said H1-receptor antagonist of said abdominal pain.

Specific embodiments of these methods relate to patients with irritable bowel syndrome (IBS). In specific embodiments of these methods, the abdominal pain is caused by a visceral sensitivity disorder, especially by visceral hypersensitivity. In specific embodiments, the histamine metabolite is imidazole acetaldehyde, imidazole-4-acetaldehyde, imidazole-4-acetic acid or N-ribosylimidazole-4-acetic acid level.

In specific embodiments, the H1 -receptor antagonist is ebastine.

In specific embodiments, the metabolite is determined by immunofluorescence or by a spectrophotometric assay.

In specific embodiments, the tissue sample is a peripheral blood sample or a biopsy of the intestine.

In specific embodiments, the body sample is a stool sample, or a urine sample, or a biopsy from the patient.

Another aspect of the present invention relates to methods for identifying whether a patient with irritable bowel syndrome (IBS), a non-ulcer (functional) dyspepsia, a Crohn's disease or diverticulosis is eligible to receive anti- H1 -receptor therapy comprising:

(a) providing a biopsy from said patient;

(b) determining the level of said at least one histamine metabolite in the biopsy sample;

(c) classifying the level relative to levels in normal tissue of a healthy individual of said metabolite; and

(d) identifying the patient as eligible for anti- H1-receptor therapy where the patient's sample is classified as having an elevated level of said histamine metabolite, compared to the level in normal tissue of a healthy individual.

Specific embodiments are for identifying whether a patient with irritable bowel syndrome (IBS) is eligible to receive anti- H1-receptor therapy.

Typically, the histamine metabolite is selected from the group consisting of imidazole acetaldehyde (lmidazole-4-acetaldehyde), imidazole-4-acetic acid and N- ribosylimidazole-4-acetic acid.

The histamine metabolite can be determined by e.g. an immunoassay or by a spectrophotometric assay.

Sensitization of the nociceptor TRPV1 by histamine is proposed as a mechanism of visceral hypersensitivity in preclinical models. Here, we aimed to investigate its role in irritable bowel syndrome (IBS) and evaluated if blockade by histamine 1 receptor (H1-R) antagonism may represent a treatment for IBS.

Using live calcium imaging, we compared activation of submucosal neurons in rectal biopsies of IBS patients and healthy subjects by the TRPV1 agonist capsaicin. Moreover, sensitization of TRPV1 by histamine, its metabolite imidazole acetaldehyde and biopsy supernatants was assessed by calcium imaging of dorsal root ganglion neurons.

Next, fifty-five IBS patients (31 yrs IQR[25-46], 34 F) were enrolled in a randomized double-blind placebo-controlled trial evaluating the effect of 12 weeks treatment with the H1 -R antagonist ebastine (20mg/day) on rectal distension-induced sensation and clinical symptoms.

TRPV1 responses of submucosal neurons from I BS patients are potentiated compared to healthy volunteers. This effect is mimicked in healthy volunteers by pre-incubation with histamine and is blocked by the H1-R antagonist pyrilamine. In a proof-of- concept study, treatment of IBS patients with ebastine reduced visceral hypersensitivity, improved symptom relief (ebastine 46% vs placebo 13% , P=0.024) and reduced abdominal pain scores (ebastine 39±23 vs placebo 62±22, P=0.0004). While in mammals, methylation of the imidazole ring by histamine N- methyltransferase (HMT) and oxidative deamination of the primary amino groups catalysed by diamine oxidase (DAO) to form N-methylhistamine and imidazole-4- acetaldehyde, respectively (figure 9) is considered an inactivation of histamine. Present invention demonstrates that H1-R mediated sensitization of TRPV1 is involved in IBS and may represent a new target for treatment of abdominal pain. Histamine levels in the supernatants were not different between IBS and human volunteer (HV) and were extremely low. Present invention, however, demonstrates that (secondary) histamine metabolites have a sensitizing on TRPV1 and that such histamine metabolites, such as imidazole acetaldehyde (lmidazole-4-acetaldehyde) and its derivatives such as imidazole-4-acetic acid and N-ribosylimidazole-4-acetic acid, which mimics the TRPV1 sensitizing effect of histamine, is a companion diagnostic biomarker.

The present invention illustrates that histamine not only has an effect on visceral hypersensitivity via activation of the histamine receptor, but that histamine and especially histamine metabolites sensitize the TRPV1 receptor. This TRPV1 receptor is also known to be involved with visceral hypersensitivity.

Histamine receptor antagonists have a beneficial effect on visceral hypersensitivity in IBS patients. It has been found that treatment with ebastine has no effect on peripheral blood cell count or cytokine production, and did not alter the expression of histamine receptors or TRPV1 in the intestine. Thus the effect of ebastine is not mediated by a potential anti-inflammatory effect but rather due to its effect on TRPV1 sensitization. Comparison of histamine metabolite levels in a patient prior to treatment with Histamine receptor antagonists indicates whether a patient would benefit from such treatment. Indeed histamine metabolites in the digestive tract will trigger the TRPV1 receptor and cause discomfort. A decrease in histamine metabolites will accordingly alleviate the disease.

Patient with symptoms of IBS are a heterogenous populations and not all of them show TRPV1 sensitisation. The present invention allows identifying those patients wherein TRPV1 sensitisation occurs, via the presence of histamine metabolites. Further, treatment of IBS can result in a relief by placebo effects. The present invention allows following up the efficacy of the method by determining an objective parameter (histamine metabolites) correlated with TRPV1 sensitization (and thus activation).

Brief description of the figures

Figure 1. is a graphic display demonstrating TRPV1 on human submucosal neurons of IBS patients is more sensitive compared to healthy volunteers due to bioactive mediators in the local micro-environment.

(a) Representative traces of the intracellular Ca ²⁺ response of submucosal neurons in biopsies of healthy volunteers (HV, blue) and IBS patients (orange) to acute application of capsaicin (0.1 nM). (b) Data showing the amplitude of the Ca ²⁺ flux and the number of responding neurons to capsaicin in IBS patients (n=9) and HVs (n=12). Data are presented as mean +_ SEM. ^* P<0.05, 2-way ANOVA repeated measures within subjects, (c) Representative response profiles (rate histogram, wavemarked neurogram and neurogram) of mouse single serosal afferents to supernatants from rectal biopsies of HV or IBS patients. Spontaneous activity (total number of action potentials normalised to baseline activity) was significantly increased during the 12 minutes incubation with IBS supernatants (n=8) compared to HV supernatants (n = 7). ^* P< 0.05, Mann Whitney U test.

Figure 2. is a graphic display demonstrating that Histamine sensitizes TRPV1 through H1-R activation.

(c) Representative traces of Ca ²⁺ flux of submucosal neurons in biopsies of healthy subjects upon capsaicin (0.1 nM) application before and after incubation with histamine (10 μΜ) in the presence or absence of the H1 -R antagonist pyrilamine. (d) Histamine application resulted in a significant increase in both the amplitude of Ca ²⁺ signal and the number of responding neurons to capsaicin using rectal biopsies of HVs (n=7) that was abolished in the presence of pyrilamine. ^* P<0.05, Wilcoxon signed rank-test, (e) I mmunohistochemical staining of submucosal plexus in rectal biopsies of a HV showing colocalisation of the pan-neuronal marker PGP9.5 (green), histamine 1 receptor (H1-R, red) and TRPV1 (blue). Scale bar: 10 μιτι upper panel. Figure 3. TRPV1 sensitization by histamine and its metabolite imidazole acetaldehyde is mediated via H1-R

Representative tracings of the effect of histamine and pyrilamine on the Ca ²⁺ response of DRG neurons evoked by 10 nM capsaicin. Histamine (5 μΜ) potentiates the effect of capsaicin, an effect that is completely abolished in the presence of the H1-R antagonist pyrilamine (1 μΜ) and in DRG neurons lacking H1-R (H1-R7 ). Figure 4. TRPV1 sensitization by histamine and its metabolite imidazole acetaldehyde is mediated via H1 -R

(a) The effect of histamine, pyrilamine (Pyr), the H2-R antagonist ranitidine (Ran, 10 μΜ), H3-R antagonist clobenpropit (Clob, 30 nM) and H4-R antagonist JNJ7777120 (JNJ, 1 μΜ) and DRG neurons lacking H1-R (H1-R7 ) on the amplitude of the Ca ²⁺ response and the percentage DRG neurons responding to 10 nM capsaicin. Pre: capsaicin response prior to incubation, Veh: vehicle, Hist: histamine, ^* P<0.05, ^{* *} P< 0.01 , ^{* * *} P< 0.0001 ; % responding neurons was analysed using a Fisher's exact test; amplitudes were analysed by 1-way ANOVA with Dunn's multiple comparison correction, (b) The effect of histamine and its enzymatically prepared metabolite imidazole acetaldehyde on TRPV1 sensitization. Imidazole acetaldehyde (Im Ac, 5 μΜ) potentiates the effect of capsaicin, an effect that is prevented by pyrilamine (1 μΜ) and in DRG neurons lacking H1 -R (H1 -R7 ) . The same results were obtained with chemically synthesized imidazole acetaldehyde (data not shown).

Figure 5. Schematic representation of the study protocol (a) and trial profile (b). Figure 6. Effect of ebastine on global symptom relieve and abdominal pain.

(a) Data showing the effect of 12 weeks treatment (week w1-w12) with ebastine or placebo on the percentage of responders or patients with at least considerable relief of global symptoms. Ebastine resulted in a gradual increase in the percentage of responders, although not statistically different from placebo using a logistic generalized estimating equation model including week as a linear variable including interaction (P=0.16). The % of responders at the end of treatment was significantly greater in the ebastine compared to the placebo treated group (46% (11/24) vs 13% (3/23), P=0.024, Fisher's exact test). Note that during the run-out period (w13-w14, grey area), there was a significant drop in the % of responders in ebastine treated patients with a return to baseline value. ^* P= 0.024, Fisher's exact test at each week. (b) Effect of 12 weeks (w1-w12) treatment of placebo or ebastine on Subjects Global Assessment (SGA) of abdominal pain assessed on a 100 mm visual analogue scale. Ebastine treatment resulted in a significant reduction in abdominal pain score compared to placebo (P= 0.002, repeated measures ANOVA with baseline as covariate, week as continuous linear variable, treatment as factor and including interaction between week and treatment). The symptom score at week 12 was significantly lower in the ebastine compared to the placebo treated patients.

(c) Effect of ebastine and placebo on quality of life. Data are shown as mean changes from baseline at the end of treatment (week 12). Ebastine resulted in improvement of sleep, diet, social role and emotional role categories. Data are presented as mean± SEM ^* P< 0.05, ^{* *} P< 0.01 , ^{* * *} P< 0.001 ebastine vs placebo, Mann-Whitney-U test.

Figure 7. Effect of ebastine on rectal sensation evoked by isobaric rectal distensions, (a) Effect of 12 weeks treatment with ebastine or placebo on the change in summated symptom score (Delta Sum score, difference in score before and at the end of treatment) for all patients (Total, n=44), hypersensitive (HS, n=19) and normosensitive patients (NS, n=25). In the total group of IBS patients, the change in summated symptom score was significantly larger in the ebastine vs placebo group ( ^* P= 0.046, ANCOVA using baseline total score as covariate and including factor for treatment and type). A trend for reduction in summated score was observed in both the HS and NS subgroups following ebastine treatment (HS: P=0.35, NS: P= 0.067, ANCOVA). (b, c) Effect of ebastine or placebo on the summated symptom score (Sum score) at baseline and at the end of 12 weeks treatment for the total patient group, HS and NS patients, (d, e) Data showing the symptom scores, assessed on a 100 mm visual analogue scale (VAS score), evoked by 3, 9 and 21 mmHg at baseline and at the end of the treatment period with ebastine (d) or placebo (e) for all (Total), HS and NS patients. A reduction in the urge score evoked by the lower distension steps (3 and 9 mmHg) was observed in the ebastine, but not in the placebo group. Data are presented as medians and IQR. Comparisons in b and e were performed using the Wilcoxon signed rank test. ^* P< 0.05, # P= 0.06.

Figure 8. PBMC im munophenotypes before and after 12 weeks ebastine treatment. T cell phenotypes and B cell numbers were assessed and compared before and after ebastine or placebo treatment.

Figure 9 shows an overview of histamine metabolism.

Detailed description of the invention.

The following detailed description of the invention refers to the accompanying drawings. The same reference numbers in different drawings identify the same or similar elements. Also, the following detailed description does not limit the invention. Instead, the scope of the invention is defined by the appended claims and equivalents thereof. The following detailed description of the invention refers to the accompanying drawings. The same reference numbers in different drawings identify the same or similar elements. Also, the following detailed description does not limit the invention. Instead, the scope of the invention is defined by the appended claims and equivalents thereof.

Several documents are cited throughout the text of this specification. Each of the documents herein (including any manufacturer's specifications, instructions etc.) are hereby incorporated by reference; however, there is no admission that any document cited is indeed prior art of the present invention.

The present invention will be described with respect to particular embodiments and with reference to certain drawings but the invention is not limited thereto but only by the claims. The drawings described are only schematic and are non-limiting. In the drawings, the size of some of the elements may be exaggerated and not drawn to scale for illustrative purposes. The dimensions and the relative dimensions do not correspond to actual reductions to practice of the invention.

Furthermore, the terms first, second, third and the like in the description and in the claims, are used for distinguishing between similar elements and not necessarily for describing a sequential or chronological order. It is to be understood that the terms so used are interchangeable under appropriate circumstances and that the embodiments of the invention described herein are capable of operation in other sequences than described or illustrated herein.

It is to be noticed that the term "comprising", used in the claims, should not be interpreted as being restricted to the means listed thereafter; it does not exclude other elements or steps. It is thus to be interpreted as specifying the presence of the stated features, integers, steps or components as referred to, but does not preclude the presence or addition of one or more other features, integers, steps or components, or groups thereof. Thus, the scope of the expression "a device comprising means A and B" should not be limited to the devices consisting only of components A and B. It means that with respect to the present invention, the only relevant components of the device are A and B.

Reference throughout this specification to "one embodiment" or "an embodiment" means that a particular feature, structure or characteristic described in connection with the embodiment is included in at least one embodiment of the present invention. Thus, appearances of the phrases "in one embodiment" or "in an embodiment" in various places throughout this specification are not necessarily all referring to the same embodiment, but may. Furthermore, the particular features, structures or characteristics may be combined in any suitable manner, as would be apparent to one of ordinary skill in the art from this disclosure, in one or more embodiments. Similarly it should be appreciated that in the description of exemplary embodiments of the invention, various features of the invention are sometimes grouped together in a single embodiment, figure, or description thereof for the purpose of streamlining the disclosure and aiding the understanding of one or more of the various inventive aspects. This method of disclosure, however, is not to be interpreted as reflecting an intention that the claimed invention requires more features than are expressly recited in each claim . Rather, as the following claims reflect, inventive aspects lie in less than all features of a single foregoing disclosed embodiment. Thus, the claims following the detailed description are hereby expressly incorporated into this detailed description, with each claim standing on its own as a separate embodiment of this invention.

Furthermore, while some embodiments described herein include some but not other features included in other embodiments, combinations of features of different embodiments are meant to be within the scope of the invention, and form different embodiments, as would be understood by those in the art. For example, in the following claims, any of the claimed embodiments can be used in any combination. In the description provided herein, numerous specific details are set forth. However, it is understood that embodiments of the invention may be practiced without these specific details. In other instances, well-known methods, structures and techniques have not been shown in detail in order not to obscure an understanding of this description.

Other embodiments of the invention will be apparent to those skilled in the art from consideration of the specification and practice of the invention disclosed herein.

It is intended that the specification and examples be considered as exemplary only. Each and every claim is incorporated into the specification as an embodiment of the present invention. Thus, the claims are part of the description and are a further description and are in addition to the preferred embodiments of the present invention.

Each of the claims set out a particular embodiment of the invention.

The following terms are provided solely to aid in the understanding of the invention. IBS ("irritable bowel syndrome") refers to abdominal discomfort and/or pain associated with altered defecation pattern in the absence of a known organic cause. Although the predominant defecation pattern may vary from diarrhoea to constipation, abdominal pain or discomfort is present in all patients and represents the most disturbing and therapy-resistant symptom in IBS. It is linked to abnormal perception of visceral stimuli such as intestinal distension or nutrient infusion, also referred to as visceral hypersensitivity [Ohman L, & Simren M. (2010) Nature rev. Gastroent. hepatol. 7, 163-73; Kuiken SD, et al. (2005) Aliment. Pharmacol, ther. 22, 157-64; Cam illeri M et al. (2008) Clin. Gastroent. Hepatol. 6, 772-81]. Clinical management of IBS largely consists of symptomatic treatment aimed at restoring the dominant defecation pattern, but often leaves visceral pain unaffected. Drugs targeting visceral hypersensitivity may thus represent an important new pharmacological approach to treat IBS.

"Visceral sensitivity disorder" or "Visceral hypersensitivity" refers to hypersensitive visceral pain perception, which is commonly experience by individuals with functional gastrointestinal disorders. The pain results from the activation of nociceptors of abdominal organs.

"Non-ulcer dyspepsia" or "functional dyspepsia" relates to indigestions other than those caused by ulcer. Mast cells and histamine are also involved in this disease such that H1 receptor antagonism could be a method to treat dyspepsia.

"H1 -receptor antagonists", also known as "antihistamines" are compounds that block the action of histamine at the H1 receptor, helping relieve allergic reactions. A specific group of H1 -receptor antagonists are second-generation and third generation compounds antihistamines that are much more selective for peripheral H1 receptors. These compounds have the advantage that they do not cross the blood- brain barrier. Examples hereof are Astemizole, Cetirizine, Loratadine, Rupatadine, Mizolastine, Acrivastine, Ebastine, Bilastine, Bepotastine, Terfenadine and Quifenadine.

H1-receptor activation is also involved in abdominal pain in disease such as Crohn's disease or diverticulosis. The methods of the present invention are thus equally applicable to those disorders.

"Body sample" refers to a sample which is collected via non invasive methods and encompasses e.g. urine, stool, sweat, tears. The easiest accessible body samples are urine and stool.

"Tissue sample" can refer to any part of the body, but typically refers to (intestinal) biopsies, peripheral blood cells or a cell fraction thereof such as blood cells (erythrocytes), white blood cells (leucocytes) and platelets. Tissue sample also relates to a biopsy such as a biopsy of the duodenum for functional dyspepsia, the small intestine and colon for IBD, and IBS, and colon for diverticulosis.

Based on these data, we hypothesized that visceral hypersensitivity in IBS results from TRPV1 sensitisation induced by mast cell mediators, in particular by histamine. To this end, the response to TRPV1 activation of submucosal neurons was studied in rectal biopsies of IBS patients and healthy volunteers. Moreover, the ability of IBS and HV supernatant of biopsies to activate murine extrinsic afferent nerves and to sensitize TRPV1 on dorsal root ganglia was compared. Finally, based on these results, a proof-of-concept clinical trial was designed evaluating the effect of 12 week treatment with the H1 -R antagonist ebastine.

Imidazole acetaldehyde (4-imidazolylacetaldehyde) can be measured by a spectrophotometric assay. Imidazole acetaldehyde reacts with 2,4- dinitrophenylhydrazine (light orange) to form a hydrazone derivative (shades of red) which are distinctively colored solids and can be isolated easily, allowing spectrophotometric analysis, the so called DNP method.

Exam pies

Exam pie 1 : Methods

Processing of rectal biopsies

All patients gave informed consent and the Leuven University Hospital. For live recording of human submucosal neurons, fresh rectal biopsies were collected from 16 HVs and 9 IBS patients. Supernatants were generated by overnight incubation of biopsies as previously described [ Valdez-Morales EE, et al. (2013) American J Gastroent 108, 1634-43].

Randomised proof-of-concept clinical trial

The study protocol was approved by the Ethics Committee of the University Hospitals Leuven and all patients gave informed consent.

Patients who fulfilled the Rome III criteria for IBS [Longstreth GF, et al. (2006) Gastroenterology 130, 1480-91] were included in a placebo controlled double-blind randomised proof-of-concept trial consisting of a 2 weeks run-in, 12 weeks of treatment and 2 weeks of follow-up (Fig. 5A). After an initial rectal barostat study, hypersensitive and normosensitive patients were treated with a daily dose of 20 mg ebastine or placebo. After 12 weeks, a second barostat was performed to evaluate the effect of ebastine on rectal distension-evoked urge to defecate. Clinical IBS symptoms were assessed before and at the end of the 12 weeks treatment period using the validated Gastrointestinal Symptom Rating Scale (GSRS). Abdominal pain, symptom relief and health-related quality of life were assessed on a weekly basis. Rectal biopsies were collected after the barostat before and at the end of the treatment period. The primary endpoint of the study was the effect of ebastine on the symptom score evoked by rectal distension. To date, no data are available using this distension paradigm to determine the magnitude of improvement in symptom score that is clinically relevant. Based on our previous ketotifen study suggesting a beneficial effect of H1-R antagonism in IBS [Klooker et al (2010) cited above], we selected a comparable sample size of 55 patients.

The secondary endpoints of the study were: the effect of treatment on quality of life (QOL), individual clinical symptoms and the percentage of responders according to the FDA criteria (http://www.fda.gov/downloads/ Drugs/Guidances/ UCM205269.pdf). A responder for global relief was defined as a patient having at least considerable relief during 6 or more of the 12 study weeks. A responder for relief of abdominal pain was defined as a patient having at least 30% reduction of abdominal pain scored on a 100 mm VAS scale during 6 or more of the 12 weeks.

Statistical analyses

All statistical analyses of the clinical data were performed using SAS software version 9.2. Continuous data were summarised by their mean and standard deviation. When serious deviations from normality were observed, medians and interquartile values were presented. Comparisons between groups were made using a t-test or Wilcoxon rank-sum test, as appropriate. For categorical variables, observed frequencies and percentages were presented. Comparisons between groups were made using a Chi- square test or a Fisher's exact test, as appropriate.

All randomised patients were included in the analyses using available data. No effort was made to impute missing data. Since, whenever possible, longitudinal models were used to estimate the effect of treatment with ebastine, the estimates were unbiased in the presence of missing data if it was assumed that data were missing at random.

The primary endpoint and all continuous variables for which a baseline measurement was done were analysed using an analysis of covariance (ANCOVA) that includes the baseline value as a covariate. The VAS pain scores were analysed using a mixed linear model that included a variance-covariance matrix to account for correlations between the weeks. Exploratory analyses revealed that it was appropriate to include time as a continuous linear variable in the model. Baseline values were included as covariates, in addition to a factor for randomised treatment and its interaction with time. There was some evidence to suggest that the interaction was significant (P=0.06), so it was kept in the model. The effect of treatment was assessed globally and at each week. SGA relief response rates were analysed using a logistic generalized estimating equation model. It was appropriate to include time as a linear variable and the interaction between time and treatment was kept in the model, despite being non-significant (P= 0.6297). The effect of treatment was assessed globally and at each time point.

All statistical tests were 2-sided and performed at a significance level of 5%.

Statistical analyses of the animal experiments are described in Supplementary Material.

Detail on methods

PART I. Study on TRPV1 sensitisation in IBS: role of H1-R activation

Processing of rectal biopsies:

Biopsies were collected after the barostat procedure before and after the 12 weeks treatment period. Two biopsies were processed for immuno-histochemical staining, and 2 biopsies were stored in RNA later (Qiagen Benelux, Venlo, the Netherlands) for real time PCR.

For live recording of human submucosal neurons, fresh rectal biopsies were collected from 16 healthy volunteers (9 M, median age of 30.5 yrs IQR [24-48]) and 9 IBS patients (2 M, median age of 35 yrs IQR [25-58] ; 7 IBS-D, 1 IBS-C and 1 IBS-U). Healthy volunteers were recruited by public advertisement and none of them had symptoms or a history of gastrointestinal disease, previous gastrointestinal surgery or were taking any gastrointestinal medication. IBS patients were recruited from the outpatient clinic of the University Hospitals Leuven and were different from those included in the clinical trial.

I mmuno histochemistry:

For immunohistochemical analysis, biopsies were fixed overnight in 4% formaldehyde and embedded in paraffin. c-Kit (1 :700, Dako, Heverlee, Belgium) positive mast cells were identified in six-micrometer paraffin sections after antigen retrieval in ethylene- diamine tetra-acetricacid (EDTA, pH 8.0; 1mM (0.37g/l) and Mach3 detection (Biocare Medical, Klinipath BVBA, Olen, Belgium) as described by Garrity MM et al Histopathology 2009;54:286-294.

Submucosal preparations from the calcium imaging experiments were fixed in 4 % paraformaldehyde and stained with mouse anti-panneuronal HuC/D (PGP9.5, 1 :500; Molecular Probes, Life Technologies Europe BV, Gent, Belgium), goat anti-H1-R (VR1 antibody P-19, 1:100; Santa Cruz Biotechnology, USA) and chicken antineurofilament 200 kD (NF200, 1:500; Abeam, Cambridge, UK) or rabbit anti- TRPV1 (1:1000, Santa Cruz Biotechnology, USA) overnight at 4 °C. After washing, tissues were incubated with Alexa fluor 488 conjugated donkey ant i- mouse (1 : 1000, Life Technologies, Gent, Belgium), donkey anti-rabbit Cy5 (1:400, Jackson ImmunoResearch, West Grove, PA, USA), donkey anti-goat Cy3 (1:500, Jackson ImmunoResearch) and donkey anti-chicken AMCA (1:250, Life Technologies). Confocal images were taken with a Zeiss LSM510 confocal microscope in the Cell Imaging Core (CIC), University of Leuven.

Real-time PCR

Total RNA was extracted by RNeasy Minikit (Qiagen) and reverse transcribed by qScript cDNA Supermix (Quanta Science, Gaithersburg, MD, USA) according to the manufacturer's instructions. Real-time PCR was performed on a LightCycler® 480 Real-Time PCR System (Roche Applied Science, Belgium) using the Lightcycler480 probes master mix as indicated by the manufacturer.

Ca2+ imaging of human submucosal neurons

After collection, the biopsies were processed to obtain the submucosal plexus, which was loaded with ΙμΜ Fluo-4 AM (Molecular Probes, Invitrogen, Merelbeke, Belgium) for Ca2+ imaging as described by Cirillo C, et al Gut 2013;62:227-235. Images were acquired at 2 Hz and collected by TILLVision software (TILL Photonics, Oberhausen, Germany). Data were analysed by custom-written macros in IGOR PRO (Wavemetrics, Lake Oswego, Oregon, USA). Regions of interest were drawn over each neuronal cell, fluorescence intensity was normalized to the basal fluorescence at the onset of the recording for each region of interest, and peaks were analysed. High background autofluorescence, which was bleaching during the recordings, was corrected for using a Runge-Kutta iterative deconvolution algorithm assuming monoexponential fluorescence decay.

The response of submucosal neurons to capsaicin (0.1 , 1 and 10 nM) (perfusion of 1 ml/min for 5 seconds) (Sigma-Aldrich, Diegem, Belgium) was compared between healthy volunteers (n= 12, 9 M, median age of 45 yrs IQR [28-54]) and IBS patients (n=9, 2 M, median age of 35 yrs IQR [25-58]). In addition, the effect of preincubation with 10 μΜ histamine (10 min) on the capsaicin response was evaluated in 9 of the healthy volunteers (7 M, median age of 44 yrs IQR [25.5-51.5]). Finally we also evaluated the TRPV1 response after pre-incubation with 10 μΜ histamine combined with 1 μΜ pyrilamine in 5 healthy volunteers (2 M, median age of 25 yrs IQR [22-36].

Ca2+ imaging of murine dorsal root ganglia (DRG)

All animal experiments were carried out in accordance to the European Community Council guidelines and were approved by the local ethics committee of Leuven University. For animal experiments performed in Leuven, the lumbosacral (L5-S2) DRGs of 3 to 4 adult C57BI6 mice (Janvier Labs, France, 10-12 weeks) or H1-R KO (Oriental Bioservice, INC, Kyoto, Japan) were bilaterally excised under a dissection microscope. The ganglia were washed in 10% fetal calf serum Neurobasal A medium (basal medium) and then incubated (95% air, 5% C02) at 37°C in a mix of collagenase of 1 mg/ml (Gibco, Gent, Belgium) and dispase of 2.5 mg/ml (Gibco, Gent, Belgium) for 45 minutes. Digested ganglia were gently washed twice with basal medium and mechanically dissociated in B27-supplemented (2%) Neurobasal A medium (Invitrogen, Gent, Belgium) containing GDNF of 2 ng/ml (Invitrogen, Gent, Belgium, NT4 of 10 ng/ml (Peprotech, London, UK), 100 pg/ml penicillin/streptomycin

(Invitrogen, Gent, Belgium) and Glutamax (Invitrogen, Gent, Belgium) (complete medium). Neurons were seeded on poly-L-ornithine/laminin-coated glass coverslips and cultured for 12-18 h at 37°C. Cultured DRG neurons were subsequently loaded with 2 μΜ Fura-2AM for 20 min at 37°C. Dose-response curves to 10, 30 and 100 nM capsaicin were constructed in the presence or absence of 5 μΜ histamine. Neurons responding to 10 nM capsaicin were more sensitized by histamine than neurons responding to 30 and 100 nM capsaicin. In a second set of experiments, DRG neurons were exposed to 10 nM capsaicin before and after histamine (5 μΜ) application, and in the presence of 1 μΜ pyrilamine. TRPV1 positive neurons were identified by application of 1 μΜ capsaicin at the end of the experiment.

Preparation of imidazole acetaldehyde

Imidazole acetaldehyde can be synthesized in various ways and was prepared enzymatically by incubation of 10 μΜ histamine with 22.5 mU diamine oxidase (DAO, Sigma Adrich) for 1 h r at 37°C followed by 5 mins heating to 95°C. In addition, imidazole acetaldehyde was chemically prepared from L-histidine by oxidation with hypochlorite (modified from Kapeller-Adler R, Fletcher M. The enzymic destruction of histamine in vitro. Biochim Biophys Acta 1959; 33: 1 - 9.) . Purity was monitored by TLC (Sil G Alugram; butanol/acetic acid/water 6/2/2-v/v) and different stainings (iodine (general detection), ninhydrin (absence of amino group), 4-amino-3-hydrazino-5- mercapro-1 ,2,4-triazole (Purpald; presence of aldehydic group) 8. Derivatisation with cyclohexadione-1 ,3- dione9, followed by RP-HPLC (Symmetry C18 column ; 4.6 χ 150 mm; 5 pm; 100 A; Waters; 20 to 80% methanol containing 0.1% TFA; fluorescence em/ex 390/460), showed one single peak (when corrected for background). To protect the aldehyde from degradation/rearrangement (at and above neutral pH) or polymerization (concentrated or dry state), it was stored as a 0.2 M aqueous solution at pH 3 at 4°C. Under these conditions it appeared stable for 3 months

Analysis of imidazolacetaldehyde content in biopsy supernatants

To 15μΙ_ of biopsy supernatant, 45μΙ_ of methanol supplemented with 0.1% formic acid was added Subsequently, samples were thoroughly vortexed followed by centrifugation at 14000 rpm (4°C, 5 min). After centrifugation, 50μΙ_ of supernatant was transferred to a HPLC vial.

lmidazole-4-acetaldehyde was analyzed using a Waters Acquity UPLC coupled to a Xevo TQ-S micro mass analyzer equipped with an electrospray interface (ESI) (Waters, Milford, USA). For separation, an Acquity UPLC BEH Amide column (1.7 pm, 2.1 x 50 mm) was used (Waters, Milford, USA) and kept at 35°C. After injection (O.lpL), separation was carried out under isocratic conditions using a mobile phase consisting of 45:55 acetonitril/H20 + 0.05% formic acid (v/v) at a flow rate of 0.6 mL/min. The total run time amounted to 2 min and the retention time of imidazoleacetaldehyde was 0.44 min.

Mass spectrometry analysis was performed in the positive electrospray ionization mode using the following parameters: capillary voltage: 0.50 kV, source temperature 150 ° C, desolvation temperature 500 ° C, desolvation gas flow: 600 L/hr, cone gas flow 10 L/hr. The mass analyzer was operated in MRM mode with the parent mass of 111.190 and the daughter ion, generated by a collision energy of 15 and a cone voltage of 35 V, of 81.960.

The described method was linear over the concentration range of 25000 to 12 nM. Analysis of histamine content in biopsy supernatants

To 50 uL of medium 450 uL of methanol was added, the solution was stored for 3 hours at -80° C. Next, samples were centrifuged for 10 min at 20x10 ³ x g (cooled at 4°C). The supernatant was transferred to an eppendorf and dried using vacuum centrifugation. The pellet was re-dissolved in 100 uL 80% acetonitrile (80:20 acetonitrile:H20).35 uL of the mix was loaded onto an Ultimate 3000 UPLC (Thermo Scientific, Bremen, Germany) equipped with a ZIC-pHILIC column (2.1 x 150 mm, 5 urn particle size, cat# 1.50460.0001, Merck, Darmstadt, Germany) in line connected to a Q Exactive mass spectrometer (Thermo Fisher Scientific).

A linear gradient was carried out using solvent A (acetonitrile, LC-MS Chromasolv, Sigma-Aldrich) and solvent B (10 mM ammoniumcarbonate, pH 9.2). Practically, samples were loaded at 80% solvent A and from 5 to 30 min a ramp to 80% solvent B was carried out. From 30 min to 31 min the gradient returned to 80% A and this was maintained until 47 min. The flow rate was kept constant at 100 uL/min and the column temperature was set at 25°C throughout the run. The mass spectrometer operated in targeted MS2 mode following the ion with m/z 112.08692 (histamine) and 111.05529 (imidazole-4-acetaldehyde). Normalized collision energy was set to 20. The mass spectrometer ran in positive polarity, the source voltage was 4.0 kV, and the capillary temperature was set at 300°C.

Additional sheat gas flow was put at 60 and auxilary gas flow rate at 20. Auxilary gas heater temperature was put at 270°C. AGC target was set at 5e4 ions with a maximum ion injection time of 200 ms acquired at a resolution of 17.500 .

For the data analyses we integrated the peak areas using the Thermo XCalibur Quan Browser software (Thermo Scientific).

Study subjects

Patients who fulfilled the Rome III criteria for IBS (Longstreth GF, et al Gastroenterology 2006;130:1480-1491) were recruited at the outpatient clinic of the University Hospitals Leuven from November 2009 until April 2012. In addition to careful history taking, all patients underwent a physical examination, biochemistry (including thyroid stimulating hormone levels and antibodies to antitissue transglutaminase), stool analysis and sigmoidoscopy or colonoscopy to exclude organic disease. Patients who had relevant concomitant disease and active treatment for psychiatric disorders were excluded. Medication likely to interfere with gastrointestinal tract function or visceral perception was discontinued before the study. The study protocol was approved by the Ethics Committee of the University Hospitals Leuven and all patients gave informed consent.

Study design

Patients were included in a placebo controlled double-blind randomised proof-of concept trial consisting of a 2 weeks run-in, 12 weeks of treatment and 2 weeks of follow-up (Fig. 5A). After an initial rectal barostat study (details below), hypersensitive and normosensitive patients were treated with a daily dose of 20 mg ebastine or placebo. After 12 weeks of treatment, a second rectal barostat study was performed to evaluate the effect of ebastine on rectal distension-evoked urge to defecate. In addition, clinical IBS symptoms were assessed before and at the end of the 12 weeks treatment period using the validated Gastrointestinal Symptom Rating Scale (GSRS): the intensity of gastrointestinal symptoms is scored on a 7-graded Likert scale with descriptive anchors (0=no and 6= very severe symptoms). Furthermore, abdominal pain (assessed by the Subjects' Global Assessment (SGA) of Abdominal Pain and Discomfort), symptom relief (monitored by the SGA of relief) and healthrelated quality of life (measured by the IBS-QOL) were assessed on a weekly basis. Finally, rectal biopsies were collected after the barostat study before and at the end of the treatment period (Fig. 5) for mRNA extraction and immunohistochemistry. Barostat studies

To assess visceral sensitivity, symptoms evoked by rectal distension were evaluated. To this end, an electronic barostat, automatically correcting for the compressibility of air (Synetics Visceral Stimulator, Stockholm, Sweden) was connected to a polyethylene barostat bag (500 ml_, with a maximal length of 13 cm). The bag was tightly wrapped on the distal end of a double lumen polyvinyl tube to connect it to the barostat device (Salem Sump tube 14 Ch.; Sherwood Medical, Petit rechain, Belgium). Before the study, subjects received a tap water enema, followed by a 20- min period of rest. The barostat bag was lubricated and positioned into the rectum. Subjects were studied in the left lateral decubitus position. After a 10-min adaptation period, minimal distending pressure (MDP) was determined. We defined MDP as the minimum pressure at which the corresponding intrabag volume was at least 30 ml_. The distension protocol consisted of a series of 3 phasic, ascending isobaric distensions, of 3, 9 and 21 mmHg increment above MDP respectively. These distension pressures correspond with the thresholds for first sensation, first urge and first discomfort in IBS patients, as reported by us earlier (Klooker TK, et al Gut 2010;59:1213-1221 and Kuiken SD, et al Aliment Pharmacol Ther 2005;22:157- 164). The inflation rate was 38 mL/s and each distension step lasted 2 min, separated by 1-min intervals at baseline (MDP). Sensations were scored at the end of each distension step. A 100mm Visual Analogue Scale (VAS) with verbal descriptors (0 = no sensation; 20 = first sensation; 40 = first sense of urge; 60 = normal urge to defecate; 80 = severe urge to defecate and 100 = discomfort/pain) was used to score evoked sensations. The barostat bag was instantaneously deflated if a subject reported discomfort or

pain

Randomisation and masking

Hypersensitive and normosensitive IBS patients were separately randomised in a 1 : 1 ratio to placebo or ebastine, according to a computer-generated block randomisation list without stratification. The allocation sequence was generated by an independent pharmacist at the University Hospitals Leuven (www.randomization.com) and not available to any member of the research team until the study had been completed.

Placebo and ebastine packaging was identical and executed by the University Hospitals Leuven pharmacy. Patients, study staff, specimen and data analyses were masked to assignment.

Study endpoints and sample size The primary endpoint of the study was the effect of ebastine on the symptom score evoked by rectal distension (individual steps and summated score = sum of the 3 distension steps). To date, no data are available using this distension paradigm to determine the magnitude of improvement in symptom score that is clinically relevant. Hence, based on our previous ketotifen study suggesting a beneficial effect of H1-R antagonism in IBS (Klooker TK, et al Gut 2010;59:1213-1221), we selected a comparable sample size of 55 patients.

The secondary endpoints of the study were: the effect of treatment on quality of life (QOL), individual clinical symptoms and the percentage of responders according to the recent FDA criteria (http://www.fda.gov/ downloads/ Drugs/ Guidances/ UCM205269.pdf). A responder for global relief was defined as a patient having at least considerable relief during 6 or more of the 12 study weeks. A responder for relief of abdominal pain was defined as a patient having at least 30% reduction of abdominal pain scored on a 100 mm VAS scale during 6 or more of the 12 weeks. PBMC I m m une-phenotyping

Whole blood was collected following a protocol approved by the Ethics Committee of the University Hospital Leuven. Heparinized blood was rested for 2 hours at 18°C before extraction using ficoll separation protocol (LSM; MP Biomedicals, Belgium). PBMCs were frozen immediately in 10% dimethylsulfoxide (DMSO, Sigma-Aldrich, Diegem, Belgium) 90% fetal bovine serum (FBS) at -80°C for a maximum of 2 months.

Thawed cells were stained using antibodies (purchased from eBiosciences, Vienna, Austria, unless mentioned elsewise) against CD3 (SK7) , CD4 (RPA-T4), CD8a (RPA- T8), CD14 (61D3), CD19 (HIB19), CD25 (BC96), CD56 (MEM188) , CD11c (3,9), CD123 (6H6), CD45Ra (H1100) , CD31 (WM-59), CCR7 (3D12), HLADR (LN3), Va24Jal8 (6B11), CXCR5 (IgG23, R&D) to identify CD4 T cell subsets (naive, RTE, TEM, TCM, Th1, Th2, Th17, Tfh, I L-2-producing, Tregs), CD8+ T cell subsets (naive, RTE, IFNY-producing, IL-2 producing), B cell subsets (Naive, switched, memory- B cells), γδ T cells, DC, NK and NKT. Intracellular staining was performed for Foxp3 (206D, Biolegend, ImTec Diagnostics N.V., Antwerp, Belgium) using the eBioscience fix/perm buffers. Intracellular cytokine expression was measured following 5 hour stimulation at 37°C with phorbol myristate acetate (PMA, 50 pg/ml, Sigma-Aldrich) and lonomycin (500 ng/ml, Sigma-Aldrich) in RPMI1620 in the presence of Golgistop (1 :250, from BD kit). Following stimulation, cells were treated with BD fixative and permeabilisation buffers and stained for IFNY (4S.B3), IL-4 (8D4-8), IL-17 (64DEC17) and IL-2 (MQ1-17H12). Acquisition was performed on a BD FACSCantoll (BD Bioscience) with analysis by Flowjo (version 7.6.5, Ashland Or, USA).

Cytokine analysis in supernatants of stimulated PBMCs Freshly isolated PBMCs were stimulated for 72 hour at 37°C and 5% C02 with CD3/CD28 (Sigma-Aldrich). PBMC supernatant was collected and stored at -80°C until analysis. Cytokine levels of IL- 13, IL-8, I L- 1 b , IL-10 and TNFa were quantified by cytometric bead array (BD Bioscience, Erembodegem, Belgium). Samples were acquired on FACSCanto flow cytometer (BD Bioscience) and analysed by FCAP v3.0 analysis software (Soft Flow Inc., Pecs, Hungary).

Statistical analyses

All statistical analyses of the clinical data were performed using SAS software version 9.2. Continuous data were summarised by their mean and standard deviation. When serious deviations from normality were observed, medians and interquartile values were presented. Comparisons between groups were made using a t-test or Wilcoxon rank-sum test, as appropriate. For categorical variables, observed frequencies and percentages were presented. Comparisons between groups were made using a Chi- square test or a Fisher's exact test, as appropriate.

All randomised patients were included in the analyses using available data. No effort was made to impute missing data. Since, whenever possible, longitudinal models were used to estimate the effect of treatment with ebastine, the estimates were unbiased in the presence of missing data if it was assumed that data were missing at random.

The primary endpoint and all continuous variables for which a baseline measurement was done were analysed using an analysis of covariance (ANCOVA) that includes the baseline value as a covariate. The VAS pain scores were analysed using a mixed linear model that included a variance-covariance matrix to account for correlations between the weeks. Exploratory analyses revealed that it was appropriate to include time as a continuous linear variable in the model. Baseline values were included as covariates, in addition to a factor for randomised treatment and its interaction with time. There was some evidence to suggest that the interaction was significant (P=0.06), so it was kept in the model. The effect of treatment was assessed globally and at each week. SGA relief response rates were analysed using a logistic generalized estimating equation model. It was appropriate to include time as a linear variable and the interaction between time and treatment was kept in the model, despite being non-significant (P= 0.6297). The effect of treatment was assessed globally and at each time point.

All statistical tests were 2-sided and performed at a significance level of 5%. Statistical analyses of the Ca2+ imaging experiments were performed with Graphpad Prism. Statistical analyses of the peak F340/380 ration for the Ca2+ imaging experiments were performed after correction for the individual baseline Ca2+ . All values are expressed as means± SEM. from n mice or n cells treated with each of the individual supernatants. Statistical comparisons between 2 groups were performed by a Wilcoxon signed rank test or Mann-Whitney U test as appropriate or ANOVA when comparing more than 2 groups. Categorical data were analysed by the Fischer's exact test.

Results

Example 1. Sensitization of TRPV1 in neurons of I BS patients

To evaluate TRPV1 sensitization in IBS, the intracellular Ca ²⁺ response of submucosal neurons to capsaicin was studied in rectal biopsies of IBS patients (n=9) and healthy volunteers (HV, n= 12) [Cirillo C, et al. (2013) Gut 62, 227-35]. Exposure to capsaicin (0.1 and 1 nM) induced significantly higher Ca ²⁺ responses (Fig. 1a-b) and activated more neurons in biopsies of IBS patients compared to HV (Fig. 1b). TRPV1 mRNA levels were however sim ilar in I BS and HV ¹² , indicating sensitization of TRPV1 on I BS submucosal neurons, possibly by mediators released in the extracellular milieu. To directly test the role of TRPV1 in the context of nociceptive signalling, we recorded responses from single afferent nerves innervating the mouse colon and tested their responses to mucosally applied human supernatants obtained from mucosal colon biopsies. Because long-term incubation is not technically feasible with this model, the effect of prolonged incubation with supernatant on TRPV1 activation was then studied using isolated murine dorsal root ganglion (DRG) neurons. Taken together, our data indicate that TRPV1 is sensitized in IBS by mediators in the submucosa that directly activate nociceptive nerve fibres and sensitize TRPV1.

Example 2. TRPV1 sensitization is mediated by histamine interacting with H1-R

Recently, we observed significant clinical improvement including reduced pain perception in IBS patients receiving ketotifen [Kook et al (2010) cited above]. This benefit appeared to result from ketotifen's H1 -R antagonist properties rather than its action as a mast cell stabilizer. Hence, we postulated that histamine could be involved in the TRPV1 sensitization observed in our IBS patients, in particular via interaction with H1-R. Moreover, pre-incubation of DRGs with histamine increased the Ca ²⁺ response to capsaicin, an effect that was absent in mice deficient in H1-R (H1-R7-, Fig. 4a) and blocked by the H1-R antagonist pyrilamine (Fig. 3), but not by H2-R (ranitidine, 10 μΜ), H3-R (clobenpropit, 30 nM) and H4-R (JNJ7777120, 1 μΜ) antagonists (Fig. 4a). RT-qPCR experiments revealed that murine DRG sensory neurons express H1-R, H2-R and H4-R but not H3-R (data not shown). Taken together, these data indicate TRPV1 sensitization via H1-R activation. Next, we evaluated if this mechanism could be replicated in human submucosal neurons. Biopsies of HV pre-treated with histamine showed increased amplitude of the Ca ²⁺ response to capsaicin and an increased number of responding neurons, an effect that was prevented in the presence of the H1 -R antagonist pyrilamine (Fig.2c- d). I m munohistochem ical staining revealed co-expression of H1-Rwith TRPV1 on the submucosal neurons in IBS and HV biopsies (Fig. 2e). No difference in mRNA expression of H1-R, H2-R, TRPV1 or the panneuronal marker PGP9.5 was found between IBS and HV in rectal biopsies further supporting TRPV1 sensitisation rather than upregulation of TRPV1 in IBS [Van Wanrooij et al (2014) cited above]. H4-R mRNA expression levels were below detection limit.

Histamine levels in the supernatants were not different between IBS and HV and were extremely low compared to previous studies [Barbara et al (2004) cited above]. Hence, we speculated that histamine must have been degraded, and hypothesized that the sensitizing effect observed by the supernatant might result from a (secondary) histamine metabolite.

Example 3. The histamine metabolite imidazole acetaldehyde mimics the TRPV1 sensitizing effect of histamine

The major route of inactivation of extracellular histamine in mammals is via oxidative deamination of the primary amino group, catalyzed by diamine oxidase (DAO). DAO is mainly located in intestinal epithelium, where it degrades endogenous (mast cell) and exogenous histamine into imidazole acetaldehyde, ammonia and hydrogen peroxide. Imidazole acetaldehyde is then converted to the acetic acid derivative by aldehyde dehydrogenase before its subsequent ribosylation for transport and excretion. As imidazole acetaldehyde contains the aromatic ring that confers high H1-R affinity [Wieland K, et al. (1999) J Biol Chem 274, 29994-30000; ter Laak AM, et al. (1995) J Comput Aided Mol Des 9, 319-30; Bruysters M, et al. (2004) Eur J Pharmacol 487, 55-63], we assessed the capacity of the histamine metabolite imidazole acetaldehyde (5 μΜ) to sensitize TRPV1. Chemically and enzymatically synthesized imidazole acetaldehyde (see Methods section) sensitized TRPV1 on DRG neurons to the same extent as histamine, an effect that was blocked by the H1-R antagonist pyrilamine and prevented in DRG neurons lacking H1-R (Fig. 4b). No differences in imidazole acetaldehyde levels were however found between the supernatants of IBS vs HV biopsies (IBS (n=10): 2.2±1.5; HV (n=10): 2.5±1.4 μ9/ιη l/mg tissue), suggesting that another metabolite or reaction product of histamine interacts with H1-R sensitizing TRPV1. Identification of this compound is of great clinical relevance as it may represent a biomarker of IBS. The complexity and instability of the degradation products of histamine will however seriously hamper this process. Nevertheless, our data indicate that not only histamine but also its metabolite imidazole acetaldehyde can sensitize TRPV1 in a H1-R dependent manner, thereby further contributing to the development of visceral hypersensitivity in IBS.

Example 4. Treatment of I BS with the H1-R antagonist ebastine reduces visceral hypersensitivity, increases global relief and reduces pain scores

Given our strong evidence that histamine and its metabolite sensitize TRPV1 on human neurons in IBS patients and could play an important role in nociception, we designed a proof-of-concept clinical trial evaluating the effect of the selective H1-R antagonist ebastine (Fig. 5a). Fifty-five patients (34 female, median age 31 yrs IQR [25-46]) were enrolled and randomized to receive placebo (n = 27) or ebastine (n = 28; 20 mg day ¹ ) and underwent barostat studies to determine their sensory thresholds. Of these, 25 patients (45%) were unable to tolerate the 21 mmHg distension pressure step and were classified as visceral hypersensitive ²⁰ . Fifty-one patients completed the 12 week treatment period and 44 patients agreed to have a second barostat study at the end of treatment (Fig. 5b). No significant side effects were reported during treatment with ebastine or placebo.

Ebastine treatment, but not placebo, resulted in a steady increase in the proportion of responders (= patients with at least considerable relief) during the 12 weeks treatment period leading to a significantly higher proportion of responders at the end of treatment compared to placebo (week 12: 46% (11/24) vs 13% (3/23), P=0.024, Fisher's exact test). Notably, the beneficial effect of ebastine was lost in the washout period (Fig. 6a). In addition, there was a gradual and significant decrease in abdominal pain scores during the treatment period in the ebastine, but not in the placebo group (ebastine: from 56±19 to 39±24 mm at 12 weeks vs placebo: from 61 ± 15 to 62±22 mm, P=0.002, repeated measures ANOVA) (Fig. 6b). At week 12, abdominal pain scores were significantly decreased by ebastine compared to placebo (mean change:-17±25 vs +3±23, P=0.0004). Using the latest FDA definition (see Methods), ebastine treatment resulted in increased percentage of responders with symptom relief (= 6 weeks or more of the 12 weeks with at least considerable relief) (25 % (6/24) vs 4 % (1/23), P= 0.0971, Fisher's exact test), and abdominal pain relief (41% (9/22) vs 20% (4/20), P=0.19, Fisher's exact test) when compared to placebo. Of note, also flatulence and bloating were significantly reduced by ebastine. Based on the quality of life (QoL) questionnaires, ebastine resulted in a significant improvement in sleep, diet, social and emotional roles (Fig. 6c).

To obtain insight into the mechanism of action of ebastine, we evaluated its effect on visceral sensitivity to rectal distension after 12 weeks of treatment compared to baseline. Ebastine treatment resulted in a significantly larger reduction of the summated urge score compared to placebo (placebo -1[-30 - +32] vs ebastine -18[- 65 - +27], P= 0.046 ANCOVA using baseline total score as covariate and including factor for treatment and type). This effect resulted mainly from a reduction in symptom scores evoked by the lower distension pressures of 3 and 9 mmHg, especially in the hypersensitive IBS patients (Fig. 7). The response to the highest distension was unaffected in the hypersensitive group, presumably because this distension pressure reflects a supramaximal stimulus.

Example 5. The H1-R antagonist ebastine does not exert immune modulatory effects in I BS patients

As some reports suggest that ebastine may exhibit anti-inflammatory properties [Rico S, et al. (2009) J Asthma Allergy 2 , 73-92] and microscopic inflammation may be involved in IBS [Ohman & Simren cited above] , we sought to evaluate the potential effects of ebastine on immune function. No statistically significant changes were found in the proportions of peripheral blood T and B lymphocyte subtypes, NK cells, NT cells, and γδ T cells in patients with IBS, at baseline and 12 weeks after ebastine or placebo treatment (Fig.8). Similarly, no differences could be detected in cytokine levels in supernatants of stimulated PBMCs of placebo versus ebastine treated patients. Moreover, no differences in mRNA expression could be detected for inflammatory markers, TRPV1 or H1-R and H2-R in rectal biopsies. Also the number of mast cells in rectal biopsies was not changed comparing pre- versus post- treatment (data not shown). Taken together, these data indicate that the beneficial effect of ebastine is not due to an anti-inflammatory effect, but rather can be explained by its H1-R antagonistic properties interfering, at least in part, with TRPV1 sensitization.

Example 6. Discussion

In the present study, we provide for the first time evidence in IBS patients for sensitization of TRPV1 in the rectal submucosal plexus, an effect mediated by histamine and/or its metabolite imidazole acetaldehyde. We obtained similar results in the context of pain signalling in murine nociceptive DRG neurons, where histamine, imidazole acetaldehyde and IBS biopsy supernatants sensitized TRPV1 via H1 -R. The significance of this mechanism in IBS was further confirmed in a proof-of-principle clinical trial evaluating the effect of 12 weeks treatment with the H1-R antagonist ebastine. Global symptom relief was significantly increased while abdominal pain scores were significantly decreased by ebastine compared to placebo. Taken together, our study indicates histamine (or metabolite)-induced sensitization of TRPV1 via H1-R as a main pathophysiological mechanism underlying visceral pain in IBS and demonstrates that selective pharmacological targeting of H1-R is a new treatment for this disorder.

Neuropathic pain models and in vitro studies have previously demonstrated TRPV1 sensitization by pro-inflammatory mediators including histamine, mediated by phosphorylation and increased transport of TRPV1 to the cell membrane [Cenac (2010) cited above; Numazaki M, et al. (2002) J Biol Chem 277, 13375-13378]. Here, we show sensitization of TRPV1 on human submucosal neurons of IBS patients by mucosal histamine or its metabolite imidazole acetaldehyde. Supernatant of mucosal biopsies collected from IBS patients, but not that of HV, not only activated murine nociceptive nerve fibers but most importantly sensitized TRPV1 on nociceptive DRG neurons. This effect was blocked by H1-R antagonism and reproduced by histamine. A new and intriguing finding is the fact that imidazole acetaldehyde, the metabolite of histamine, possesses a comparable sensitizing effect as histamine on TRPV1 via H1-R activation. This is most likely explained by the aromatic ring in both molecules that is responsible for their high H1-R affinity. This property of imidazole acetaldehyde is of great importance as it implies that even after degradation of histamine, TRPV1 continues to be sensitized by its metabolite. Of note, no increased levels of histamine or imidazole acetaldehyde were detected in the biopsy supernatant of IBS patients indicating that other metabolites or reaction products of histamine must be responsible for the activation of H1-Rand subsequent sensitization of TRPV1 on DRG neurons. For example, under physiological conditions, histamine can be converted into 4-methylspinaceamine, a molecule that contains an aromatic ring like histamine and thus may potentially possess H1 -R agonistic properties [Ohya T, & Niitsu M. (2003) Biol Pharm Bull 26, 1215-1218]. The search for this compound will be complex however in view of the chemical instability of histamine metabolites or reaction products.

To further prove the clinical relevance of H1-R mediated TRPV1 sensitization in IBS, we examined the effect of H1-R antagonism in a proof-of-concept clinical trial. Ebastine resulted in a gradually increased proportion of patients with at least considerable relief at week 12 and an increased percentage of responders compared to placebo. Moreover, abdominal pain scores at the end of treatment were reduced by ebastine, mirrored by an increased percentage of abdominal pain responders. In line with these clinical data, symptoms evoked by mild and moderate rectal distension were significantly reduced by ebastine. The effect of ebastine on distension-evoked rectal sensation was especially observed in the "sensory enriched" or the visceral hypersensitive patient group. Taken together, our study shows that treatment with ebastine affects perception of rectal distension and improves clinical symptoms. It should be emphasized however that our study was not powered for the clinical endpoints and thus the magnitude of the therapeutic effect of ebastine should be confirmed in larger clinical trials. Nevertheless, the therapeutic gain with respect to responders for degree of relief (21%) and abdominal pain (21%) reported here is comparable to that of linaclotide (19-22% and 15% respectively) [Quigley EM, et al. (2013) Aliment Pharmacol & ther 37, 49-61], a guanylate cyclase agonist recently registered in the US as treatment of constipation-predominant IBS [Chey WD, et al. (2012) Am. J. Gastroent. 107, 1702-1712. Rao S, et al. (2012) Am. J. Gastroent. 107, 1714-1725] , supporting our conclusion that H1 -R antagonism should indeed be further explored as a treatment for IBS.

Finally, selection of patients for a specific treatment is of particular importance in I BS as this patient population is very heterogeneous and includes patients with different underlying mechanisms. This likely explains why not all patients revealed TRPV1 sensitization. Other mechanisms, such as PAR2-mediated activation of TRPV4 by metabolites of arachidonic acid, may be involved in these patients [Cenac N, et al. (2015) Gastroenterology 49, 433-444]. Clearly, identification of the underlying mechanism involved will be of crucial importance to select the appropriate therapeutic strategy. Identification of the TRPV1 sensitizing compound present in the supernatant is therefore of great interest as it may represent a biomarker to identify patients responding to H1-R antagonism.

In summary, this is the first evidence in humans using IBS patient biopsies and supernatants showing that TRPV1 is sensitized in IBS patients. This effect is mediated via H1 -R activation by histamine and its metabolite imidazole acetaldehyde and may lead to increased visceral pain perception in at least a subgroup of patients. Moreover, it provides the first demonstration that peripheral H1-R antagonism can serve as a new treatment for I BS, resulting in a reduction of symptoms and improved quality of life.

Particular and preferred aspects of the invention are set out in the accompanying independent and dependent claims. Features from the dependent claims may be combined with features of the independent claims and with features of other dependent claims as appropriate and not merely as explicitly set out in the claims. Thus, the claims following the detailed description are hereby expressly incorporated into this detailed description, with each claim standing on its own as a separate embodiment of this invention.