Irritable bowel syndrome: towards biomarker identification

Review

Irritable bowel syndrome: towards biomarker identification Gerard Clarke1,2, Eamonn M.M. Quigley1,3, John F. Cryan1,4,5 and Timothy G. Dinan1,2 1

Alimentary Pharmabiotic Centre, University College Cork, Cork, Ireland Department of Psychiatry, University College Cork, Cork, Ireland 3 Department of Medicine, University College Cork, Cork, Ireland 4 Department of Pharmacology and Therapeutics, University College Cork, Cork, Ireland 5 School of Pharmacy, University College Cork, Cork, Ireland 2

Irritable bowel syndrome (IBS), the most common functional gastrointestinal disorder referred to gastroenterologists, affects 7–10% of the general population worldwide. The lack of suitable disease-defining biological markers coupled with a poorly understood underlying pathophysiology complicates patient diagnosis and seriously hampers drug discovery efforts. Over the past few years, a number of potential biomarkers have emerged, and in this review we critically evaluate such candidates. In particular, we highlight the increasing number of studies supporting a low-grade immune activation in IBS and consider how the latest preclinical developments can contribute to the development of more robust and reliable biological markers of this disorder. The successful identification of biomarkers is critical to progressing our understanding of IBS and addressing the unmet therapeutic needs of this debilitating condition. Introduction Although the prognosis for irritable bowel syndrome (IBS) is benign, it is a disorder that places a considerable burden both on the individual sufferer and society as a whole. Patients typically present with chronic abdominal pain and an altered bowel habit, symptoms that are accompanied by a plethora of additional features including bloating, distension and disturbances in defaecatory function. It is often a lifelong affliction with flares of activity followed by periods of remission, and whose incidence commonly peaks in the third and fourth decades of life. Recent estimates suggest a worldwide prevalence of 7–10%, although in industrialised countries it has been identified both as one of the leading causes of work absenteeism and presenteeism and a significant drain on healthcare resources [1,2]. Despite the high prevalence of this functional gastrointestinal disorder (FGID), it is still poorly understood and diagnosis is commonly dependant solely on symptombased criteria such as the Rome criteria (Table 1: Rome III criteria, Box 1) following the exclusion of organic diseases of the gastrointestinal tract (GIT). The pathophysiology of the disorder has traditionally focused, on one hand, on the primacy of visceral hypersensitivity in the development of pain or discomfort and, on the other the impact of gut dysmotility on the underlying bowel habit [3]. These separate theories have now been largely integrated within

a more holistic concept of IBS aetiology: a dysregulated brain–gut axis [4]. Despite these advances, IBS is still largely characterised by the lack of a reliable biological marker and inadequate treatment options [5]. In this review, we highlight the importance of biological markers in modern translational drug discovery efforts. The concept of a dysregulated brain–gut axis in IBS will be discussed and the challenges facing biomarker discovery scientists in this heterogeneous disorder outlined. We will evaluate the key recent findings in terms of putative biological markers within the context of a dysregulated brain– gut axis, assessing both clinically-derived candidates and those generated from animal models of the disorder. Biological markers Although traditional biomarker studies relied on the assaying of accessible biological fluids, a broader base is now more routinely considered. Current biomarker discovery approaches embrace psychological rating scales, physiological recordings, imaging studies, morphological data and behavioural outputs in addition to the genomic, proteomic or metabolomic information garnered from targeted or unbiased approaches to the analysis of biological samples [6,7]. Glossary Biological marker (biomarker): Any characteristic that is objectively measured as an indicator of normal biological processes, pathogenic processes or pharmacologic responses to a therapeutic intervention (Biomarker Definitions Working Group 2001). State marker: A biomarker that is only detectable when symptoms are evident and is consequently regarded as being causally related to the development of those symptoms or a direct result of them. Trait marker: A biomarker that is present during the active and quiescent phase of the disorder and can reflect the vulnerability of an individual to the development of a particular disease. HPA axis: Hypothalamic-pituitary-adrenal axis, the core stress response system in man. Visceral hypersensitivity: An abnormal perception of essentially normal visceral sensations. CRD: Colorectal distension, a technique, usually using a barostat, to assess visceral hypersensitivity. Motility: A broad term encompassing myoelectrical activity, tone, compliance and transit. Somatisation: The chronic and persistent reporting of physical symptoms of unexplained origin. GIT microbiota: The microbial composition of intestinal flora. Brain–gut axis: A theoretical construct describing the transmission of information through the autonomic nervous system (ANS) and the neuroimmune and neuroendocrine systems. Gnotobiotic animal: One that is born under aseptic conditions.

Corresponding author: Clarke, G. ([email protected]).

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Review Table 1. Rome III criteria Recurrent abdominal pain or discomfort at least three days per month in the past three months with two or more of the following: Improvement with defecation; 1 Onset associated with a change in frequency of stool; or 2 Onset associated with a change in form (appearance) of stool. 3

Regardless of their source, biological markers can also be considered in terms of state and trait markers. The unearthing of these disease correlates is essential to advancing our understanding of a particular disease and once validated they can be used as diagnostic tools, predictive aids and in the identification of at-risk individuals. Moreover, they can potentially be employed over time as translational indicators of novel drug action and efficacy [8]. Despite the growing appreciation of the important role that biomarker discovery plays in drug development (Box 2), biomarkers have an equally important function in defining and refining disease pathophysiology. This process requires that they should not be associated with harm to the subjects under evaluation, and attempts to establish a clear divide between healthy and affected individuals should not employ invasive sampling or measurement protocols [6]. Whereas the ideal biomarker should exhibit accuracy, reproducibility, sensitivity, specificity and patient acceptability, the reality is that not all disease states permit total adherence to all of these tenets [8]. Biomarker discovery challenges in IBS IBS places a number of obstacles in the path of the biomarker discovery scientist. The first, and perhaps, greatest originates from the heart of the disorder and relates to the intrinsic heterogeneity and temporal instability of its symptom profile; putting it simply, symptoms can vary considerably over time both within and between patients. Furthermore, the same symptoms employed to define the disorder are notoriously non-specific and can originate from a host of other disorders such as inflammatory bowel disease and celiac sprue, for example. To complicate matters further, the symptom set that waxes and wanes in IBS is routinely encountered in the non-IBS population, albeit less frequently and without the potentially disabling consequences.

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The assessment of any putative biomarker in IBS is further compounded by the frequent overlap of IBS with other FGIDs including functional dyspepsia and gastroesophageal reflux disease. Psychiatric comorbidity and the occurrence of a variety of extra-gastrointestinal somatic symptoms are common in IBS sufferers. When added to the absence of a clear understanding of disease pathophysiology, an unexplained female predominance and an incomplete description of its global epidemiology [3], the extent of the challenge that confronts those who attempt to define useful disease correlates in IBS can be readily appreciated [9]. These challenges are largely reflected in the treatment approaches and available therapeutic options. These approaches vary and options are often employed in a trial and error manner that begin with reassurance, lifestyle and dietary modifications and often lead to psychological or pharmacological therapy [2]. General pharmacological treatment options aimed at alleviating the altered bowel habits do little to affect the underlying pain [3]. Similarly, pain-directed modalities are often ineffective against the altered bowel habit making monotherapy problematic. Other strategies that have been advanced include antidepressants, modulators of the serotonergic system or nonmedicinal probiotic options [10]. Clearly, a validated biomarker in IBS that was relevant to pathophysiology, responsive to pharmacological intervention and capable of predicting drug success in clinical trials would be of immense value to research in this illdefined area. The challenges outlined above make it difficult to rigorously apply the biomarker principles discussed. Nevertheless, significant advances have been made in recent years, aided in no small part by the successful application of the brain–gut axis model in IBS. The biopsychosocial model of IBS and dysregulation of the brain–gut axis The links between psychosocial and physiological factors in IBS form the basis for the biopsychosocial model, a concept that views IBS as an illness resulting from the disturbed interaction between physiological, psychosocial, behavioural and environmental factors [11]. In healthy individuals, the components of the brain–gut axis interact to control the motor, sensory and secretory functions of the GIT [12]. The dysregulation of these interactions provides

Box 1. The Rome process and IBS subtypes The Rome diagnostic criteria, which have integrated many features of the earlier IBS classification systems such as the Manning criteria, Kruis criteria and the American College of Gastroenterology Guidelines, are now viewed as the gold standard in the field [94]. They have been revised since their inception as the Rome I criteria to the current Rome III version (Table 1). Although each revision has generated controversy along the way, even critics of the process acknowledge that they have made a positive contribution and allowed for the evolution of a framework in which to classify IBS in the scientific community. They have provided a set of guidelines that can be employed as inclusion criteria for clinical trials and established a solid platform for standardised scientific studies [95,96]. However, a number of limitations exist and need to be considered in the context of the findings discussed in this review. Firstly, there is evidence to suggest that the guidelines are not as widely employed in

the clinical setting as in the research domain [2]. The implications of this are serious because drug development strategies are largely based on recruitment policies adhering to the Rome guidelines. Although the less restrictive Rome III guidelines should rectify this disparity somewhat, a second problem is noteworthy. The approach to subtyping IBS differs between Rome II and Rome III with debate over the agreement between the resulting patient populations [97,98]. This is compounded by the fact that the method of subtyping in the Rome II guidelines is still deemed acceptable, if not preferable, in the Rome III update [99]. It is conceivable then that two studies with the same subjects carried out on the basis of the Rome III criteria but with different subtyping strategies could yield different biomarker and drug efficacy conclusions. This needs to be borne in mind when evaluating scientific evidence, especially where subgroup-specific biomarker candidates or treatment efficacies have been proposed.

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Box 2. Biological markers in translational drug discovery The escalating costs of drug discovery have certainly helped focus the minds of those involved in a process described as slow and inefficient and associated with high drug failure rates. With efficacies in the region of 40–60% and multiple adverse effects, even drugs that have survived the vagaries of the selection procedure are not all considered a resounding success. Nowhere is this more apparent than in the treatment of IBS where tegaserod and alosetron, interventions that showed some efficacy in the management of the disorder, are now either withdrawn from the market or severely restricted due to their adverse side effect profile. This is by no means unique to IBS, and can be regarded as a general indictment of the trial and error process used to identify disease states and develop therapeutic options [100]. Traditionally, biomarkers have been used as surrogate endpoints in the clinical testing and validation phase of drug development. However, biomarkers are also important in the assessing the efficacy and safety of new drugs in preclinical studies. Translational research bridges these development phases and FDA policy now dictates that biomarkers are incorporated into all strands of future drug development strategies

a rich source from which putative biological markers can be mined (Table 2). The role of initiating or perpetuating factors, including stress and infection (Box 3), and their potential consequences, in terms of measurable outputs, are illustrated in Figure 1. Targeting brain–gut axis dysfunction for biomarker discovery in IBS Visceral hypersensitivity In addition to its proposed pathophysiological role in IBS, visceral hypersensitivity has also long been mooted as a biological marker of the condition [13]. It continues to be an intensely studied area in IBS with reports claiming an incidence of up 80–90% [14]. More conservative estimates put this figure closer to 60% and this seems to be borne out by a recent study, employing the barostat technique, of 136 unselected IBS patients classified according to the Rome II criteria and in the active phase of the disease. In this study, Kanazawa and colleagues reported the presence of visceral hypersensitivity in only 57% of their patient cohort [15].

Box 3. Post-infectious IBS The heterogeneous nature of IBS has prompted researchers to seek well-defined subsets with demographic features conducive to the identification of potential aetiological factors. PI-IBS is one such subgroup and refers to the sudden onset of IBS symptoms after a bout of gastroenteritis in individuals with a previously healthy GIT. Various infections such as Campylobacter jejuni and Salmonella enteritidis induce short-term diarrhoea and abdominal pain. In a minority of these cases these symptoms do not resolve and patients go on to develop chronic IBS symptoms that can account for 6–17% of the general IBS population. Risk factors for the development of PIIBS include the severity of the initial insult, gender, age and comorbid depression or anxiety [103]. The role of infection in IBS has gained acceptance in the literature and is validated by the presence of visceral hypersensitivity and altered gut motility during bacterial gastroenteritis [13]. Nevertheless, the universal application of the theory is not without controversy, and the true role of prior infection as a causative factor in IBS remains to be fully elucidated [89]. The paradigm of PI-IBS has, however, been central to the more general concept of a link between an environmental trigger, a sustained inflammatory response and the development of IBS in general. This latter hypothesis forms the basis for many of the studies discussed in the main text.

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[100]. The implementation of this requirement is seen as critical to reducing costs, improving efficiency and yielding safer, more efficacious therapies. Nonetheless, problems remain and the inability of poorly chosen biological markers to accurately track both human and animal responses is regarded as a major impediment to drug development. Although animal models have been implicated in the high attrition rate of drug discovery because of the lack of translational models and reliance on those with poor predictive power [101], for behavioural pharmacologists the inaccurate description of clinical phenotypes and associated biomarkers is sometimes just as detrimental to the drug development process [102]. Nevertheless, once biomarkers share construct validity between clinical and animal models, the drug discovery process should become a more rational and successful process. All the current indications are that, with the increasing and appropriate incorporation of truly translational biomarkers into all facets of the bench-to-bedside model, the drug discovery penny has finally started to drop.

Although visceral hypersensitivity can be evoked through a variety of methods, most experts in the field rely on the relatively invasive and technically demanding barostat technique for an accurate and reproducible definition of visceral hypersensitivity. Technical issues persist, however, not least the realisation that both control subjects and IBS patients ‘‘adapt’’ to repeated study [16]. Despite these and other reservations about its use as a diagnostic marker [17], recent reports suggest a role as a surrogate endpoint in drug development in certain subsets of the overall IBS population. In a study that specifically selected IBS patients with a reduced sensory threshold to rectal distension, pregabalin was shown to restore the thresholds to normal values [18]. In a reverse translational approach, subsequent animal studies confirmed the data from this earlier clinical study by showing that pregabalin reduced the viscerosomatic and autonomic responses associated with colorectal distension (CRD)-induced visceral pain in rats [19]. Recent noxious CRD studies in rats have highlighted the role of peripheral cannabinoid receptor 1 (CB1) in mediating the analgesic effects of cannabinoids [20], an interesting finding in the light of current interest in the cannabinoid system in IBS. Similar studies have suggested a role for the positive allosteric modulation of GABA (B) receptors in the treatment of visceral pain [21]. It should, of course, be pointed out that noxious CRD studies should be regarded as models of visceral pain and not of IBS per se, and the studies highlighted above should be interpreted accordingly. The study of visceral hypersensitivity has also been extended to animal models of the disorder. In particular, the rodent maternal separation model, a neonatal stress model (Box 4), has produced a viscerally hypersensitive phenotype [22]. Moreover, utility for the model in the assessment of analgesic effects has been demonstrated [23]. Despite the apparent ubiquity of visceral hypersensitivity in some studies of IBS and the ability to reproduce this phenomena in animal models where it can be positively influenced by pharmacological interventions, the major limitation of visceral hypersensitivity as a putative biomarker in IBS, its invasive nature and technical complexities notwithstanding, has been its inability to

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Figure 1. A conceptual framework for IBS and prospective biomarker targets. The effects of either stress or infection result in a dysfunctional brain–gut axis. The impact of these insults can be measured in the many systems that comprise and impact on the brain–gut axis. The disturbances in these systems offer a rational basis for their utility in biomarker discovery and as a target for therapeutic intervention.

consistently predict clinical responses in human studies. This has most recently been exemplified by the experience with visceral pain-related readouts in studies of a novel 5HT1A receptor antagonist; in an all too familiar fashion efficacy in animal studies did not translate into therapeutic benefits in man [24]. Motility Disturbances in motility have long been regarded as the primary source of disturbed bowel function, be it diarrhoea or constipation in IBS [3] and, in the past, were also

considered relevant to the pathogenesis of pain and discomfort, leading to the use of terms such as ‘‘spastic colon’’. Although motility abnormalities have been described throughout the GIT in IBS, many of these findings were non-specific, some were not reproduced and most required highly specialised and invasive testing [17]. Despite these shortcomings regarding the use of motility measurements as biological markers, they continue to be reported in the literature. Of the various measures employed, gut transit is the simplest and most widely available. Studies employing the breath hydrogen methodology, a non-invasive but

Box 4. Animal models of IBS Animal models are critical to unravelling the pathophysiology of human diseases and play an integral part in the development of new treatments. Adherence to the requirements for face, construct and predictive validity should ensure that they are reasonably analogous to the human disorder, provide parameters that can be monitored and reversed by pharmacological interventions and allow for a greater level of reproducibility between laboratories. Disorders presenting with a wide spectrum of disruptions cause problems that do not easily allow the fulfilment of these guidelines. However, if a particular model can mimic even specific features of a disorder and show good predictive validity, then the research benefits would be great [104]. The application of this realisation in IBS has facilitated the introduction of a number of models based on the induction of IBS symptoms using CNS-directed stressors, GIT stressors, enteric inflammation and enteric infection [105]. The latter examples, however, could been criticised on the basis that visceral hypersensitivity models based on a severe initiating inflammatory insult do not reflect the biology of IBS and have been of limited utility in translational drug discovery. Indeed, it is generally considered that these models have not been useful in the provision of new molecular insights or predicting downstream clinical efficacy. This has

prompted the search for more robust and reliable models. The natural visceral hypersensitivity exhibited by the high anxiety WistarKyoto rat has seen it adopted as the potential genetic model of IBS [106]. Of the models proposed, it seems that those based on neonatal stress offer the most hope for the translation of basic research to successful pharmacological interventions [107]. The links between early life stress and subsequent development of IBS in adulthood provides solid construct validity for these models [16]. Indeed, a recent study has cited a rate of abuse history of almost 51% among a patient cohort, confirming the relevance of these models to the clinical situation [108]. Face validity has been confirmed by studies showing that rodents subjected to early stressful interventions develop many of the phenotypic characteristics of IBS, including visceral hypersensitivity and altered GIT transit [22] and altered reactivity to stress [109]. Moreover, the alterations described offer potential as endpoints and suggest a predictive validity for these models. Recent indications are that neonatal stress models might be to the fore in the development of future therapeutic agents [23]. The caveat that remains is whether lessons from other fields will be learned and a battery of screening tests employed so that the drugs in development are not overly reliant on a specific mode of action.

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Review often unreliable technique [25], indicate, as have more formal approaches, the presence of delayed transit in CIBS and accelerated transit in D-IBS [17]. The use of scintigraphy or radiopaque markers, more reliable if less generally available transit measurement techniques, confirm such findings [16]. A recent study employing radioscintigraphic measurements demonstrated abnormal colonic transit in 32% of a female IBS population after 48 hours, with 46% of the D-IBS patient cohort exhibiting accelerated transit at this time point [26]. Advocates of motility measurements correctly cite the successful use of colonic transit endpoints, measured using these more reliable techniques, in the assessment of novel therapeutics [27]. It has also been argued that the motility response obtained in pharmacodynamic studies can reliably be used to predict the efficacy or otherwise of novel medications in clinical trials [28]. In addition, the apparently selective delay of gas transit in IBS, as demonstrated by Serra and colleagues and reviewed elsewhere [17], needs to be considered despite the invasive methodology and limited availability. Although the use of motility biomarkers seems to satisfy many of the criteria we have set out, in our view a caveat remains that greatly undermines its overall utility in that it has led to an overemphasis on tailoring specific drugs for these bowel habit stratified markers: prokinetic drugs, such as tegaserod, for the constipated variety and motility-suppressing compounds, such as alosetron or cilansetron, for those with predominant diarrhoea. Both compounds encountered problems which reflect the complexity of IBS. Given the fact that many IBS patients can oscillate between diarrhoea and constipation and that what IBS patients describe as diarrhoea and constipation rarely equates with the classical definition of these bowel habits, the troubling frequency of the side effects of diarrhoea and constipation with tegaserod and alosetron could have been predicted. Bowel symptoms in IBS must be interpreted with caution and simple extrapolations of transit or motility findings avoided because they oversimplify a complex disorder. Interestingly, a recent clinical study linked delayed transit in a C-IBS cohort to an elevated bile acid metabolism [29]. Also of interest are reports linking the cannabinoid system to the regulation of gastrointestinal motility in rodents [30]. Faecal pellet output from the rodent maternal separation model of IBS is increased [22], also suggesting increased motility. It remains to be seen whether this alteration in the animal model is open to modulation by pharmacological agents and how this might be translated to the clinical setting. Such a development might well represent the optimal future use of motility measurements in IBS. Neurotransmitters and neuropeptides Of the neurotransmitter systems thought to be involved in IBS, the serotonergic system is the most studied. Many of the studies that have focused on the serotonin (5-HT) content of biological samples, on the basis that alterations in 5-HT levels might explain the predominant bowel habit through its influence on GIT motility, are contradictory in nature [31]. Interestingly, in an animal model of postinfectious (PI)-IBS, elevated intestinal 5-HT concentration 482

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and release was shown to persist after the infection resolved [32]. It is also noteworthy that both IBS patients and maternally-separated rodents have shown an exaggerated plasma prolactin release following a challenge with buspirone, a challenge test that monitors central 5-HT1A receptor responsivity [33]. The precise mechanism whereby this receptor contributes to the aetiology of IBS or its manifestations remains to be elucidated, and this test should be regarded as a means to explore the pathophysiology of this disease rather than a bona fide surrogate endpoint. Tryptophan, the dietary precursor of 5-HT, has also been studied in IBS. An elevated degradation of tryptophan was found in a female IBS cohort that was most pronounced in those with the most severe symptoms [34]. This increased degradation was also found in a male cohort and correlated with the degree of immune activation [35]. An examination of serotonin transporter (SERT) function in mucosal biopsies has yielded conflicting results with a recent study finding no alteration in SERT levels in either C-IBS or D-IBS patients [36]. This result contradicted an earlier report that showed reduced mucosal SERT immunoreactivity in both subtypes [37]. Interestingly, an association has been described between SERT polymorphisms and drug response [38]. Brain activation following CRD has also been found to be related to this polymorphism in a recent neuroimaging study [39], whereas a functional single nucleotide polymorphism of the transporter was recently shown to be more common in IBS patients compared with controls in a genotyping study [40]. It should be noted, however, that the most recent genotyping study on this polymorphism did not replicate some of the earlier findings, and additional studies are required to fully elucidate the relevance of these interesting studies [41]. Whether a distinctive biological marker will be derived from the studies described above is open to debate because 5-HT alterations have been reported in a variety of other GI disorders including inflammatory bowel disease, chronic constipation and celiac disease. However, based on the efficacy shown for alosetron and tegaserod, the therapeutic modulation of the serotonergic system is a strategy that continues to attract attention [31]. Recent evidence suggesting a role for the micro RNA-regulated expression of the serotonin receptor-type 3E gene in D-IBS suggests exciting developments lie ahead [42]. Of the number of neuropeptides being examined with a view to unearthing a suitable biological marker, the most relevant might be a study that showed elevated basal and postprandial cholecystokinin (CCK) levels in IBS compared with healthy controls [43]. Moreover, the importance of a polymorphism in the CCK receptor gene has recently been noted [44]. Interestingly, CCK antagonists are now considered promising therapeutic agents in the treatment of IBS [45]. No consistent findings for neuropeptide Y (NPY) or vasoactive intestinal peptide (VIP) have been found, and would seem that there are no alterations in somatostatin or substance P [4]. Recently decreased serum leptin concentrations have been described in IBS [46], whereas neither plasma nor tissue extract levels of ghrelin showed any alteration between patients and controls [47].

Review This latter study did demonstrate an interesting lowering in the density of ghrelin immunoreactive cells in the oxyntic mucosa in C-IBS and an increased density in DIBS respectively compared with controls. Clearly, further exploration of these agents is required before any firm conclusions can be established despite the current interest in neuropeptide modulation as a therapeutic option in IBS [48]. Again it will be important to differentiate between the non-specific effects of the associated bowel habit, be it diarrhoea or constipation, and those of IBS per se. Measures of endocrine function A variety of endocrine measures can be used to assess hypothalamic-pituitary-adrenal (HPA) axis function, both under normal conditions and following various challenge tests [8]. Because of the difficulties associated with the measurement and interpretation of peripheral corticotrophin-releasing hormone (CRH) measurements, peripheral adrenocorticotrophic hormone (ACTH) and glucocorticoid measures have chiefly been used to assess HPA axis activity [49]. Although historical reports of alterations in basal cortisol levels in IBS have been contradictory in nature [4], the more recent literature seems to support a hyperactivated HPA axis. There are a number of studies showing elevated plasma cortisol levels in IBS patients compared with controls [50], an overall trend towards higher plasma cortisol levels over a 24-hour period [51] or elevated plasma cortisol in the morning [52]. However, baseline serum cortisol levels were not found to be different between a female D-IBS cohort and healthy controls [53]. Basal plasma ACTH levels have been reported to be unchanged [50], reduced [51] or slightly elevated [54]. Serum chromogranin A levels, a neuroendocrine secretory protein, were recently reported to be transiently elevated in a subset of D-IBS patients [55]. Interestingly, elevated plasma levels of corticosterone, the rodent equivalent of cortisol, was reported to be elevated in the rat maternal separation model [22]. HPA axis responses to various stressors and challenge tests have also been examined and yielded some interesting results. Public speaking stress-induced neuroendocrine measures yielded no differences between plasma cortisol levels and ACTH in IBS or controls [54]. A study examining the neuroendocrine response to sigmoidoscopy showed higher stimulated plasma cortisol levels in a D-IBS cohort [51]. The cortisol response to a 35% carbon dioxide stress challenge showed no differences between serum measures of the hormone in a female D-IBS cohort compared with controls [53]. An exaggerated release of both plasma cortisol and ACTH has been demonstrated following CRH infusion in IBS patients compared with controls, with no differences in the hormone response to the dexamethasone suppression test [50]. The results of this latter test suggest that the negative feedback control of the HPA axis is intact in IBS [49]. In summary, measures of endocrine function have yielded interesting, if at times conflicting, results. Differences between studies might be accounted for by the varied study designs, patient populations and measurement strategies employed [4]. Caution should be exercised, in particular, in the interpretation of single time point

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measurements of stress hormones that exhibit a diurnal variation, peak 30 minutes after waking or are responsive to the anticipation of stress [56]. Moreover, it should be noted that HPA axis alterations are not specific to IBS and are also present in metabolic and psychiatric disorders such as Cushing syndrome, anxiety and depression. Dissection of the relative contribution of HPA axis dysfunction to the emergence of comorbid psychiatric symptoms in IBS is now warranted. Immune function The growing view that inflammation plays an important part in IBS has led to recent interest in immune system measures as biological markers of the condition. In particular, many studies have focused on various cytokines as indicators of immune activation. Thus, plasma levels of both the proinflammatory cytokine IL-6 and its soluble receptor sIL-6R have been shown to be elevated in an IBS population [50]. The same study reported a reduction in basal plasma levels of the anti-inflammatory IL-10 and an unaltered TNF-a profile. Baseline levels of TNF-a, IL-1b and IL-6 in peripheral blood mononuclear cells (PBMCs) were later shown to be elevated in IBS with patients with the D-IBS subtype presenting with higher levels of these cytokines than the C-IBS subtype [57]. In addition, the authors of this study reported an increased secretion of IL6 from LPS-stimulated PBMCs and an increased LPS stimulated release of IL-1b in their C-IBS subgroup only. Of further interest is the report of elevated in vitro production of IL-6 and TNF-a in LPS-stimulated whole blood in the rat maternal separation model of IBS [22], and the correlation between this IL-6 release and a proinflammatory polyunsaturated fatty acid profile in the plasma of these animals [58]. Genetic input from the high producer TNF genotype is also worthy of note [44]. Furthermore, symptom improvement following probiotic treatment has been associated with a normalisation of the IL-10/IL-12 ratio in unstimulated PBMCs, one of the few demonstrations of immune marker responsiveness to a therapeutic intervention [59]. The biological consequences of such a cytokine imbalance, as they pertain to the development of IBS symptomatology and the role of the normalisation of the ratio in treatment response is still unclear. These and other outstanding questions have prompted much interest in the likely origin of these alterations and the mechanisms behind them. Speculation that they were of intestinal mucosal origin has not yet been demonstrated with the secretion of IL-6, TNF-a and IL-1B from ex vivo biopsy cultures not shown to be different between IBS sufferers and controls [60], although reduced secretion of IL-8, CXCL-9 and MCP-1, as well as reduced colonic mRNA expression of genes related to chemokine function, was noted. This would seem to be borne out by a study that evaluated cytokine mRNA levels in mucosal biopsies from the sigmoid colon and found that the expression of IL-2, IL6 and IL-10 was reduced in a D-IBS population [51]. From a mechanistic standpoint, it has recently been suggested that the enhanced release of IL-6 is mediated by the cholinergic system [61]. Of further note is that the pyridostigmine-induced release of IL-6 in this study correlated with an IBS symptom score. 483

Review Cells of the immune system have also been evaluated in the context of an activated immune system in IBS. Mast cells have attracted considerable attention with most, but not all, studies reporting increased mast cell numbers in various parts of the GIT though, for obvious reasons, the colon and rectum have been the areas most extensively studied [4]. A more recent study found increased mast cell activation in addition to hyperplasia in the jejunum in IBS [62]. Some recent studies illustrate the possible relevance of these findings to specific IBS symptoms. In one study, increased mast cell numbers were reported to correlate with visceral hypersensitivity in a D-IBS cohort [63]. A direct link between immune activation and symptoms has also been provided by Barbara and colleagues who demonstrated not only an increased prevalence of mast cell degranulation in the colon in IBS, but also a direct correlation between the proximity of mast cells to neuronal elements and pain severity [64,65]. Information garnered from neonatal stress models of the disorder also implicates mast cell degranulation in maternally-separated rodents and reported decreased visceral hypersensitivity following treatment with doxantrazole, a mast cell stabiliser [66]. A closer association between mast cells and enteric neurons has also been reported following maternal deprivation [67] as has mast cell hyperplasia in the maternal separation model [68]. Enterochromaffin (EC) cells, natural killer cells and lymphocytes have all been studied in IBS cohorts with contradictory reports of increases, decreases or no alterations [4]. However, the most recent studies in this area seem to favour either an increased number of immune cell types or their increased activation. These reports include increased B-cell activation [69] and increased T-cell activation [70] in IBS. Moreover, it has recently been demonstrated that IBS patients have a 72% increase in immune cell numbers in the gut compared with controls, an increase that was present in 50% of their IBS cohort [71]. Of special interest in this study is that the authors also included patients with microscopic colitis and ulcerative colitis and found that their IBS cohort immune cell increases were intermediate between these disorders and control levels. A number of problems have been identified that are relevant to the interpretation of these studies. These include variations in biopsy site, the extent of the overlap between patients and controls and the laborious quantitative methods required to demonstrate abnormalities not obvious during routine histological examination [4]. Furthermore, whereas such studies have been invaluable in establishing an immune pathology in IBS, their reliance on obtaining biopsies, an invasive process, does not readily lend themselves to use in a diagnostic biomarker capacity or as surrogate endpoints in clinical trials. This has prompted the investigation of immune cell products in more accessible biological samples. Elevated tryptase, a marker of mast cell activation, has been found in the jejunal luminal fluid but not the serum of an IBS population [62]. However, some studies examining tryptase levels in stool samples from IBS patients have found no alterations [72,73]. Cenac and colleagues, by contrast, not only described increased concentrations of 484

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mast cell products, such as proteases, in the lumen, but also demonstrated the ability of such proteases to induce visceral hypersensitivity in appropriate models [64,65]. Faecal serine protease activity was increased in a D-IBS cohort compared with other subtypes and controls [73]. Unfortunately, the D-IBS cohort could not be distinguished from inflammatory bowel disease (IBD) patients. However, a study from the same laboratory did show that mucosal application of colonic supernatants from these D-IBS patients elicited allodynia and colonic epithelial barrier dysfunction in mice [74]. Plasma neopterin levels, a marker of macrophage activation, have also been reported to be increased in a male IBS cohort [35]. Of the other markers of immune activation that have been studied few have shown specificity in the discrimination of IBS patients from controls. These include S100A12 [75], lactoferrin [76], calprotectin [77] C-reactive protein (CRP) [76] and myeloperoxidase (MPO) [72]. However, these markers do distinguish IBS from IBD, which might be of great utility in the clinical setting. It is also noteworthy that human beta-defensin 2 (HBD2), an antimicrobial peptide and marker of intestinal barrier function, has been found to be elevated to a level that is intermediate between those of control subjects and those with ulcerative colitis levels in faecal samples from an IBS population [78]. Interestingly, the permeability of colonic biopsies from IBS patients has been shown to be greater than in healthy controls, and soluble mediators in supernatants from these biopsies increase the in vitro permeability of Caco-2 cells [79]. Even though data gathered from Ussing chamber studies does not directly reflect in vivo colonic permeability, the findings offer an interesting mechanistic insight. Indeed, it was recently demonstrated in the clinical setting that a subset of patients with D-IBS had increased intestinal permeability using the lactulose/mannitol method [80]. In summary, the majority of the studies described above provide strong evidence for the presence of a low grade inflammation in IBS. Much work remains, however, before these studies’ utility as biomarkers can be validated. It is still unclear, for example, how PBMC IL-10/IL-12 ratios relate to tissue levels. Moreover, the use of biopsy tissue in attempting to answer these questions suffers from intrinsic flaws such as sampling error, variability of the biopsy content and the stress of the procedure itself. Similarly, although the evidence for a cytokine imbalance in IBS is gaining momentum, further work is required to establish the mechanism whereby this imbalance leads to the development of IBS or of specific symptoms therein. It has not been fully established whether alterations in mast cells or their activation products can be ascribed to the cause of IBS or is incidental to its development. In this regard, a recent report describing the activation of human enteric neurons by supernatants from the colonic biopsy specimens of IBS patients and the inhibition of this activation by antagonists at receptors responsive to mast cell products is enlightening [81]. It remains to be seen if the disturbances that have been described will resolve following successful therapeutic intervention, but irrespective of the difficulties outlined the potential for the future development of these biomarker candidates is considerable.

Review Microbiota The bacterial composition of intestinal flora is considered important for both the maintenance of good health and development of disease states such as IBD and IBS [4]. It has recently been shown that faecal samples from IBS patients have quantitatively different numbers of various bacterial strains [82,83] as well as a greater instability in the composition of their intestinal microflora over time [84]. Additional support comes from the maternal separation model of IBS, where alterations in the faecal microbiota have also been found compared with non-separated animals [22]. Studies on intestinal microbiota are certainly intriguing and provide a rationale for the use of probiotics and antibiotics in the treatment of IBS. However, the interpretation of studies of microbiota in IBS must be tempered by the great challenges presented by any study of gut flora. For example, modern molecular techniques have revealed that culture-based techniques, the norm in the past, can identify as little as 40% of the species and strains present in the normal colonic microbiota. The complete delineation of the normal microbiota continues; it would be premature, therefore, to jump to conclusions about the role of the flora, be it primary or secondary, in IBS. Although the presence of microbiota disturbances in both the clinical setting and in animal models suggests that a suitable screening method for characteristic changes in IBS could be developed, the logistical challenges presented by this approach must not be underestimated. Behavioural changes The fact that dysregulation of the brain–gut axis is the most accepted current model of IBS requires that behavioural alterations be considered possible markers of the condition. Factors to be considered and that can determine illness behaviour include symptom perception and evaluation [11]. The utility of behavioural and complementary treatment approaches in the management of IBS more than hint at the importance of these parameters [85]. Sleep problems, somatic abnormalities, high levels of illness behaviour and anxiety have been reported as independent risk markers for the development of IBS [86]. Catastrophic thinking specific to pain has also been reported, as has an abnormal level of somatisation [87]. Abnormal behaviours have also been reported in the rat maternal separation model of IBS, with increased anxiety behaviours frequently being demonstrated [22]. It is unclear how these findings will be advanced and a rigorous application of the biomarker criteria we have established does not favour the use of these parameters in the diagnostic or therapeutic arenas. However, the role they have played in cementing the position of the brain–gut axis model within the field of IBS should not be too readily discounted. Therapeutic implications The importance of biological markers in drug discovery can be gauged from the number of therapeutic modalities evaluated on the basis of the biomarker candidates discussed. Continuing emphasis on traditional measurements such as visceral hypersensitivity and motility is evident as exemplified by the current trials evaluating

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Table 2. Biomarker candidates in IBS Biomarker class Visceral hypersensitivity

Motility

Neurotransmitters and neuropeptides

Endocrine function Immune function

Microbiota

Behavioural changes

Supporting evidence Barostat studies Responsive to therapeutic intervention Animal models of IBS Gut transit measurements (including scintigraphy, radio-opaque markers and breath tests) Responsive to therapeutic intervention Animal models of IBS Alteration in serotonin levels Abnormalities in serotonin reuptake transporter Responsive to therapeutic intervention Alterations in CCK levels Use of CCK antagonists Ghrelin Leptin Hyperactivated HPA axis Animal models of IBS Elevated IL-6 levels Animal models of IBS Mast cell alterations B-cell activation T-cell activation Alterations in immune cell numbers Elevated tryptase Serine protease activity Elevated neopterin levels Elevated beta-defensin II S100A12 Lactoferrin Calprotectin CRP MPO Bacterial strains Microflora instability Animal model of IBS Symptom perception Alternative treatment approaches Animal models of IBS

References [13,15] [18] [22] [2,16,17]

[27] [22] [31] [38,40] [10] [43] [45] [47] [46] [49–51] [22] [50,57,61] [22,58] [64,65,68] [69] [70] [71] [72] [72] [35] [78] [75] [76] [77] [76] [72] [82,83] [84] [22] [11] [85] [22]

the efficacy of pregabalin and recent approval of lubiprostone for the treatment of C-IBS respectively. Similarly, the demonstrated utility of serotonergic modulators such as alosetron and tegaserod has stimulated the development of more selective agents acting on this system. This is evident in the consideration being given to prucalopride, a 5-HT4 agonist, for the treatment of C-IBS in the light of its apparent success in treating chronic constipation [88]. Questions remain over the efficacy of such options in treating global symptoms of IBS, but it should equally be recognised that alleviation of specific symptoms does offer some relief to sufferers and will probably remain a strategy for some time. Indeed, some of the recent preclinical findings of visceral hypersensitivity discussed in this study will see the imminent arrival of more paindirected therapies. The groundswell of reports identifying immune activation in IBS cannot be ignored, and the fact that many immune-modulating therapies are already in existence 485

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Table 3. Drugs targeting potential biomarkers and under investigation for the treatment of IBS (Clinicaltrials.gov) Candidate biomarker Visceral hypersensitivity

Motility

Neurotransmitters

Neuropeptides Immune function

Microbiota

Potential treatments Pregabalin AV608 GW876008 AGN 203818 *Lubiprostone BMS-562086 *Linaclotide DNK333 MD-1100 Acetate Rezular DDP 225 Solabegron YM060 Prucalopride Mosapride Citalopram Ondansetron Dexloxiglumide AV608 Mesalazine

Bifidobacterium infantis 35624 Bifidobacterium breve Lactobacillus plantarum MF 1298 VSL #3 E. coli strain M17 Saccharomyces boulardii Rifaximin

Comment Majority target pain relief, might not be effective in treating global symptoms

References [17–19]

Majority aimed at either increasing or decreasing transit, might not be effective in treating global symptoms *primary mode of action is via an increase in fluid secretion and it is thought that both accelerate colonic transit

[17,27]

Based on relative success of tegaserod and alosetron, most of these agents target the serotonergic system

[31,88]

Targets require further evaluation

[43,45]

Based on suppression of low grade immune activation, earlier trial with prednisolone did not show efficacy in the treatment of PI-IBS Majority are probiotics with multiple effects and unknown mechanism of action

[89]

argues for interesting short-term developments. The potential pitfalls in the application of the findings discussed are underlined by the fact that prednisolone showed no efficacy in the treatment of PI-IBS [89]. However, prednisolone is a potent immunosuppressive drug that might not be appropriate for the low level of inflammation described in IBS and this needs to be taken into account in the assessment of therapeutic candidates. The challenge it seems will lie in applying the same ingenuity displayed in identifying these parameters to the therapeutic manipulation of drug candidates. It is likely that facing up to this challenge will mean the use of existing immunomodulatory agents with the channelling of resources to the development of newer agents being the long-term response to evidence of short-term success. Furthermore, the proposed role for probiotic treatment in the alleviation of IBS symptoms is an exciting development that requires further attention, and a definitive, well-designed clinical trial to adequately evaluate specific probiotic candidates is urgently required. Targeting the human microbiome is likely to be an important future strategy and is the focus of much current comment [90]. Of course, the evaluation of potential therapies has to contend with the high placebo response rate among the IBS population, a factor that can lead to the demise of many promising interventions [91]. Clearly, challenging times lie ahead in the treatment of IBS, and it will be of interest to see whether future developments can merge clinical and preclinical findings in a way that translates into improved therapeutic options in IBS. Table 3 summarises some of the drug candidates currently under evaluation in clinical trials (Clinicaltrials.gov). 486

[90]

Concluding remarks Although considerable hurdles remain in the validation of biomarker candidates in IBS, key aspects of the brain–gut axis have been identified that have augmented our knowledge of the disorder and offer promise both in diagnosis and outcome measurement (Table 2). In particular, evidence continues to increase supporting a low-grade immune activation in IBS. Similarly, a growing emphasis on the intestinal microflora and its manipulation by probiotic preparations seems to offer novel insights, although more studies are required to address exactly what differentiates a normal microbiota from that of IBS patients (Box 5). On the preclinical front, the study of gnotobiotic animals offers the means to address some of these issues. Box 5. Outstanding questions  What is the origin of and mechanism behind immune activation in IBS?  How do immune alterations relate to the aetiology and symptoms of IBS?  Can existing immunomodulatory therapies be exploited to treat IBS?  How do probiotics alleviate symptoms in IBS?  Will more selective pain- and motility-directed therapies be effective in alleviating the global symptoms of IBS?  Can technological advances allow the complete characterisation of normal and diseased microbiota?  Can biomarkers identified using preclinical animal models translate to the development of suitable treatments for IBS?  Can a biomarker panel be established that will allow diagnosis of IBS?  Are there other genetic polymorphisms that might predict disease severity and treatment outcome?

Review Substantial efforts are ongoing in an attempt to bridge these gaps in knowledge (Box 5) and will occupy major research efforts in the coming years. In addition, our understanding of the traditional concepts of visceral hypersensitivity and altered gut motility have been reassessed and refined. Furthermore, the findings from animal models of IBS have, in many cases, mirrored clinical findings and shown considerable potential in the establishment of a bench-to-bedside model for the development of new therapies. In the short-term, this is likely to be reflected in additional pain- and motility-directed therapeutic modalities. However, the heterogeneous nature of the disorder suggests that it is unrealistic to expect a single biomarker candidate to be applicable to all facets of the disease. At the outset, we identified this heterogeneity, in addition to the temporal instability of the non-specific symptom profile and a poorly understood disease pathophysiology, as significant obstacles in the biomarker discovery process. We reiterate those sentiments here and suggest that future strategies pay more attention to comprehensively defining the disease phenotype. This will require studies that are sufficiently powered to detect potentially subtle subgroup alterations. An innovative approach utilising panels of biomarkers as opposed to single entities is likely to be a more informative option. This approach would also allow the unravelling of IBS genotypes, an advancement that would in turn allow for the further refinement of potential biomarkers and their interactions with that genotype. Indeed, this strategy has recently been embraced, albeit not without controversy and with a predictive value that is inferior to the Rome criteria, in a proposed diagnostic test for IBS [92,93]. The integrated use of imaging technology and information on the IBS genotype could be employed to answer some of the key remaining questions [16]. Ultimately, it is likely that the success of recent innovations will be determined by their inclusion in future criteria. Until that happens, the current symptom-based approach will remain the standard in the diagnosis of IBS. References 1 Spiegel, B.M. (2009) The burden of IBS: looking at metrics. Curr Gastroenterol Rep 11 (4), 265–269 2 Spiller, R. et al. (2007) Guidelines on the irritable bowel syndrome: mechanisms and practical management. Gut 56 (12), 1770–1798 3 Talley, N.J. (2006) Irritable bowel syndrome. Intern Med J 36 (11), 724–728 4 Ohman, L. and Simren, M. (2007) New insights into the pathogenesis and pathophysiology of irritable bowel syndrome. Dig Liver Dis 39 (3), 201–215 5 Quigley, E.M. (2006) Changing face of irritable bowel syndrome. World J Gastroenterol 12 (1), 1–5 6 Mueller, C. et al. (2008) Biomarkers: past, present and future. Swiss Med Wkly 138 (15–16), 225–229 7 Schwarz, E. and Bahn, S. (2008) The utility of biomarker discovery approaches for the detection of disease mechanisms in psychiatric disorders. Br J Pharmacol 153 (Suppl 1), S133–136 8 Connor, T.J. and Leonard, B.E. (2004) Biological Markers of Depression, in Handbook of Experimental Pharmacology, SpringerVerlag, pp. 117–148 9 Huett, A. and Xavier, R.J. (2008) Neither hide nor hair: the difficulty of identifying useful disease biomarkers. Gastroenterology 134 (7), 2164– 2168 10 Snelling, N. (2006) Do any treatments work for irritable bowel syndrome? International Journal of Osteopathic Medicine 9, 137–142

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