Guanylate Cyclase-C agonists as peripherally acting treatments of chronic visceral pain

Abstract

Irritable Bowel Syndrome (IBS) is a chronic gastrointestinal disorder characterized by abdominal pain and altered bowel habit that affects ~11% of the global population. Over the last decade, pre-clinical and clinical studies have revealed a variety of novel mechanisms relating to the visceral analgesic effects of guanylate cyclase-C (GC-C) agonists. Here we discuss the mechanisms by which GC-C agonists target the GC-C/cyclic guanosine-3’,5’-monophosphatec (GMP) pathway, resulting in visceral analgesia as well as clinically relevant relief of abdominal pain and other sensations in IBS patients. Due to the preponderance of evidence we focus on linaclotide, a 14 amino acid GC-C agonist with very low oral bioavailability that acts within the gut. Collectively, the weight of experimental and clinical evidence supports the concept that GC-C agonists act as peripherally acting visceral analgesics.

Chronic Visceral Pain: A global unmet medical need

Irritable Bowel Syndrome (IBS) is a common chronic functional gastrointestinal disorder characterized by abdominal pain and altered bowel habit that affects ~11% of the global population[1]. Current diagnosis of IBS is based on symptoms enshrined in the multiple iterations of Rome criteria, and exclusion of pathophysiology based on standard clinical testing[2]. Sub classifications of IBS are determined by the nature of stool irregularities, such as those experiencing constipation (IBS-C), diarrhea (IBS-D), or alternating constipation and diarrhea (IBS-M)[2]. Additionally, chronic idiopathic constipation (CIC) affects approximately 14% of the global population. Symptoms associated with CIC diagnosis include reduced bowel movements, hard stools, straining during defecation, as well as bloating and discomfort. Thus CIC patients have some symptoms which overlap with those of IBS-C. Given the clinical burden of these conditions, efficacious therapies are necessary. There are multiple efficacious treatments for bowel dysfunction, as illustrated in systematic reviews and network meta-analyses in patients with chronic constipation[3,4] or IBS-C[5]. However, there is a major unmet need in the management of symptoms such as pain and bloating. Although there are many candidate approaches in development as visceral analgesics[6], one of the more attractive pharmacological approaches involves the peripherally-acting guanylate cyclase-C (GC-C) agonists (see Glossary) linaclotide and plecanatide. These agonists ‘tap’ into the intrinsic mechanisms controlling intestinal fluid homeostasis that are regulated by the endogenous hormones uroguanylin and guanylin [Box 1]. Importantly, linaclotide and plecanatide have limited bioavailability [Box 2] and hence have clinical safety as well as being bereft of central adverse effects, unlike traditional analgesics such as μ-opioids, or centrally acting neuromodulators and most available cannabinoid agents[6].

Box 1: Uroguanylin and guanylin: Endogenous GC-C agonists that increase intracellular cGMP.

Uroguanylin and guanylin are endogenous hormones of the guanylin peptide family of cGMP-regulating peptides, which regulate fluid secretion in response to meals, aiding in the digestive process. Uroguanylin is a 16 amino acid peptide expressed by tuft-like epithelial cells, which are primarily located within the proximal small intestine, with fewer cells in stomach, distal small intestine and colorectum[61]. Uroguanylin activates GC-C, which is abundantly expressed in intestinal epithelial cells [19,22], with greatest potency in the slightly acidic (pH 6) environments of the duodenum and proximal jejunum. In contrast, guanylin is a 15 amino acid peptide that is predominantly expressed in the colorectum by goblet cells. Guanylin activates GC-C in neutral to slightly basic pH environments[61]. Upon binding by the natural hormones uroguanylin and guanylin, the intrinsic guanylate cyclase activity of GC-C rapidly elevates the intracellular concentration of the second messenger cGMP via the enzymatic conversion of cytoplasmic guanosine triphosphate (GTP) to cyclic GMP (cGMP)[7-9]. Cyclic GMP is involved in the regulation of a broad range of physiological processes, including the activation of cGMP-dependent protein kinase II (PKG-II), and a direct interaction with cyclic nucleotide-gated ion channels and cGMP-regulated phosphodiesterases [9,10]. The role of cGMP in PKG-II phosphorylation and protein kinase A (PKA) activation is important for intestinal secretion, as they regulate the activity of the cystic fibrosis transmembrane conductance regulator (CFTR), which is co-localized with PKG-II at the apical surface of intestinal epithelial cells. Increased activity of CFTR leads to the efflux of Cl⁻ and HCO₃⁻ ions into the intestinal lumen and promotes water secretion into the gastrointestinal tract[8,62]. Furthermore, cGMP activated PKG-II inhibits the activity of the sodium/hydrogen exchanger 3 protein (NHE3), blocking absorption of sodium ions from the intestinal lumen and inhibiting fluid absorption[62]. The important role of GC-C in intestinal fluid homeostasis is confirmed in studies using GC-C gene knockout (gucy2c^−/−) mice. Furthermore in humans, gain of function mutations in GC-C are associated with chronic diarrhea[63], whilst loss of function mutations in GC-C have been associated with meconium ileus, which is characterized by neonatal intestinal obstruction[64]. The mechanisms by which GC-C regulates intestinal secretion are reviewed in depth elsewhere[65] which contains references to the original discoveries.

Box 2: Chemistry and Molecular Properties of Linaclotide and Plecanatide.

Linaclotide is a synthetic, selective 14 amino acid peptide agonist of GC-C, composed of naturally occurring amino acids that shares some sequence similarities with guanylin and uroguanylin. Linaclotide’s structure was designed with three disulfide bonds (Cys¹–Cys⁶,Cys²–Cys¹⁰,Cys⁵–Cys¹³) to stabilize its molecular structure, enhance its resistance to degradation and lock it into a constitutively active conformation[7,18]. Linaclotide exhibits high affinity and pH independent binding to GC-C on monolayers of human colonic carcinoma T84 cells that occurs, unlike guanylin and uroguanylin, across the broad range of physiological conditions (pH 5-8) found in the gastrointestinal tract[7,8]. Linaclotide has also binds to rat intestinal epithelial cells in a pH independent manner[11]. When combined, these beneficial actions enhance the ability of linaclotide to bind to GC-C and mediate the pharmacological actions of linaclotide[7,18]. Upon binding, GC-C rapidly elevates the intracellular concentration of cGMP via the conversion of cytoplasmic GTP to cGMP[7-9]. Notably, linaclotide has a higher potency of activation of cGMP production compared to guanylin/uroguanylin when studied in vitro in T84 cells[7,8]. Linaclotide is relatively stable in gastric fluid, showing minimal metabolism during a 3 hour in-vitro incubation with simulated gastric fluid[11]. The single pharmacologically active metabolite of linaclotide, MM-419447 (Des-Tyr14), is produced following the proteolytic cleavage of its C-terminal tyrosine by incubation with carboxylpeptidase A[11]. This process occurs more rapidly in duodenal and jejunal loops than in ileal loops[11]. MM-419447 binds to GC-C in T84 cells or rat intestinal epithelial cells in a pH-independent and equipotent manner to linaclotide[7,8,11].

Orally administered peptides are generally poorly absorbed across the intestinal epithelium due to their size and hydrophilicity[66], rendering them unable to passively traverse the lipid bi-layer of the luminal cell membrane of enterocytes. Consistent with this, linaclotide has a low permeability coefficient, as determined in Caco-2 cells[11]. Furthermore, the absolute oral bioavailability of linaclotide and MM-419447 (evaluated in rats and mice administered up to a dose of 10 mg/kg of linaclotide [ca. 1,000-2,000 times the therapeutic dose in humans] was ≤0.1%[11]. The minimal absorption of linaclotide into the systemic circulation holds true in clinical studies. Patients orally given 290μg of linaclotide once daily for seven days showed no quantifiable concentrations of linaclotide or MM-419447 in plasma samples[11]. Stool samples from healthy volunteers showed recovery of the active peptide was almost exclusively in the form of the metabolite MM-419447, representing 3-5% of the orally administered linaclotide dose[20].

Plecanatide is a 16 amino acid peptide analogue of uroguanylin, in which aspartic acid is replaced with glutamic acid in the third position of the N-terminus[38]. Accordingly, plecanatide has the same mechanism of action as uroguanylin, and has been shown to stimulate cGMP production in vitro in T84 cells[38]. Plecanatide preferentially binds GC-C with high affinity in the slightly acidic environments (pH 5-6) of the duodenum[67], making it somewhat distinct from linaclotide which binds throughout the gastrointestinal tract in a pH-independent, but concentration dependent manner. Similar to linaclotide, plecanatide is not absorbed systemically, eliminating the potential for off-target effects[38].

Anti-nociceptive effects of GC-C agonists in pre-clinical animal models of IBS

Upon binding by GC-C agonists, the intrinsic guanylate cyclase activity of GC-C rapidly elevates the intracellular concentration of the second messenger cyclic guanosine-3’,5’-monophosphate (cGMP) via enzymatic conversion of cytoplasmic guanosine triphosphate (GTP) to cGMP[7-9]. Cyclic GMP is involved in the regulation of a broad range of physiological processes, including activation of cGMP-dependent protein kinase II (PKG-II), and a direct interaction with cyclic nucleotide-gated ion channels and cGMP-regulated phosphodiesterases[9,10]. The endogenous hormones guanylin and uroguanylin are GC-C agonists and key regulators of intestinal fluid homeostasis. In vivo studies in rodents show linaclotide evokes fluid and cGMP secretion within intestinal loops and accelerates gastrointestinal transit[7,8,11-14]. This GC-C/cGMP pathway is now an emerging target for the treatment of visceral pain[15-18], as activation of this pathway by linaclotide [19-27] and plecanatide[28,29] reduces nociception and abdominal pain in numerous pre-clinical studies and clinical trials (Figure 1).

Figure 1: Proposed mechanism of action of GC-C agonists in modulating secretion and visceral pain via the GC-C/cGMP signaling pathway.

Schematic diagram describing the key elements of GC-C activation leading to relief of constipation or analgesia. The pathway starts with GC-C expression on the apical membrane of intestinal epithelial cells. Activation of GC-C results in the production of cGMP, via the enzymatic conversion of cytoplasmic guanosine triphosphate (GTP) to cGMP. Cyclic GMP then activates the cGMP-dependent protein kinase II (PKG-II) with resultant activation of CFTR. MRP4 is also involved in the apical release of cGMP into the intestinal lumen to enhance fluid secretion. Also shown are the proposed anti-nociceptive actions of cGMP. This mechanism involves transport of cGMP through a basolateral cGMP transporter or efflux pump allowing the release of cGMP out of intestinal epithelial cells and into the underlying layers of the colon wall. This extracellular cGMP then acts on an extracellular target on colonic nociceptors to inhibit their function. CFTR: Cystic fibrosis transmembrane conductance regulator, cGMP: cyclic guanosine-3’,5’-monophosphate. cGMP-dependent protein kinase II (PKG-II), GC-C: guanylate cyclase-C. GTP: guanosine triphosphate. MRP4: multidrug resistance-associated protein 4. PKG-II: cGMP-dependent protein kinase II. Created with BioRender.com.

To understand the analgesic mechanisms induced by GC-C activation it is important to understand the sensory pathways contributing to nociception. The gastrointestinal tract is innervated by sensory afferents of vagal and spinal origin, providing a three-neuron chain to transfer sensory information from the gut to the brain, with synapses at the spinal cord dorsal horn and the brain stem or thalamus[30]. Generally speaking, spinal afferents are responsible for signaling nociceptive stimuli, particularly from the colorectum[30,31] (for details see Box 3). Visceral hypersensitivity, in particular the enhanced responses of colonic afferents to mechanical and/or chemical stimuli, is implicated in the development and maintenance of visceral pain in IBS[31-33].

Box 3: Sensory Afferent Innervation of the Gastrointestinal Tract.

The gastrointestinal tract is innervated by vagal and spinal sensory afferent terminals that innervate the gut wall. These afferents convey sensory information from the gastrointestinal tract to the CNS, where the information is coordinated and integrated, leading to reflex secretory and motor responses and to conscious perception of visceral events[30,31]. Vagal afferents have their cell bodies located within the nodose and jugular ganglia, whilst spinal lumbar splanchnic and sacral pelvic nerves have cell bodies in the dorsal root ganglia (DRG). These afferents provide a three-neuron chain to transfer sensory information from gut to brain with synapses at the dorsal horn of the spinal cord and in the brain stem or thalamus[30]. Generally speaking, vagal afferents more densely innervate the upper gastrointestinal tract, whilst spinal afferents more densely innervate the distal gastrointestinal tract. In rodents at least, spinal afferents innervating the colon are almost exclusively C-fibres; thus, pain and discomfort from the intestine are signaled by C-fibers[31,68]. Elegant studies have shown that the peripheral projections of vagal and spinal afferents have specialized sensory structures in the wall of the gut, with ~13 morphologically distinct endings identified to date[69]. These include intraganglionic laminar endings, which serve the function of proprioceptors (stretch or tension receptors). Other types of endings include mucosal endings, submucosal endings, intramuscular arrays and vascular afferents that wrap around blood vessels[70]. These endings correlate with different functional classes of afferents found within the splanchnic and pelvic innervations of the rodent colon. This includes afferents that reside throughout the wall of the intestine, and on the mesentery, that have low- or high-thresholds to circular stretch or distension or detect very low-threshold events in the mucosa[71]. Importantly, these functional classes of afferent have been confirmed in recordings from human colon[72,73]. Importantly, nociceptive afferents that normally have high-thresholds for distension in healthy naïve conditions become sensitized and are activated at much lower stimulation intensities in animal models of IBS. Moreover, mechanically insensitive afferents or ‘silent nociceptors’ in naïve conditions become active and behave like high-threshold nociceptors following sensitization[31]. These functional afferent subtypes correspond with 7 different molecular subtypes of afferents[74]. Afferent sensitivity can also be profoundly affected by interactions with the mucosal epithelium, motility patterns [75-79] and by ‘bottom up’ and ‘top down’ sensitization[32,33]. Overall, these data suggest that the ascending pathways may diverge according to the nature and region of the stimulus[60].

Evidence of GC-C mediated anti-nociceptive effects on spinal afferents were first demonstrated pre-clinically following oral linaclotide administration in a rodent trinitrobenzene-sulfonic-acid (TNBS) model of colitis-induced visceral hypersensitivity[20]. Linaclotide reduced TNBS-induced colonic hypersensitivity, measured as a decrease in abdominal contractions (visceromotor response [VMR]) during colorectal distension (CRD)[20]. The efficacy of linaclotide in reducing visceral pain has also been assessed in various animal models of stress, the rationale being that stress is a known IBS risk factor, and stress exacerbates pain in IBS[1,34,35]. Oral administration of linaclotide in adult animal models of water avoidance or partial restraint stress reduced colonic hypersensitivity, as measured by a decrease in abdominal contractions during CRD[20]. Similar anti-nociceptive actions of linaclotide (daily for 7-days) occur in neonatal rats exposed to unpredictable early life stress (ELS)[36], which also display increased mucosal permeability, an effect also reversed by linaclotide[36]. GC-C agonism also modulates corticolimbic activation and corticotropin-releasing factor signaling in a rat model of stress-induced colonic hypersensitivity[37].

Although far less studied, rat visceral hypersensitivity models show a single oral dose of plecanatide suppresses TNBS-induced increases in abdominal contractions to CRD without affecting colonic elasticity[28]. Oral plecanatide also reduces colonic hypersensitivity in a partial restraint stress rat model[28]. The precise mechanisms by which plecanatide reduces visceral hyperalgesia in these studies have not been specifically investigated. In fact, pre-clinical studies have predominantly focused on the anti-inflammatory effects of plecanatide, which show once-daily treatment for 7 days ameliorates colitis in both dextran-sodium-sulphate (DSS) and TNBS-induced models of acute colitis. These anti-inflammatory effects were determined by a reduction in colitis severity scores, changes in body weight and disease activity. However, amelioration of colitis by plecanatide treatment was not dose-dependent[38]. Plecanatide also ameliorated colitis in a model of spontaneous colitis[38] and reduced inflammation-induced carcinogenesis in a mouse model of colon cancer[39].

Studies show the anti-nociceptive actions of linaclotide are mediated at the level of afferent endings innervating the colon. Ex vivo studies show a single application of linaclotide to the colonic mucosa inhibits high-threshold pain sensing afferents (nociceptors) to mechanical stimulation, an effect lost in recordings from mice lacking GC-C. Removal of the colonic mucosa prior to these recordings attenuated linaclotide-induced nociceptor inhibition [19], implicating a key role for the epithelium in this process. Similarly, the cGMP transporter inhibitor, probenecid, prevented linaclotide-induced nociceptor inhibition [19] implicating cGMP in linaclotide’s inhibitory action (Figure 1).

Interestingly, linaclotide exhibited greater efficacy in reducing nociceptor mechanosensitivity in a post-inflammatory model of chronic visceral hypersensitivity (CVH)[19]. In this model, 4-weeks after TNBS-colitis, colonic nociceptors exhibit sustained hypersensitivity to mechanical stimulation, whilst CVH mice display increased numbers of activated dorsal horn neurons in the spinal cord and hyperalgesia to noxious CRD [22,30,31,40]. In ex vivo experiments, single applications of linaclotide dose-dependently reduced colonic nociceptor firing to mechanical stimuli. These ex vivo findings were confirmed in vivo, as a single intra-colonic dose of linaclotide in CVH mice reduced the abnormally high numbers of activated dorsal horn neurons in response to noxious CRD observed in vehicle-treated CVH mice[19]. Chronic (2-week) daily oral linaclotide (3μg/kg) attenuates colonic nociceptor firing ex vivo in the same CVH model[22]. Furthermore, linaclotide mediated nociceptor inhibition ex vivo correlates with in vivo studies, as chronic linaclotide (3μg/kg) treatment in CVH mice reduces sprouting of colonic afferent central terminals within the dorsal horn, whilst decreasing the number of dorsal horn neurons activated by noxious CRD[22]. Correspondingly, chronic oral linaclotide (3μg/kg) treatment in CVH mice normalizes the VMR to CRD[22](Figure 2).

Figure 2: Proposed mechanism of action of GC-C agonists in modulating secretion and visceral pain via the GC-C/cGMP signaling pathway.

A) Schematic diagram describing indicating how transient colitis induces long-term changes in colonic sensory pathways following resolution of intestinal inflammation. This neuroplasticity in colonic sensory pathways occurs at the level of the afferent ending, cell soma and spinal cord resulting in elevated pain response to CRD. This neuroplasticity in colonic sensory pathways subsequently results in neuroplasticity in bladder sensory pathways and bladder voiding dysfunction. B) Schematic diagram describing key elements of the GC-C/cGMP signaling pathway starting with GC-C activation of GC-C to cGMP production, stimulation of secretion, actions on CFTR, the role of a cGMP transporter in the apical release of cGMP to enhance secretion. Also shown is the role of a basolateral cGMP transporter or efflux pump allowing the release of extracellular cGMP that acts on an extracellular target on colonic nociceptors to inhibit their function. Studies have shown that chronic oral linaclotide treatment, acting upon GC-C expressed on epithelial cells within the gut, can normalize colonic sensory signaling and pain responses to CRD, and subsequently normalize bladder afferent function and bladder voiding. CFTR: Cystic fibrosis transmembrane conductance regulator, cGMP: cyclic guanosine-3’,5’-monophosphate. CRD: colorectal distension, GC-C: guanylate cyclase-C, GTP: guanosine triphosphate, MRP4: multidrug resistance-associated protein 4, PKG-II: cGMP-dependent protein kinase II. Created with BioRender.com.

These findings support the concept that linaclotide decreases peripheral drive, or the transmission of pain signaling along the ascending gut-brain pathway. Whilst a single application of linaclotide has anti-nociceptive actions, chronic linaclotide (3μg/kg) appears to have additional benefits by reducing CVH-induced neuroplasticity within central pathways, including reducing nociceptor hypersensitivity and reducing sprouting of afferent central terminals in the spinal cord. These findings may help explain observations in clinical trials, whereby the percentage of patients reporting a ≥30% improvement in abdominal pain compared to baseline during the first week of treatment, continues to increase over the ensuing 10-12 weeks and is maintained throughout the remainder of the 26-week treatment[19].

Chronic daily (2-week) intracolonic linaclotide (3μg/kg; to simulate a clinically tested linaclotide delayed release formulation[41]) in CVH mice also inhibits colonic nociceptors to mechanical stimuli ex vivo, as well as normalising in vivo VMR to CRD[42]. Notably, chronic intra-colonic linaclotide reduces the sprouting of colonic afferent central terminals within the spinal cord, reduces the number of dorsal horn neurons activated by noxious CRD, as well as reducing CVH-induced glial activation in dorsal root ganglia (DRG)[43]. These studies suggest the predominant analgesic site of action of linaclotide occurs within the colon, as similar findings were observed comparing chronic oral and intra-colonic dosing paradigms[22,42,43]. These findings reinforce the observation that linaclotide inhibits colonic nociceptors to reduce ‘peripheral drive’ from the colon to the spinal cord. The manner in which linaclotide inhibits nociceptor function is discussed below.

Extracellular cGMP inhibits colonic nociceptors from mice and DRG neurons from humans

Recent studies demonstrate linaclotide itself does not inhibit nociceptors, as linaclotide applied directly to isolated DRG neurons does not alter their excitability[22]. This functional evidence confirms expression studies using in situ hybridization showing GC-C is absent from the DRG and spinal cord[19]. Furthermore, linaclotide treatment also has no effect on colonic compliance during graded CRD[22], nor affects electric field stimulation-induced colonic contractions[19], which is consistent with the absence of GC-C on smooth muscle cells. These expression profiles are in contrast to the colonic epithelium where GC-C is extensively expressed in rodents and humans [19,22,44].

It was hypothesized that cGMP produced in intestinal epithelial cells upon GC-C activation could mediate the analgesic actions of linaclotide by acting on sensory afferents in the colon wall. It is known that cGMP is actively transported out of cells through membrane-bound transport proteins (cGMP transporters or efflux pumps)[45] and that in the CNS, extracellular cGMP has direct inhibitory effects on neurons causing reduced excitability and neurotransmitter release. Accumulating in vitro, ex vivo and in vivo evidence support the concept that the anti-nociceptive actions of linaclotide are mediated by activation of the GC-C/cGMP pathway and extracellular cGMP secreted from intestinal epithelial cells acting on afferent endings.

Firstly, in vitro exposure of intestinal Caco-2 cells to GC-C agonists stimulates bidirectional, active extracellular transport of cGMP into luminal and basolateral spaces that are significantly and concentration-dependently decreased by probenecid, an inhibitor of cGMP efflux pumps[18,19,45]. This cGMP secretion after GC-C activation is also observed from the basolateral side of rat colonic epithelium into the submucosal space[18]. This implicates energy dependent transport of cGMP by cGMP efflux pumps, such as the multidrug resistant proteins 4 (MRP4) or 5 (MRP5). Under normal conditions, cGMP phosphodiesterases are considered the major elimination pathway for intracellular cGMP. However, MRP4/5-mediated extracellular cGMP transport is consistent with their function as overflow pumps, allowing cGMP to be released extracellularly when concentrations are supra-physiological, such as during activation of GC-C by linaclotide. For example, MRP4 is expressed on the apical membrane of intestinal epithelial cells and inhibition of MRP4 suppresses apical but not basolateral cGMP efflux[11,46]. As discussed above, the cGMP transporter inhibitor probenecid prevents linaclotide-induced inhibition of colonic nociceptors[19], implicating cGMP in linaclotide’s inhibitory effect.

Confirmation of cGMP-induced modulation of colonic hyperalgesia comes from studies directly dosing or applying cGMP in animal models. In rats with TNBS-induced visceral hypersensitivity, oral cGMP administration prior to CRD dose-dependently increases colonic distension thresholds, and decreases the number of abdominal contractions to CRD in a partial restraint-induced stress model[18]. These effects of cGMP mimicked the anti-hyperalgesic effects of uroguanylin. Furthermore, mice with visceral hypersensitivity treated intra-duodenally with cGMP exhibited decreased colonic afferent firing to increasing CRD pressures[18].

The use of ex vivo single-unit recordings of colonic nociceptors confirmed the anti-nociceptive effects of cGMP and linaclotide[19]. These studies showed cGMP applied to the surface of the colonic epithelium concentration-dependently decreased nociceptor activity with greater efficacy in CVH mice, mimicking the effects of linaclotide[18,19]. Importantly, the anti-nociceptive effect of cGMP was greater following removal of the colonic mucosa, allowing cGMP to bypass the epithelial barrier. These findings provide further evidence that cGMP is acting directly on the underlying afferent nerve endings following basolateral release from epithelial cells[19]. cGMP also reduces the number of action potentials fired by low-threshold stretch-sensitive pelvic afferents [16],[18]. Most recently, patch-clamp recordings of isolated DRG neurons, completely independent of any colonic tissue, demonstrated cGMP causes dose-dependent inhibition of action potential firing. This occurs in populations of colon-innervating DRG neurons from mice, as well as a mixed population of DRG neurons from human donors[22]. Fluorescence resonance energy transfer (FRET)/cGMP measurements showed conclusively for the first time that cGMP acts on a membrane target that is accessed from an extracellular site to inhibit nociceptors[22]. The precise molecular target that cGMP acts on extracellularly remains elusive but is the subject of continued investigation.

Analgesic efficacy of GC-C agonists across distension paradigms in experimental animals

Importantly, the analgesic efficacy of linaclotide occurs across a broad spectrum of distension paradigms, which are usually stepwise, graded distensions called ‘ascending method of limits’ or ‘rapid phasic’ distensions. Overall, these distension paradigms would simulate rapid mechanical stimulation and pressure levels observed during high amplitude colonic contractions. VMR studies of controlled isobaric colonic distensions 0-60mmHg at 15mmHg steps[20] or controlled isobaric colonic distensions 0-80mmHg at 10-20mmHg steps[22] have shown orally administered linaclotide reduces visceral hypersensitivity in rodents in vivo when performing CRD with the ascending method of limits. Moreover, controlled isobaric colonic distensions at randomized pressures of 0,20,40 and 60mmHg, also shows orally administered linaclotide reduces visceral hypersensitivity in rats[23,36]. Studies have used single intra-colonic [19], daily chronic oral (14 days)[22] or chronic intra-colonic (14 days)[43,47] linaclotide treatment followed by rapid phasic CRD and then examined dorsal horn neuron activation within the spinal cord. In all of these studies, acute or chronic linaclotide reduces the number of CRD-activated dorsal horn neurons[19,22,43,47]. These studies show that irrespective of the method of distension (ascending method of limits/randomized phasic distensions) that linaclotide is analgesic in models of rat and mouse acute and CVH.

Studies also show a single oral dose of plecanatide in healthy naïve rats did not alter the rate of abdominal contractions to CRD. However, in rats 4 days post-TNBS lower doses (0.01 and 0.05mg/kg), but not higher doses (>0.1mg/kg) of plecanatide attenuated the TNBS-induced increase in the number of abdominal contractions with increasing distending pressures up to 60mmHg[28]. The precise mechanisms by which plecanatide induces visceral hyperalgesia in these studies have not been specifically investigated.

Effects of GC-C agonists on abdominal pain in patients with CIC or IBS-C

Numerous clinical trials have documented benefits of GC-C agonists on pain and other sensory symptoms such as bloating in patients with CIC or IBS-C. In a double-blind, placebo-controlled, parallel-group phase II study of 310 patients with CIC (assessing 75,150,300,600μg Linaclotide or placebo), Linaclotide reduced abdominal symptoms such as discomfort and bloating as well as constipation severity while improving adequate relief of constipation, treatment satisfaction and quality of life, for each dose of linaclotide[48]. A more recent phase II randomized, double-blind, placebo-controlled dose finding study of Japanese CIC patients provided similar primary endpoint efficacy in improving weekly spontaneous bowel movement (SBM) frequency as well as relief of abdominal symptoms of chronic constipation for all linaclotide doses (62.5-500μg)[49]. Furthermore, in a phase IIb randomized, double-blind, placebo-controlled study of 420 IBS-C patients meeting Rome II criteria that were given doses ranging from 75μg-600μg, the 300μg linaclotide dose most significantly reduced abdominal pain. In contrast to the rapid improvement of bowel habits (within 24hr), the improvement of abdominal pain was significant after one week of treatment and gradually increased further throughout the remaining 12-week treatment[50]. Interestingly the analgesic effect of linaclotide was most profound in patients with severe abdominal pain at baseline[50].

A randomized, double-blind, placebo-controlled, phase II dose-finding trial of a Japanese IBS-C patient cohort diagnosed using Rome III criteria evaluated once daily 62.5-500μg linaclotide during a 12-week treatment period. Monthly responder rates of complete SBMs, abdominal pain/discomfort relief, and global assessment of relief of IBS symptoms were higher than placebo during the 12-week treatment[51]. Furthermore, a phase III, randomized, double-blind, placebo-controlled trial of linaclotide has been performed in an additional patient cohort of 839 individuals in China[52]. Greater numbers of IBS-C patients randomized to once-daily oral 290μg linaclotide at centres in China, North America and Oceania met co-primary endpoints of ≥30% reduction in average weekly abdominal pain or abdominal discomfort score for 6 out of 12-weeks and IBS degree of relief ≤2 weekly responder for 6- out of 12-weeks compared to those receiving placebo. In a phase III, double-blind, parallel-group, placebo-controlled trial, IBS-C patients were randomized to placebo or 290μg oral linaclotide once daily in a 12-week treatment period, followed by a 4-week randomized withdrawal period. Notably, greater improvements were seen in linaclotide vs. placebo patients for all secondary end points throughout the 12-weeks. During the 4-week randomized withdrawal period, patients who remained on linaclotide showed a sustained improvement in abdominal pain[26]. In contrast, patients re-randomized from linaclotide to placebo showed return of symptoms[26]. Overall, in all phase III studies conducted in IBS-C or CIC patients to date, linaclotide significantly improved abdominal pain, discomfort and bloating[24-27,52].

Importantly, on September 22, 2020 the FDA approved a supplemental New Drug Application (sNDA) for linaclotide based on data from a phase IIIb, randomized, double-blind, placebo-controlled parallel-group trial evaluating the safety and efficacy of linaclotide 290μg on multiple abdominal symptoms in 614 adult IBS-C patients over 12-weeks compared to placebo. The additional data demonstrated linaclotide improved the overall abdominal symptoms score of bloating, discomfort, and pain in adult IBS-C patients[53](https://www.accessdata.fda.gov/drugsatfda_docs/label/2020/202811s016lbl.pdf). This trial was designed to highlight the impact of linaclotide on a primary endpoint based on the mean abdominal score (composite of abdominal bloating, abdominal discomfort, and abdominal pain) across 12-weeks, as well as the secondary endpoint which was a responder analysis based on at least a 2.5-point improvement in the abdominal score from baseline for >6 out of 12-weeks. In addition, individual scores of bloating, discomfort, and pain were assessed over the 12- week treatment period. Linaclotide-treated patients showed a 29.7% mean decrease from baseline in their weekly abdominal score (bloating, pain and discomfort) through the 12-week treatment, compared to 18.3% for the placebo-treated patients. In addition, the data showed improvement in individual abdominal symptoms of bloating, discomfort and pain with linaclotide [53].

A 2020 study shows 26 IBS-C patients treated daily with linaclotide (290μg) for 10-weeks displayed prolongation of rectal and anal cortical evoked potential responses (using anorectal electrical stimulations and transcranial/transspinal-anorectal magnetic stimulations) compared to 13 placebo-treated IBS-C patients. These data suggest that linaclotide may improve rectal hypersensitivity in IBS-C patients[54] and supports the observations from pre-clinical studies that linaclotide inhibits nociceptors to reduce ‘peripheral drive’. Along these lines a recent report has documented that linaclotide delayed release formulations, released in the terminal ileum (LIN-DR1) or colon (LIN-DR2: MD-7246) reduces abdominal pain in IBS-C patients compared to placebo, without altering bowel function or inducing diarrhea[55]. This may suggest the predominant analgesic site of action of linaclotide occurs within the colon, as indicated by rodent studies.

Two phase III identical randomized double blind placebo controlled trials of plecanatide in patients with IBS-C randomized 2189 individuals among whom 1879 completed the studies[29]. Using the overall responder definition, patients reporting ≥30% reduction from baseline in worst abdominal pain plus increase of ≥1 CSBM/week from baseline in the same week for ≥6- of 12-treatment weeks), both plecanatide 3mg and 6mg were superior to placebo, including the responses of abdominal symptoms such as pain, discomfort and bloating[29]. Overall, a systematic review and network meta-analysis[5] involving 12 trials composed of 7302 patients reported in 10 separate articles that reported dichotomous data for failure to achieve an abdominal pain response, all secretagogue treatments were more effective than placebo, with linaclotide 290μg once daily ranked as the most effective treatment based on 3 RCTs (RR 0.79; 95% CI 0.73–0.85).

Anti-nociceptive effects in animal models of bladder pain syndrome and endometriosis.

Neuroanatomical studies performed in rodents show sensory afferents innervating the urogenital organs and colon are predominantly peptidergic (containing substance P, calcitonin gene related peptide)[56-58]. There is also strong evidence for convergence of bladder and colon sensory innervation at the level of primary afferents, and peripheral sensitization of urinary bladder and pelvic nerve afferents in the setting of colonic irritation, which potentially contribute to disorders characterized by chronic pelvic pain[57,59]. Consistent with the mechanisms underlying the anti-nociceptive actions of linaclotide, recent studies suggest linaclotide may be useful in the treatment of bladder hypersensitivity disorders occurring concurrently with gastrointestinal disorders[31,59]. Neonatal rats exposed to unpredictable ELS exhibit increased abdominal contractions to CRD, but also exhibit bladder hypersensitivity[36]. Linaclotide (3μg/kg daily for 7 days) but not vehicle, reduced hypersensitivity from both the colon and bladder, suggesting the anti-nociceptive actions of linaclotide may extend beyond the colon. Protamine sulfate infused into the bladder, which transiently enhances mucosal permeability, induced mechanical hypersensitivity of both the bladder and colon. The 7-day linaclotide, but not vehicle, treatment attenuated bladder and colonic hyperalgesia to control levels[23]. Therefore, these studies suggest linaclotide impacts visceral hypersensitivity in adjacent pelvic organs through a novel mechanism that is likely via common shared sensory pathways. Indeed, recent evidence shows linaclotide is able to exploit the neuronal pathways responsible for visceral organ ‘cross-talk’, and resolve bladder afferent hypersensitivity that normally occurs following TNBS-induced colitis[22](Figure 2). The study first identified CVH mice, which exhibit colonic afferent sensitization ex vivo and hyperalgesia to CRD in vivo, also exhibit simultaneous bladder afferent hypersensitivity, increased excitability of bladder-innervating DRG neurons and altered urinary voiding patterns, suggestive of overactive bladder. Following chronic daily oral linaclotide (3μg/kg) administration for 2-weeks colonic hypersensitivity was reversed and bladder afferent sensitization resolved[22]. This study also demonstrated linaclotide had no direct effect on bladder afferents, with GC-C absent from bladder urothelial cells, suggesting linaclotide acts via an indirect mechanism to normalize bladder hypersensitivity. Notably, these beneficial effects of linaclotide on bladder function also occur following 2-week intra-colonic linaclotide administration, to mimic a delayed release formulation[47].

Cross-organ sensitization of the bladder via afferent mechanisms has been well characterized[31,57,59]. This includes the presence of dichotomizing neurons that have a single cell body with axons that innervate both the colon and bladder[22]. The central terminals of distinct bladder- or colon-innervating afferents also reside in close proximity to one another within the spinal cord[22]. Studies using a mouse CVH model show colonic afferent central terminals are denser and display sprouting into other regions of the dorsal horn[60], thereby increasing potential interaction between colon and bladder afferent terminals[22]. These studies suggest such interconnected neuronal pathways can be exploited to reduce comorbid hypersensitivity of adjacent organs, in particular the bladder (Figure 2).

Such mechanisms were recently exploited in a rat model of endometriosis whereby 8-weeks after endometrium transplantation into the intestinal mesentery, rats developed endometrial lesions as well as vaginal hyperalgesia to distension and decreased mechanical hind paw withdrawal thresholds[21]. Daily oral linaclotide (3μg/kg) administration increased pain thresholds to vaginal distension and increased mechanical paw withdrawal thresholds, indicating analgesic actions of linaclotide. Furthermore, using a cross-over design, administering linaclotide to rats previously administered vehicle resulted in increased paw withdrawal thresholds (reduced pain), whereas replacing linaclotide with vehicle decreased paw withdrawal thresholds (increased pain)[21](Figure 3). Retrograde tracing studies from ileum, colon and vagina identified dichotomizing dual-labelled ileal/colon innervating afferents as well as colon/vaginal dual-labelled neurons and a rare population of triple traced ileal/colon/vaginal neurons within thoracolumbar DRG [21]. The central terminals of these sensory afferents also lie in close apposition to one another within the spinal cord [21]. Notably, GC-C expression is absent in the vagina and endometrial cysts, suggesting the actions of linaclotide are shared through nerve pathways between these organs [21]. These pre-clinical studies suggest linaclotide may offer a novel therapeutic option not only for treatment of chronic abdominal pain and co-morbid bladder dysfunction, but also endometriosis-associated pain and concurrent treatment of comorbid chronic pelvic pain syndromes.

Figure 3: Endometriosis induces vaginal and somatic hypersensitivity in rats, which can be reduced by chronic oral linaclotide administration.

A) Schematic diagram describing how endometriosis induces allodynia and hyperalgesia to vaginal distension, as well as somatic hypersensitivity to mechanical stimulation of the hind paw. In rat studies, endometrial lesions within the abdominal wall release inflammatory mediators that activate visceral afferents and cause allodynia and hyperalgesia to vaginal distension. The hypersensitivity of visceral afferents then sensitizes afferent pathways innervating the skin via a process called ‘viscerosomatic convergence’. This leads to somatic hypersensitivity to mechanical stimulation of the skin. B) In rat studies, chronic oral linaclotide, acting on GC-C expressed on epithelial cells within the gut can reduce the severity of vaginal and somatic hypersensitivity. Data suggests this occurs via linaclotide acting on GC-C expressed on intestinal epithelial cells. The resultant cGMP acts on and inhibits colonic nociceptors. This reduction in peripheral drive then results in reduced viscerosomatic convergence, as common afferent sensory pathways innervate the colon, vagina and skin. Overall, chronic linaclotide treatment results in reduced allodynia and reduced hyperalgesia to vaginal distension and decreased somatic hypersensitivity to mechanical stimuli. Created with BioRender.com

Concluding remarks and final perspectives

Pre-clinical and clinical evidence supports the concept that GC-C agonists act as peripherally acting visceral analgesics. Preclinical studies show that linaclotide is a peripherally acting drug with cGMP acting as a neuromodulator and downstream effector of linaclotide that directly inhibits colonic nociceptors via a membrane target accessed from an extracellular site. However, the precise identity of this molecular target remains to be identified. Randomized controlled clinical studies of GC-C agonists have demonstrated beneficial treatment effects on abdominal symptoms including pain and bloating in adult IBS-C patients. Whether changes in the GC-C/cGMP pathway are apparent in patients and whether such changes contribute to patient symptomatology warrants further investigation. Finally, recent preclinical studies also provide the prospect that linaclotide may offer a novel therapeutic option, not only for the treatment of chronic abdominal pain in IBS-C, but also co-morbid bladder dysfunction and endometriosis-associated pain.

HIGHLIGHTS:

• Linaclotide and plecanatide are synthetic peptide agonists of guanylate cyclase C (GC-C), a transmembrane receptor that is predominantly located on intestinal epithelial cells. These medications have very low oral bioavailability, which when combined with the expression profile of GC-C, provides a mechanism of action ‘targeted to the gut’.

• Linaclotide has been shown to inhibit colonic nociceptors and reduce peripheral drive from the colon, resulting in reduced numbers of activated dorsal horn neurons within the spinal cord and reduced pain responses to noxious colorectal distension. These effects are greatest in animal models of chronic visceral hypersensitivity that drive ‘bottom-up’ and/or ‘top-down’ sensitization that are relevant to Irritable Bowel Syndrome (IBS).

• Activation of GC-C results in the generation and release of cyclic guanosine-3’,5’-monophosphate (cGMP) from intestinal epithelial cells. When released into the submucosal space through the basolateral membrane, extracellular cGMP acts as a neuromodulator to inhibit pain sensing nerve fibres innervating the colon. cGMP has also been demonstrated to inhibit pain sensing nerves from human donors.

• In all human phase III clinical studies conducted to date in patients with Irritable Bowel Syndrome with constipation (IBS-C) or chronic idiopathic constipation (CIC), linaclotide significantly improved abdominal pain, discomfort and bloating.

• Recent preclinical studies also provide the prospect that linaclotide may offer a novel therapeutic option, not only for the treatment of chronic abdominal pain in IBS-C, but also co-morbid bladder dysfunction and endometriosis-associated pain.

OUTSTANDING QUESTIONS:

• cGMP acts on a membrane target that is accessed from an extracellular site to inhibit nociceptors. However, the precise molecular target that cGMP acts on extracellularly remains elusive but is the subject of continued investigation.

• What are the precise mechanisms by which reducing peripheral drive from the colon with chronic linaclotide treatment reduces colitis-induced bladder dysfunction or endometriosis induced vaginal and somatic hypersensitivity?

• Are changes in the GC-C/cGMP pathway apparent in patients with IBS-C or CIC? Do these changes contribute to patient symptoms and does linaclotide treatment reverse such changes?

GLOSSARY:

Afferent

neuron that signals from peripheral tissue towards the central nervous system.

Agonist

a molecule or chemical that binds to a receptor and activates the receptor to produce a biological response

Allodynia

painful perception of a normally non-painful stimuli.

Anti-nociceptive

the process of blocking or reducing the detection of a painful or damaging stimuli

Cross-organ sensitization

Mechanism by which one ‘diseased’ organ can alter the function of an adjacent organ. For instance, colonic inflammation can result in profound changes to colonic sensory pathways which then affect the sensory pathways innervating the bladder, resulting in severe bladder dysfunction.

Cyclic guanosine-3’,5’-monophosphate (cGMP)

the intrinsic guanylate cyclase activity of GC-C rapidly elevates the intracellular concentration of the second messenger cGMP via the enzymatic conversion of cytoplasmic guanosine triphosphate (GTP) to cyclic GMP (cGMP).

Dorsal root ganglion (DRG)

cluster of neuronal cell bodies located in the dorsal root of spinal nerves.

DRG neurons

cell soma of peripheral peripheral sensory neurons that innervate the visceral organs and other tissues. These neurons carry peripheral information that is sent to the central nervous system.

Early life stress

Chronic and/or extreme stress in early life that can trigger long lasting negative effects on a number of neural systems.

Guanylate cyclase-C (GC-C)

a type I transmembrane receptor with intrinsic guanylate cyclase activity that is abundantly expressed in intestinal epithelial cells. The gene name of GC-C is GUCY2C.

Hyperalgesia

exacerbated perception of painful stimuli caused by chemical, thermal or mechanical stimuli.

Irritable bowel syndrome (IBS)

a chronic gastrointestinal disorder characterized by symptoms including chronic abdominal pain, bloating, diarrhea, and constipation.

Neuroplasticity

The structural, synaptic or intrinsic changes that alter neuronal function

Nociceptors

sensory neurons activated by noxious stimuli that relay pain signals and are classified based on their responsiveness to mechanical and chemical stimuli.

Peripheral drive

Amount of sensory information being signaled from the periphery to the central nervous system. There is increased peripheral drive in conditions of visceral hypersensitivity.

Peripheral sensitization

increased sensitivity of peripheral sensory afferent neurons. Sometimes referred to as ‘bottom-up’ sensitization. This contrasts with ‘top down’ sensitisation that originates in the brain and travels down to spinal and peripheral levels

Polymodal

Has the capacity to respond to a variety of different chemical and mechanical stimuli.

Responder rates

The percentage of human patients in a clinical trial who achieve a pre-defined level of improvement on the main assessment outcomes at certain time points of the trial.

Rome Criteria

Diagnostic criteria established by the Rome Foundation, an independent not-for-profit organization dedicated to supporting the creation of scientific data and educational information to assist in diagnosing and treating Disorders of Gut-Brain Interaction (DGBIs), formerly called Functional Gastrointestinal Disorders (FGIDs).

Visceral hypersensitivity

Enhanced sensory signaling from the visceral organs measured at the level of the periphery, spinal cord, brain or whole-body level.

Viscerosomatic convergence

Afferent neurons innervating viscera organs or skin that then converge upon the same dorsal horn neurons within the spinal cord.