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Keywords:

  • cisapride;
  • constipation;
  • 5-HT4 receptor;
  • prucalopride;
  • tegaserod

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. The 5-HT4 receptor as a target for prokinetic drugs
  5. 5-HT4 receptor agonists
  6. Conclusion
  7. Acknowledgements
  8. References

Abstract  5-Hydroxytryptamine4 (5-HT4) receptors are an interesting target for the management of patients in need of gastrointestinal (GI) promotility treatment. They have proven therapeutic potential to treat patients with GI motility disorders. Lack of selectivity for the 5-HT4 receptor has limited the clinical success of the agonists used until now. For instance, next to their affinity for 5-HT4 receptors, both cisapride and tegaserod have appreciable affinity for other receptors, channels or transporters [e.g. cisapride: human ether-a-go-go-related gene (hERG) is K+ channel and tegaserod: 5-HT1 and 5-HT2 receptors]. Adverse cardiovascular events observed with these compounds are not 5-HT4 receptor-related. Recent efforts have led to the discovery of a series of selective 5-HT4 receptor ligands, with prucalopride being the most advanced in clinical development. The selectivity of these new compounds clearly differentiates them from the older generation compounds by minimizing the potential of target-unrelated side effects. The availability of selective agonists enables the focus to shift to the exploration of 5-HT4 receptor-related differences between agonists. Based on drug- and tissue-related properties (e.g. differences in receptor binding, receptor density, effectors, coupling efficiency), 5-HT4 receptor agonists are able to express tissue selectivity, i.e. behave as a partial agonist in some and as a full agonist in other tissues. Furthermore, the concept of ligand-directed signalling offers great opportunities for future drug development by enlarging the scientific basis for the generation of agonist-specific effects in different cell types, tissues or organs. Selective 5-HT4 receptor agonists might thus prove to be innovative drugs with an attractive safety profile for better treatment of patients suffering from hypomotility disorders.


Abbreviations
Ach

acetylcholine

CGRP

calcitonin gene-related peptide

DAT

dopamine transporter

EC

enterochromaffin

GORD

gastro-oesophageal reflux disease

GI

gastrointestinal

hERG

human ether-a-go-go-related gene

5-HT

5-hydroxytryptamine

IBS

irritable bowel syndrome

IPAN

intrinsic primary afferent neurone

LMMPP

longitudinal muscle myenteric plexus preparation

NANC

non-adrenergic non-cholinergic

NAT

noradrenaline transporter

NO

nitric oxide

PDE

phosphodiesterase

RASSL

receptor activated solely by synthetic ligands

SERT

serotonin transporter

VIP

vasoactive intestinal peptide

Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. The 5-HT4 receptor as a target for prokinetic drugs
  5. 5-HT4 receptor agonists
  6. Conclusion
  7. Acknowledgements
  8. References

Serotonin [5-hydroxytryptamine (5-HT)], a ubiquitous signalling molecule, exerts its actions by interacting with seven receptor subtypes.1 It is involved in a variety of functions in the brain and periphery. 5-HT is synthesized by serotonergic neurones in the central nervous system but the largest amount of 5-HT in the body is found in the gut, specifically in the enterochromaffin (EC) cells of the GI mucosa and to a smaller extent in descending interneurones. After a meal, EC cells secrete large amounts of 5-HT, the excess moving into the portal circulation and intestinal lumen.2 Virtually all 5-HT in the blood originates from mucosal EC cells. Platelets avidly take up 5-HT and store it in dense granules; platelets themselves are incapable of synthesizing 5-HT.3 Since its discovery almost 60 years ago, extensive research on 5-HT has generated a plethora of therapeutic agents (agonists, antagonists and re-uptake inhibitors), some of which have had a major impact on disease management.4

The role of 5-HT in the gut is complicated by its interactions with different receptor subtypes (5-HT1, 5-HT2, 5-HT3, 5-HT4 and 5-HT7) present in the GI tract. All classes of the 5-HT receptor family, except for the ligand-gated 5-HT3 receptor, are members of the seven transmembrane-spanning G protein-coupled receptor family.1 Besides the 5-HT3 receptors, 5-HT4 receptors are the most thoroughly studied subtype with regard to gut function. Together with 5-HT6 and 5-HT7 receptors, 5-HT4 receptors are positively coupled to Gs proteins, resulting in stimulation of adenylyl cyclase and increase in cellular cyclic AMP (cAMP). Almost 20 years ago, the first description of the 5-HT4 receptor and its pharmacological characterization was made by Dumuis et al., who reported 5-HT4 receptor-mediated stimulation of adenylyl cyclase by 5-HT in cultured mouse embryonic colliculi neurones.5 After the initial discovery of 5-HT4 receptors, it was rapidly recognized that gastrokinetic benzamides such as metoclopramide, cisapride and renzapride were agonists at this receptor in mouse embryo colliculi neurones and guinea-pig hippocampus.6–8 It is now well-established that 5-HT4 receptors are widely expressed in the body.9 In the periphery, 5-HT4 receptor activation has been shown to be involved in many responses in different organs such as the GI tract, the heart and the urinary bladder [for review, see Ref. (9)]. In the brain, the highest expression of 5-HT4 receptors is found in limbic structures, hippocampus and basal ganglia, which are anatomical structures linked to cognition. Consequently, 5-HT4 receptors have been implicated in a variety of pathological disorders and constitute a valuable target for the design of new drugs.

Many C-terminal splice variants of the 5-HT4 receptor have been described that all are positively coupled to cAMP. In addition, several splice variant-specific signal transduction cascades have been described.10 To date it is not realistic to imagine the design of selective ligands for a given isoform as the variations in the receptor sequence are located intracellularly. However, the concept of agonist-directed trafficking of the stimulus response might open up new avenues in this direction.

The 5-HT4 receptor as a target for prokinetic drugs

  1. Top of page
  2. Abstract
  3. Introduction
  4. The 5-HT4 receptor as a target for prokinetic drugs
  5. 5-HT4 receptor agonists
  6. Conclusion
  7. Acknowledgements
  8. References

The effect of 5-HT4 receptor activation in the GI tract has been studied extensively and varies by the region as well as the species under study. The involvement of 5-HT4 receptors in peristalsis in humans, rat, mouse and guinea-pig is well established. Mucosal stimulation induces release of 5-HT from EC cells, activating intrinsic primary afferent neurones (IPANs), which then release acetylcholine (ACh) and calcitonin gene-related peptide (CGRP). These neurotransmitters connect via interneurones to ascending excitatory (releasing ACh and/or tachykinins) and descending inhibitory (releasing nitric oxide (NO) and/or vasoactive intestinal peptide (VIP) and/or ATP) motor neurones, resulting in ascending contraction and descending relaxation. Radioligand binding and functional studies in the intestine suggest that 5-HT4 receptors are present in all segments of the human GI tract and are expressed on EC cells, IPANs, interneurones and efferent neurones of the myenteric plexus and smooth muscle cells, and their activation can thereby affect all components of the peristaltic reflex.3,11,12

In different species, including pig and humans, inhibitory 5-HT4 receptors are expressed on EC cells, thereby mediating autoregulation of 5-HT release.13 Activation of 5-HT4 receptors on IPANs might either initiate or strengthen the peristaltic reflex.3,14–18 On their turn, presynaptically located 5-HT4 receptors on myenteric interneurones can increase reflex activity.19 5-HT4 receptors are also present on the efferent limbs of the peristaltic reflex. Activation of these 5-HT4 receptors on efferent myenteric cholinergic excitatory neurones, leading to enhanced ACh release and hence increased contraction, is probably the predominant mechanism by which 5-HT4 receptor agonists affect GI motility. This has been shown in the human, porcine, canine, rat, murine and guinea-pig stomach,20–26 in guinea-pig ileum,27 and in the human, canine and guinea-pig colon.19,28,29 In addition, 5-HT4 receptors are expressed on non-adrenergic non-cholinergic (NANC) excitatory neurones in the guinea-pig19,30 and rat23 stomach and guinea-pig ileum31 and on inhibitory neurones in the murine stomach32 and guinea-pig and human colon.33,34 5-HT4 receptors causing relaxation are present on smooth muscle in the rat oesophageal muscularis mucosae,35 stomach36 and ileum37 and in canine and human large intestine.38,39 Besides these effects on the motor pathways, 5-HT4 receptor activation affects reflex activity that regulates intestinal secretion, thereby increasing HCO3 secretion in the duodenum of mouse and rat and in Cl secretion in the human and porcine small intestine and human and rat colon.40–48

This promiscuous expression profile of 5-HT4 receptors might contribute to regional and/or time-coordinated relaxation–contraction mechanisms, which are essential for GI function. For instance, in the canine colon, 5-HT4 receptor-mediated relaxation of the circular muscle is almost absent in the ascending colon, but increases towards the rectum.39 In addition, 5-HT4 receptors on cholinergic neurones mediate contractility in the proximal to middle, but not the distal, region of the canine large intestinal longitudinal muscle layer.28 These regional differences might provide a basis for a coordinated action in the colon upon stimulation with a 5-HT4 receptor agonist.

It is clear from the above that drugs that act as 5-HT4 receptor agonists are potent prokinetic agents.3,12,49 They have considerable therapeutic potential to treat patients with GI motility disorders, such as constipation, constipation-predominant irritable bowel syndrome (c-IBS), gastroparesis and gastro-oesophageal reflux disease (GORD).50,51 These disorders are both common and debilitating, and there is a need for safe and effective treatments. To meet this need, many efforts have been directed towards the development of 5-HT4 receptor agonists (Table 1). With the exception of SL65.0155 and PRX-03140, all these agonists were selected on the basis of their potential to treat GI motility disorders, highlighting the importance of this receptor in GI research. The clinical development of the older generation of compounds, which have poor 5-HT4 receptor selectivity, has been complicated by the occurrence of rare side effects. Although these adverse events were not 5-HT4 receptor-related, they have influenced the perception on the use of 5-HT4 receptor agonists in GI disorders and created confusion as regards the risk–benefit profile of 5-HT4 receptor agonists. However, the newer generation of selective 5-HT4 receptor agonists is devoid of off-target effects. Furthermore, we show in this review that 5-HT4 receptor agonists should be considered on an individual basis, based on the many 5-HT4 receptor-related differences.

Table 1.   Receptors at which gastrokinetic and enterokinetic agents, acting primarily at 5-HT4 receptors, interact at therapeutic doses
CompoundCompanyPhaseMode of action
5-HT45-HT35-HT2*5-HT1*D2
  1. Only compounds that entered clinical trials are included.

  2. +, agonist, −, antagonist.

  3. *See text for receptor subtypes.

  4. †Compound on the market.

  5. ‡Restricted availability.

  6. §M0003 = R149402; M0004 = R199715.

  7. ¶Mainly its main metabolite.

  8. **Preregistration.

Metoclopramide (PrimperanTM)Sanofi-AventisM†+  
CleboprideAlmirall ProdesfarmaM†+  
Cisapride (PrepulsidTM)Johnson & JohnsonM‡+  
MosaprideDainipponM†+−¶   
Tegaserod (ZelnormTM)NovartisM‡+ + 
RenzaprideAlizymeIII+   
E-3620EisaiII+   
Prucalopride (ResolorTM)MovetisIII**+    
Prx-03140Epix/GlaxoSmithKlineII+    
ATI-7505ARYx TherapeuticsII+    
SL65.0155Sanofi-SynthelaboII+    
M0003§MovetisII+    
TD-2749TheravanceI+    
TD-5108TheravanceI+    
AU-228 (-224)Abbott, Hokuriku SeiyakuI+    
M0004§MovetisI+    

5-HT4 receptor agonists

  1. Top of page
  2. Abstract
  3. Introduction
  4. The 5-HT4 receptor as a target for prokinetic drugs
  5. 5-HT4 receptor agonists
  6. Conclusion
  7. Acknowledgements
  8. References

An interesting characteristic of 5-HT4 receptors is their ability to be activated by a wide range of compounds from very different chemical classes.9 The first prokinetic benzamide, metoclopramide (Fig. 1), which was later found to be a ligand for both 5-HT3 and 5-HT4 receptors, played an important role in the discovery of potent and selective 5-HT4 receptor ligands available today and has been the progenitor of the benzamide class. It was characterized as a gastric prokinetic agent with dopamine (D2) receptor antagonistic properties (Table 1). Clebopride was designed by modifiying the amino chain of metoclopramide, resulting in a strengthening of the affinity for D2 dopamine receptors. Nevertheless, the introduction of other substitutions resulted in the discovery of potent gastrokinetic compounds devoid of antidopaminergic properties, with cisapride (Fig. 1) being the first representative of this family.9 New 5-HT4 receptor agonists were also designed departing from the structure of 5-HT, as 5-HT is a potent agonist at 5-HT4 receptors. An example is tegaserod (Fig. 1) which is an indole carbazimidamide derivative of 5-HT. Compared with 5-HT, tegaserod has a guanidine moiety rather than a protonated amine in the indole side chain.52 Prucalopride (Fig. 1) is the first representative of a new chemical class, the dihydrobenzofurancarboxamide compounds. Important differences between these 5-HT4 receptor agonists are highlighted in the following sections and we show that 5-HT4 receptor agonists should be considered on an individual basis, rather than generalizing agonist-specific observations.

image

Figure 1.  Chemical structure of 5-HT and the 5-HT4 receptor agonists metoclopramide, renzapride, cisapride, prucalopride and tegaserod.

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Differences unrelated to 5-HT4 receptors: selectivity

The selectivity of 5-HT4 receptor agonists has proven to be an important determinant of the benefit/risk profile and ultimately their clinical success. Non-selectivity at 5-HT4 receptors provides an explanation for most of the differences observed between the different 5-HT4 receptor agonists on the market (Fig. 2). Metoclopramide (PrimperanTM) was the first compound to be used in GI hypomotility disorders and has been used in clinical practice for many years. However, its lack of selectivity continues to limit its more widespread use. In the same period, cisapride (PrepulsidTM) was introduced for the treatment of symptoms of severe dyspepsia, gastroparesis, pseudo-obstruction, paediatric reflux and nocturnal heartburn. After its successful use for more than 10 years, rare reporting of cardiovascular side effects started to emerge, notably in patients with predisposing conditions like a history of cardiac disease or the simultaneous use with other medications that inhibit cisapride’s metabolism; it is now only available under specific conditions. Although these adverse events were not 5-HT4 receptor-related, they have influenced the perception on the use of 5-HT4 receptor agonists in GI disorders. This was further corroborated by a recent request of the Food and Drug Administration (FDA) to suspend marketing of another 5-HT4 receptor agonist, tegaserod (ZelnormTM), which was used for the management of c-IBS and constipation since 2004. Reviewing some of the clinical and postmarketing data, the FDA got concerned about the possible link between use of tegaserod and increased risk of ischaemic adverse events. Recently, the FDA has permitted restricted use of the drug. Metoclopramide, cisapride and tegaserod are not 5-HT4 receptor-selective and the interaction with multiple receptors may contribute to their effect profile.

image

Figure 2.  Selectivity of the 5-HT4 receptor receptor agonists cisapride, tegaserod and prucalopride. Binding affinities (pKi) were obtained from the literature and from screening experiments performed at J&J Pharmaceutical Research and Development. Results are shown for binding at human (h), mouse (m) or rat (r) receptors or transporters. Each circle represents a single experimental value; for filled circles, the receptor or transporter studied is indicated. pKi values for the 5-HT4 receptor that were obtained in competitive binding experiments with a radiolabelled antagonist are shown in red, while those obtained in experiments with a radiolabeled agonist are shown in green. Because the labeled antagonist does not differentiate between high and low affinity states of the receptor, affinity values obtained in these experiments slightly underestimates the agonist’s affinity. Therefore, the affinity of tegaserod for 5-HT4 receptors is expected to be a little higher than indicated. Discrepancies exist between affinity values of tegaserod at 5-HT2A, 5-HT2B and 5-HT2C receptors that are reported in the literature or obtained from J&J.

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Dopamine receptors  Metoclopramide has clear dopamine receptor antagonistic properties. This contributes to its favourable and widely accepted antiemetic function, as dopamine produces nausea and vomiting through stimulation of the medullary chemoreceptor trigger zone.53 Additionally, as dopamine slows gastric emptying, metoclopramide might relieve symptoms in patients suffering from upper GI motility disorders such as functional dyspepsia.54 On the other hand, the compound is not free of side effects. Metoclopramide induces prolactin release by its antagonism at dopamine receptors at the level of the pituitary gland.55 Additionally, certainly with high doses, blocking striatal dopamine receptors can result in extrapyramidal side effects56 which halted the widespread acceptance of the drug.

5-HT3 In addition to being a D2 receptor antagonist, metoclopramide is a 5-HT3 receptor antagonist.57 It shares this property with renzapride and mosapride and to a lesser extent with cisapride or its major metabolite. As chemotherapeutic agents are known to elicit abnormal 5-HT release in the gut, blocking 5-HT3 receptors on intestinal afferents can decrease emesis associated with chemotherapy. On the other hand, blocking 5-HT3 receptors also has clinical relevance as in chronic diarrhoea this blockade reduces intestinal contractility, slows colonic transit and increases fluid absorption.58 Indeed, the most common side effect observed with the 5-HT3 receptor antagonist alosetron is constipation. This may explain why for example cisapride and renzapride exert potent prokinetic effects in the upper GI tract but have less clear effects on colonic motility.12 There is, however, some concern about a possible association of 5-HT3 antagonism with ischaemic colitis, as observed with selective 5-HT3 receptor antagonists including alonsetron and cilansetron.59,60 A 5-HT4 receptor agonist that also antagonizes 5-HT3 receptors might thus be less applicable in constipation-associated disorders, but might provide an additional antinausea function.

hERG  The concern about the potential for ventricular arrhythmias when using 5-HT4 receptor agonists mainly originates from the ability of cisapride to block the cardiac human ether-a-go-go-related gene (hERG) potassium channel, with associated prolongation of the repolarization phase of the ventricular action potential and QT interval. The reported IC50 values for the blocking effect of cisapride on the hERG channel vary between 6.5 and 44.5 nmol L−1 61,62 with the exception of Potet et al.63 who reported an IC50 of 240 nmol L−1. For comparison, the affinities of renzapride, mosapride, tegaserod and prucalopride for the hERG channel all are in the micromolar range which makes this unfavourable interaction most pronounced for cisapride63 (Fig. 2). Cisapride displays little (<10-fold) selectivity for 5-HT4 receptors relative to its hERG channel affinity. This lack of selectivity likely underlies the rare cardiac side effects observed with cisapride. In patients with predisposing conditions (pre-existing cardiac disease, concomitant use of CYP3A4 inhibitors), cisapride therapy has been associated with cardiac disturbances, including QT prolongation, syncope and ventricular arrhythmia. In the class of 5-HT4 receptor agonists, this property is unique to cisapride.

5-HT1 Tegaserod, in addition to its affinity for 5-HT4 receptors, has affinity for 5-HT1A, 5-HT1B and 5-HT1D receptors (only 4- to 50-fold lower than for 5-HT4; Fig. 2),9,30,64 and thereby constitutes the only 5-HT4 receptor agonist with known high affinity for 5-HT1 receptors. Tegaserod is an agonist at 5-HT1B/D receptors [results of pilot (n = 4) in vitro studies with 5-HT1B-transfected HEK 293 cells: pEC50 = 6.2, maximal effect: 77% relative to the effect of the 5-HT1B/D agonist alniditan]. Its agonism at 5-HT1B/D receptors might be beneficial by affecting its prokinetic effects, because in humans this action has been shown to cause a relaxation of the gastric fundus, a delay of gastric emptying and a stimulation of interdigestive small intestinal motility.65 However, in the rat, 5-HT causes acute gastric mucosal injury probably due to the activation of 5-HT1D receptors.66 Activation of these receptors has also been shown to cause vasoconstriction.67 Recently tegaserod has been associated with a number of possible ischaemic cardiovascular adverse events, including rare cases of myocardial infarction, unstable angina and stroke, especially in patients with predisposing conditions.68 It is postulated that these effects may be related to vasoconstriction mediated by 5-HT1B receptors in the vascular wall. This is also observed with 5-HT and triptans, compounds with an indole nucleus similar to tegaserod.

5-HT2 Tegaserod is also a high-affinity 5-HT2A and 5-HT2B receptor antagonist69, a property it shares with cisapride, an antagonist at 5-HT2A receptors. In the human colon, 5-HT2B receptor activation by exogenous 5-HT increases colonic muscle contractions.69 In addition, animal studies have reported the contraction of gastric muscle through 5-HT2A and 5-HT2B receptors.3,70 Therefore, it is possible that the antagonistic properties at 5-HT2A/2B receptors influence their prokinetic activity. Antagonism at 5-HT2B receptors might also have contributed to the ischaemic cardiovascular events of tegaserod because endothelial 5-HT2B receptors mediate vasorelaxation.71,72 However, to the best of our knowledge, no ischaemic events have been reported with other clinically used compounds with 5-HT2B antagonistic properties.

Monoamine transporters  Tegaserod has recently been shown to inhibit the 5-HT, dopamine and noradrenaline transporters, serotonin transporter (SERT), dopamine transporter (DAT) and noradrenaline transporter (NAT), respectively, albeit with low potency.73 This is also reflected in the binding affinity of tegaserod at these transporters (Fig. 2). As tegaserod has a bioavailability of only 10% and approximately two-thirds of tegaserod is excreted unchanged in faeces, most of the drug is available to act locally on EC cells and local high concentrations of the drug might therefore be reached to inhibit SERT, which might add to the prokinetic action of tegaserod. An increase in the availability of 5-HT might, however, induce receptor desensitization, reducing the efficacy of tegaserod with chronic usage.

The emerging link between the benefit–risk profile of 5-HT4 receptor agonists and their selectivity resulted in the synthesis of selective agonists. Prucalopride, Prx-03140, ATI-7505, SL65.0155 and M0003 are the clinically most advanced compounds. The selectivity of these compounds for the 5-HT4 receptor differentiates them from other 5-HT4 receptor agonists. Prucalopride is the first selective high-affinity 5-HT4 receptor agonist. Affinity for other receptors was detected only at concentrations which exceed its Ki value for the 5-HT4 receptor with a factor of at least 200.30 The selectivity of prucalopride is also underscored by the observation in vivo that all its induced effects could be blocked by a 5-HT4 receptor antagonist.74 Therefore, prucalopride and other selective 5-HT4 receptor agonists may offer an attractive, effective and safe alternative for patients suffering from hypomotility disorders such as chronic constipation.

Differences related to 5-HT4 receptors

In vitro

Agonist–receptor interaction.  The structural features that define the 5-HT4 ligands are an aromatic moiety, a coplanar hydrogen acceptor group such as the carbonyl function, and a basic nitrogen atom. Despite these common features, it has been demonstrated that triptamines (5-HT, tegaserod) and benzamides (metoclopramide, renzapride, cisapride) have different pharmacophores.52,75 The mutation of the conserved Asp100 generates a 5-HT4 receptor activated solely by synthetic ligands (RASSL). The 5-HT4 RASSL is insensitive to 5-HT and tegaserod, which can be explained by the lack of binding of these compounds to the mutant receptor.76 The 5-HT4 receptor antagonist GR113808 is still able to bind to the mutant receptor, which can also be stimulated by numerous synthetic agonists from different chemical classes, including the benzamides metoclopramide, renzapride and cisapride. These compounds all have a bulky substitute of the basic nitrogen, most of them with the basic nitrogen included in a structured ring (Fig. 1), necessary to interact in a hydrophobic pocket of the receptor.77 In contrast, the highly conserved Asp100 is known to make an ionic bond with the protonated amine of 5-HT derivatives. Taken together, this suggests that tegaserod on the one hand and the benzamides and prucalopride on the other hand, use different binding pockets on the 5-HT4 receptor.

It is becoming increasingly recognized that seven-transmembrane receptors are allosteric proteins that can adopt numerous conformations which can activate different transduction pathways by agonist-directed stimulus trafficking (ligand-directed signalling).78,79 Therefore, a linear view of efficacy whereby a single receptor activation state (two-state model, where the receptor is either on or off) triggers all possible receptor interactions with a cell is no longer supported. The existence of different receptor conformations has been shown for constitutively active mutants of the 5-HT4 receptor80 and for the full, partial and inverse agonist-liganded 5-HT4 receptor.81,82 In addition, the efficacy of a partial 5-HT4 receptor agonist is likely due to the stabilization of an agonist-specific conformational state.82 The efficacy of a certain agonist is then defined as the ability to stabilize these specific conformations. This also implies that some 5-HT4 receptor agonists might only partially activate the library of potential signalling systems in a cell. Indeed, in HEK293 cells, 5-HT4(a) receptor activation triggers a strong [Ca2+]i increase that is dependent on the compound structure because benzamide-related compounds trigger higher responses than indoleamines.83

The existence of many C-terminal splice variants of the 5-HT4 receptor adds even further complexity. Several splice variant-specific alternative signal transduction cascades have been described in recombinant expression systems.83–87 The expression of different splice variants in different tissues (or species) might therefore contribute to differences in overall coupling in the different tissues. These variant and therefore tissue- or species-dependent differences in coupling are expected to influence the response of all agonists unless agonist-directed trafficking of the stimulus response occurs. Different agonists may then be able to activate different alternative signalling cascades through the different splice variants. In support of this, the calcium response observed with benzamidic compounds seems to be splice variant-specific, being only observed in 5-HT4(a) and not in 5-HT4(b).83

Agonist-induced effects.  Quite often, prucalopride is referred to as a full agonist while tegaserod is regarded as a partial agonist [see for instance, Refs (88,89)]. This clearly is an oversimplification and one really should avoid extrapolating the degree of agonism of a substance in a given tissue. For a start, there is the inherent difficulty in defining what a full agonist is.90 For reasons of simplicity we refer to full agonism when the agonists elicit the same maximal response as 5-HT, the endogenous ligand of the 5-HT4 receptor. When using this criterium in the literature, it is clear that both prucalopride and tegaserod have been reported as either full or partial agonist [see, e.g. Refs (21,39,69)].

These differences may originate from different grounds. First, a given agonist can behave as a full agonist in certain tissues, while it can express partial agonism in others. Indeed, the expression of partial vs full agonism by a drug largely depends on the system in which it is evaluated as the receptor density or coupling efficiency can differ. Some agonists will thus behave as partial agonists in low efficacy systems, while they behave as full agonists in high efficacy systems (e.g. with high receptor reserve). Therefore, based on differences in receptor density or coupling efficiency (which might be influenced by the nature of the splice variants), the degree of agonism of a given agonist in a given tissue can vary between species. Even within a single species, these properties can lead to a differential degree of agonism for a given substance between tissues.21 This may explain the many differences in benzamide-induced 5-HT4 receptor-mediated responses between different tissues or species.9

One example of a low efficacy system is the heart, which is reflected in the relatively low stimulation potency of 5-HT4 receptor agonists in this tissue. Because of its nature of low efficacy, this tissue provides a useful method for evaluating differences in the pharmacological profile of 5-HT4 receptor ligands. Substituted benzamides like renzapride, cisapride and metoclopramide or dihydrobenzofurancarboxamides like prucalopride, M0003 and M0004 behave as partial agonists at porcine sinoatrial and/or human right atrial 5-HT4 receptors91–93 while most tryptamines are full agonists compared with 5-HT.91,92 Prucalopride also acted as a partial agonist on the L-type Ca2+ current through 5-HT4 receptors in human atrial cells.94 Therefore, different organ-specific behaviour can be expected between these classes of compounds. Recently a porcine in vitro model has been developed to describe and explain organ-specific behaviour of 5-HT4 receptor agonists.21 In this model, the effects of 5-HT4 receptor agonists in a gastric assay (5-HT4 receptors on cholinergic neurones) were compared with their effects on the right and left atrium (low 5-HT4 receptor density 95). The model quantified the tissue-dependent partial agonism of prucalopride, M0003 and M0004 with prucalopride behaving as a full agonist in the gastric assay, and as a partial agonist in the left and right atrium whereas tegaserod was shown to be a full agonist in all assays.21 Tissue-selective expression of partial agonism might thus result in organ selectivity of a certain drug, as illustrated for prucalopride above. Interestingly, because of their low efficacy, benzamides and dihydrobenzofurancarboxamides actually antagonize the effects of exogenously administered 5-HT, as demonstrated for prucalopride, cisapride, renzapride and metoclopramide in human and/or porcine right atrium.91,93,96 A similar antagonizing and possibly protective behaviour might also occur vs endogenously released 5-HT as demonstrated in a porcine in vivo model in which renzapride reduced the 5-HT-induced tachycardia.97

Secondly, the situation is further complicated by the concept of agonist-directed trafficking of receptor signalling. This hampers the interpretation and extrapolation of in vitro tissue experiments since different agonists might couple to different effectors (which might be even more complicated by the differential expression of splice variants between tissues or species, see above) and consequently, the order of efficacy between agonists might be reversed in different tissues (or species). Several examples of reversed efficacy order between 5-HT4 receptor agonists can be found in the literature. In many systems where both agonists were tested, prucalopride has been described as the agonist with a lower efficacy compared with tegaserod,15,21,92,98 while in others prucalopride is classified as a full and tegaserod as a partial agonist.34,39 Interestingly, at the guinea-pig 5-HT4 receptor expressed in HEK293 cells, tegaserod behaved as a full agonist and prucalopride as a partial agonist. This order was reversed in the guinea-pig isolated colon longitudinal muscle myenteric plexus preparation (LMMPP).99 In addition, compared to 5-HT, cisapride has been shown to behave as a superagonist in mouse colliculi neurones, while in most GI preparations it is a partial agonist.9 Furthermore, the effect of prucalopride on substance P release in the guinea-pig colon (NK1-mediated rebound contraction) has not been observed with cisapride (30, personal observation). Although speculative, all these observations might result from the stabilization of a different conformational state of the 5-HT4 receptor by the different agonists.

Taken together, based on mixed drug-specific and tissue-dependent properties (e.g. the expression of partial agonism or agonist-specific signal transduction pathways), different agonists might differentially activate the multiple 5-HT4 receptor populations in the GI tract, thereby resulting in distinct overall effects in different tissues.

The tissue selectivity of agonists could also be affected by the degree of receptor desensitization. It is well established that 5-HT4 receptors can desensitize upon stimulation by an agonist as shown in recombinant expression systems, but also with native neuronal and non-neuronal receptors.100,101 It is often generalized that a partial agonist is less likely to induce desensitization.102 This general paradigm that the activity of an agonist and its ability to cause desensitization are coupled is based on studies on β2-adrenergic and muscarinic receptors demonstrating that partial agonists cause less desensitization than full agonists.103 However, for several 5-HT4 receptor agonists, the desensitization power was shown to be a function of the activation potency of the drugs rather than of their efficacy (partial agonistic character).100 The dependence on potency and not on efficacy reflects the influence of the agonists’ binding affinity. This is underscored by the fact that antagonists can also induce desensitization104 which even challenges the involvement of G protein activation and second messenger generation in the triggering of the desensitization. Indeed, for the motilin receptor it has been shown that desensitization can be decoupled from activation and desensitization is not only determined by potency.105 The existence of agonist-specific receptor conformations (see above), each unfolding and exposing different regions to the intracellular apparatus and each associated with specific function may render different desensitizing properties to two agonists despite similar activation potencies. Furthermore, since the desensitization process depends on the cellular environment of the 5-HT4 receptor,106 different agonists might induce different degrees of desensitization in different tissues, resulting in agonist-specific tissue selectivity; this also complicates extrapolation of data obtained in recombinant receptor systems. Above that, there are also splice variant-specific differences in desensitization.107,108 As shown in heterologous expression systems, 5-HT4(d) and 5-HT4(b) desensitize more rapidly than do 5-HT4(e) and 5-HT4(a). Therefore, different organ systems might exert different degrees of desensitization after stimulation with the same concentration of agonist if they express different 5-HT4 receptor splice variants. Differential effects on desensitization may have important therapeutic implications because desensitization can limit the biological effects of 5-HT4 agonists in vivo.

Differences with 5-HT at cardiac 5-HT4 receptors.  The QT prolonging effect of cisapride (see above) together with the cardiac location of 5-HT4 receptors did establish an atmosphere of confusion on the cardiac effects of 5-HT4 receptor agonists. Some authors still associate the cardiac effects of cisapride with cardiac 5-HT4 receptors.109 This is, however, completely wrong. 5-HT4 receptors are expressed in human atria and ventricles, albeit at very low densities.110 Therefore, the effects of many 5-HT4 receptor agonists (benzamides and dihydrobenzofurancarboxamides; see above) in the heart are particularly modest compared to 5-HT. These effects include an increase in contractile force (inotropic effect), a hastening of the onset of muscle relaxation (lusitropic effect) or for sinoatrial receptors an increase in beating rate (chronotropic effect). The very low efficacy compared with 5-HT is particularly relevant for the hypothetical association between 5-HT4 receptor activation and atrial arrhythmias, for which no evidence was found using renzapride and prucalopride96,111 but only using 5-HT at high concentrations and in specific in vitro conditions or preconditioned tissues. Prior treatment with ß-adrenoreceptor antagonists enhances the inotropic and arrhythmogenic potential of 5-HT by increasing the maximal response of ICaL to 5-HT112 without affecting expression levels of 5-HT4 receptors or L-type Ca2+ channel subunits.113 Interestingly, even in cells from β-adrenoreceptor antagonist-treated patients, prucalopride was devoid of any effect on arrhythmic activity.94 Proarrhythmic effects of 5-HT have been suggested to be related to Ca2+ overload. Prucalopride induces a smaller increase in L-type Ca2+ current compared with 5-HT and it would therefore appear unlikely that prucalopride could elicit atrial arrhythmias in patients with constipation.94,96 Prucalopride would actually block cardiac 5-HT4 receptors (see above), thereby even preventing arrhythmogenic effects of 5-HT.96

Cardiac 5-HT4 receptor-mediated responses quickly fade because of the action of phosphodiesterases (PDEs) which catalyse cAMP breakdown. Their inhibition is often needed to unmask the effects of 5-HT4 receptor agonists and 5-HT, as illustrated for the left atrium and the ventricles respectively.92,114 In addition to quenching of the 5-HT4 receptor-mediated effect, PDEs play a role in the compartmentation and targeting of the cAMP signal in the myocytes. Because of this mechanism, activation of different Gs-coupled receptors (e.g. β-adrenoreceptors and 5-HT4 receptors), both increasing cAMP levels, may lead to different effects on cardiac function. Because PDE inhibition greatly reduces cardiac compartmentation, the inotropic effect of the 5-HT4 receptor agonists in the presence of PDE inhibitors could be a direct consequence of the inhibition of PDE action (less cAMP breakdown) and/or, as compartmentation is lost, result from the more diffuse activation of cAMP substrates. Therefore, the physiological relevance of the experimental 5-HT4 receptor-mediated responses in the presence of a PDE inhibitor is questionable.

The large species-dependent heterogeneity in 5-HT receptor subtypes expressed in the heart limits the options to study all of this in a laboratory animal. Only pigs and monkeys have functional cardiac 5-HT4 receptors under physiological conditions. In addition, 5-HT4 receptor mRNA was detected in rat hearts, but a functional inotropic response only manifested after its upregulation following the induction of post-infarction congestive heart failure.115,116 Up as well as down regulation of 5-HT4 receptor mRNA expression levels associated with cardiac dysfunction has been demonstrated in human hearts.114,117

In vivo

Based on the arguments described above, in vivo differences between 5-HT4 receptor agonists can be expected. However, the complexity of the underlying mechanisms hampers a clear-cut interpretation. Some degree of tissue-specificity has been described for cisapride, mosapride and prucalopride. Mosapride selectively enhanced upper GI motility in conscious dogs.118 Cisapride accelerates gastric emptying and facilitates gastric accommodation, but has limited effects on colonic motility in humans.119,120 In contrast, prucalopride has been suggested to selectively stimulate colonic transit in healthy humans without altering gastric emptying or small bowel transit.121 However, the effect profile of cisapride might be influenced by 5-HT4 receptor-unrelated mechanisms (see above).

The in vivo effect of prucalopride in the canine colon supports a regional coordinated relaxation–contraction mechanism upon stimulation of 5-HT4 receptors.74 Prucalopride stimulates GI motility with a pronounced effect on the large bowel. In conscious dogs, it induces a pattern of enhanced motility in the proximal colon and reduced motility in the distal colon, facilitating propulsion of luminal contents. In addition, it clearly induces propulsive waves of contractions (colonic giant migrating contractions) starting in the proximal colon and progressing all the way to the anal sphincters, i.e. the canine equivalent of human large bowel mass movements.74 This typical ‘profile’ has not been observed with cisapride and tegaserod (Schuurkes, JAJ). Although tegaserod stimulates colonic transit, it does not induce giant migrating contractions in the canine colon.122

The physicochemical properties of the agonists might also underlie certain differences observed in vivo. It has been suggested that tegaserod exerts prokinetic effects in the lumen directly because it is poorly absorbed and is largely excreted unmetabolized in stool.12 Tonini123 suggested that antagonism at the inhibitory 5-HT4 autoreceptors on EC cells by ‘the partial 5-HT4 receptor agonist’ tegaserod might lead to enhanced release of 5-HT from EC cells which might contribute to the prokinetic effect of tegaserod.123 When higher concentrations of tegaserod diffuse into the intestinal wall, also the 5-HT4 receptors on the IPANS can be antagonized by the drug, decreasing the prokinetic effect. However, this hypothesis only holds if tegaserod is indeed a partial agonist at the 5-HT4 receptor in these two cell types but the experimental data are lacking to support this hypothesis and as discussed above, tegaserod has been shown to behave as a full agonist in several studies.

Conclusion

  1. Top of page
  2. Abstract
  3. Introduction
  4. The 5-HT4 receptor as a target for prokinetic drugs
  5. 5-HT4 receptor agonists
  6. Conclusion
  7. Acknowledgements
  8. References

5-HT4 receptor agonists have clear-cut prokinetic effects in the gut. These agonists differ in many aspects that are either related or unrelated to their interaction with 5-HT4 receptors. Differences that are unrelated to the 5-HT4 receptor, such as affinity at non-5-HT4 receptors, may influence the agent’s safety and overall benefit/risk profile. 5-HT4 receptor-related differences have an impact on the agonist’s overall activity in a given tissue. Together, these differences affect the therapeutic potential for the treatment of GI motility disorders. Based on available evidence, a highly selective 5-HT4 receptor agonist, such as prucalopride, may offer improved efficacy and safety to treat patients with impaired GI motility, such as severe chronic constipation.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Introduction
  4. The 5-HT4 receptor as a target for prokinetic drugs
  5. 5-HT4 receptor agonists
  6. Conclusion
  7. Acknowledgements
  8. References

The authors are grateful to Luc Ver Donck and Lieve Heylen from Johnson & Johnson Pharmaceutical Research and Development (Beerse, Belgium) for the provision of the cisapride and tegaserod binding data.

Conflict of interests: The authors have no competing interests.

References

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  2. Abstract
  3. Introduction
  4. The 5-HT4 receptor as a target for prokinetic drugs
  5. 5-HT4 receptor agonists
  6. Conclusion
  7. Acknowledgements
  8. References
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