Epithelial 5-HT4 Receptors as a Target for Treating Constipation and Intestinal Inflammation.

Because of their importance in the regulation of gut functions, several therapeutic targets involving serotonin-related proteins have been developed or repurposed to treat motility disorders, including serotonin transporter inhibitors, tryptophan hydroxylase blockers, 5-HT3 antagonists, and 5-HT4 agonists. This chapter focuses on our discovery of 5-HT4 receptors in the epithelial cells of the colon and our efforts to evaluate the effects of stimulating these receptors. 5-HT4 receptors appear to be expressed by all epithelial cells in the mouse colon, based on expression of a reporter gene driven by the 5-HT4 receptor promoter. Application of 5-HT4 agonists to the mucosal surface causes serotonin release from enterochromaffin cells, mucus secretion from goblet cells, and chloride secretion from enterocytes. Luminal administration of 5-HT4 agonists speeds up colonic motility and suppresses distention-induced nociceptive responses. Luminal administration of 5-HT4 agonists also decreases the development of, and improves recovery from, experimental colitis. Recent studies determined that the prokinetic actions of minimally absorbable 5-HT4 agonists are just as effective as absorbable compounds. Collectively, these findings indicate that targeting epithelial receptors with non-absorbable 5-HT4 agonists could offer a safe and effective strategy for treating constipation and colitis.

Colonic 5-HT4 receptors are targets for novel prokinetic drugs.

5-HT4 receptors are G protein-coupled receptors that link to the stimulatory protein Gs which activates adenylate cyclase to increase intracellular cyclic AMP which then activates protein kinase A (PKA). 5-HT4 receptors are expressed by neurons in the central and peripheral nervous systems especially the enteric nervous system (ENS). In general, 5-HT4 receptors are stimulatory and their activation in the ENS enhances neurotransmitter release and propulsive motility patterns. 5-HT4 receptors are expressed by enterochromaffin (EC) cells, Goblet cells, and most enteric neurons. The study by Konen and colleagues in this issue of Neurogastroenterology and Motility features two novel 5-HT4 receptor agonists (5-HT4 -LA1 and 5-HT4 -LA-2) that are not absorbed from the gastrointestinal tract of mice and act locally in the colonic mucosa to stimulate propulsive motility. The authors show that 5-HT4 -LA1 and 5-HT4 -LA2 were not absorbed from the colon and that both drugs stimulated colonic transit when administered by gavage. Both agonists stimulated colonic glass bead expulsion, and 5-HT4 LA1 activation stimulated fecal output and increased fecal water content. These effects were detected in young and aged mice. 5-HT4 receptors were also localized to the epithelium of the human duodenum, ileum, and colon. These studies highlight novel 5-HT4 receptor agonists that have prokinetic actions on the GI tract. These drugs are not absorbed and act locally in the gut mucosa to stimulate propulsive motility while minimizing access to systemic 5-HT4 receptors and avoiding potential unwanted side effects.

Serotonin enhances depolarizing spontaneous fluctuations, excitability, and ongoing activity in isolated rat DRG neurons via 5-HT4 receptors and cAMP-dependent mechanisms.

Ongoing activity in nociceptors, a driver of spontaneous pain, can be generated in dorsal root ganglion neurons in the absence of sensory generator potentials if one or more of three neurophysiological alterations occur - prolonged depolarization of resting membrane potential (RMP), hyperpolarization of action potential (AP) threshold, and/or increased amplitude of depolarizing spontaneous fluctuations of membrane potential (DSFs) to bridge the gap between RMP and AP threshold. Previous work showed that acute, sustained exposure to serotonin (5-HT) hyperpolarized AP threshold and potentiated DSFs, leading to ongoing activity if a separate source of maintained depolarization was present. Cellular signaling pathways that increase DSF amplitude and promote ongoing activity acutely in nociceptors are not known for any neuromodulator. Here, isolated DRG neurons from male rats were used to define the pathway by which low concentrations of 5-HT enhance DSFs, hyperpolarize AP threshold, and promote ongoing activity. A selective 5-HT4 receptor antagonist blocked these 5-HT-induced hyperexcitable effects, while a selective 5-HT4 agonist mimicked the effects of 5-HT. Inhibition of cAMP effectors, protein kinase A (PKA) and exchange protein activated by cAMP (EPAC), attenuated 5-HT's hyperexcitable effects, but a blocker of hyperpolarization-activated cyclic nucleotide-gated (HCN) channels had no significant effect. 5-HT4-dependent PKA activation was specific to DRG neurons that bind isolectin B4 (a nonpeptidergic nociceptor marker). 5-HT's effects on AP threshold, DSFs, and ongoing activity were mimicked by a cAMP analog. Sustained exposure to 5-HT promotes ongoing activity in nonpeptidergic nociceptors through the Gs-coupled 5-HT4 receptor and downstream cAMP signaling involving both PKA and EPAC.

The novel, potent and highly selective 5-HT4 receptor agonist YH12852 significantly improves both upper and lower gastrointestinal motility.

BACKGROUND AND PURPOSE: 5-HT4 receptor agonists have been shown to be effective at treating various gastrointestinal tract disorders. However, a lack of selectivity against off-targets has been a limiting factor for their clinical use. EXPERIMENTAL APPROACH: The binding affinity and selectivity of YH12852 for human 5-HT4(a) receptor in CHO-K1 cells were evaluated using radioligand binding assays, and agonistic activity was assessed using a ß-lactamase reporter system. Contractile activity and propulsive motility were measured in the guinea pig isolated distal colon. Its prokinetic effect on the gastrointestinal tract was evaluated in guinea pigs, dogs and monkeys. Its tissue distribution was evaluated in rats. KEY RESULTS: YH12852 exhibited high affinity and potency for human recombinant 5-HT4(a) receptor with high selectivity over other 5-HT and non-5-HT receptors, ion channels, enzymes and transporters. YH12852 induced contractions and increased propulsive motility in guinea pig isolated colon. These effects were abolished by the 5-HT4 receptor antagonist GR113808. YH12852 increased defecation more effectively than prucalopride in guinea pigs and dogs and improved gastric emptying more effectively than mosapride in guinea pigs, dogs and monkeys. YH12852 was highly distributed to the gastrointestinal tract as the target organ. CONCLUSION AND IMPLICATIONS: The high in vitro potency and selectivity of YH12852 for 5-HT4 receptor translated into potent in vivo efficacy with good tolerability. YH12852 significantly improved both upper and lower bowel motility in the animal models tested and has the potential to address considerable unmet needs in patients with functional constipation, gastroparesis or both.

Desensitization of the human 5-HT4 receptor in isolated atria of transgenic mice.

In the human cardiovascular system, serotonin (5-HT) exerts positive inotropic and chronotropic effects mediated by 5-HT4 receptors. Moreover, 5-HT4 receptor stimulation can cause arrhythmias in the human heart. Response to 5-HT can fade due to desensitization of the receptor system and/or activation of phosphodiesterases. In this study, we investigated a potential desensitization of the human 5-HT4(a) receptor expressed in the mouse heart. Therefore, we have used atrial preparations of transgenic (TG) mice with cardiac myocyte-specific overexpression of the human 5-HT4(a) receptor and their non-transgenic littermates (WT). Homologous (by 5-HT) and potentially heterologous (by isoprenaline) desensitization of the 5-HT4 receptor was investigated in atria of TG mice. 5-HT increased force of contraction in isolated electrically paced left atria and beating rate in spontaneously beating right atria only in preparations from TG but not from WT. Pre-treatment of isolated atria with high concentrations (10-600 µM) of 5-HT for 60 min attenuated the positive inotropic effects and the positive chronotropic effects of 5-HT in TG atria. Several inhibitors of desensitization including Zn2+, sucrose, and paroxetine were tested. Whereas sucrose was without any effect and Zn2+ only was partially effective, paroxetine was able to inhibit desensitization favoring at least in part a G-protein receptor-coupled kinase-mediated mechanism of 5-HT4 receptor desensitization in the TG mouse heart. In addition, desensitization of ventricular 5-HT4 receptors was noted in isolated perfused hearts (Langendorff preparations) from TG mice. In summary, we show homologous desensitization of the 5-HT4 receptor in the heart of a transgenic mouse model possibly dependent on active G-protein receptor-coupled kinase. The exact mechanism and a potentially heterologous desensitization have to be elucidated by further investigations.

Metabolism of a serotonin-4 receptor partial agonist 4-{4-[4-tetrahydrofuran-3-yloxy)-benzo[d]isoxazol-3-yloxymethyl]-piperidin-1-ylmethyl}-tetrahydropyran-4-ol (TBPT): identification of an unusual pharmacologically active cyclized oxazolidine metabolite in human.

4-{4-[4-Tetrahydrofuran-3-yloxy)-benzo[d]isoxazol-3-yloxymethyl]-piperidin-1-ylmethyl}-tetrahydropyran-4-ol (PF-4995274, TBPT) is a new agent that is a partial agonist of the human serotonin-4 (5-HT4) receptor and is under investigation for neurological disorders. Metabolism of TBPT was examined in vitro in human liver microsomes and human hepatocytes. Metabolites were also identified in the plasma of healthy human subjects in a phase 1 clinical study. Human-derived metabolite profiles were compared with corresponding profiles obtained in laboratory animal species. There were two major routes of metabolism in vitro: N-dealkylation of the methyltetrahydropyran moiety (M1) and hydroxylation at the seven position of the benzisoxazole moiety (M4). These were also observed in human plasma; however, in that matrix, the major metabolite was an unusual cyclized oxazolidine entity (M2). M2 was proposed to be formed via generation of an intermediate 4° iminium ion on the piperidine ring followed by spontaneous cyclization by attack of the ß-hydroxyl substituent of the tetrahydropyran ring to form a cyclized oxazolidine product. An authentic standard of the metabolite was generated using a methylene-blue-sensitized photochemical oxidation reaction as well as microbial transformation. Further investigation of this metabolite showed that it also possessed 5-HT4 agonism activity similar to the parent. The metabolite was 150-fold more highly protein bound in human plasma than TBPT, which is consistent with its presence as a major circulating metabolite while being only a minor metabolite in in vitro systems. Overall, this illustrates the importance of understanding the complex dispositional properties of a pharmacologically active metabolite.

Effects of mosapride citrate, a 5-HT4-receptor agonist, on gastric distension-induced visceromotor response in conscious rats.

Mosapride citrate (mosapride), a prokinetic agent with 5-HT(4)-receptor agonistic activity, is known to enhance gastric emptying and alleviate symptoms in patients with functional dyspepsia (FD). As hyperalgesia and delayed gastric emptying play an important role in the pathogenesis of FD, we used in this study balloon gastric distension to enable abdominal muscle contractions and characterized the visceromotor response (VMR) to such distension in conscious rats. We also investigated the effects of mosapride on gastric distension-induced VMR in the same model. Mosapride (3-10 mg/kg, p.o.) dose-dependently inhibited gastric distension-induced VMR in rats. However, itopride even at 100 mg/kg failed to inhibit gastric distension-induced VMR in rats. Additionally, a major metabolite M1 of mosapride, which possesses 5-HT(3)-receptor antagonistic activity, inhibited gastric distension-induced VMR. The inhibitory effect of mosapride on gastric distension-induced visceral pain was partially, but significantly inhibited by SB-207266, a selective 5-HT(4)-receptor antagonist. This study shows that mosapride inhibits gastric distension-induced VMR in conscious rats. The inhibitory effect of mosapride is mediated via activation of 5-HT(4) receptors and blockage of 5-HT(3) receptors by a mosapride metabolite. This finding indicates that mosapride may be useful in alleviating FD-associated gastrointestinal symptoms via increase in pain threshold.