Secretory Diarrhoea: Mechanisms and Emerging Therapies Jay R

REVIEWS

Secretory diarrhoea: mechanisms and emerging therapies Jay R. Thiagarajah, Mark Donowitz and Alan S. Verkman

Abstract | Diarrhoeal disease remains a major health burden worldwide. Secretory diarrhoeas are caused by certain bacterial and viral infections, inflammatory processes, drugs and genetic disorders. Fluid secretion across the intestinal epithelium in secretory diarrhoeas involves multiple ion and solute transporters, as well as activation of cyclic nucleotide and Ca2+ signalling pathways. In many secretory diarrhoeas, activation of Cl– channels in the apical membrane of enterocytes, including the cystic fibrosis transmembrane conductance regulator and Ca2+-activated Cl– channels, increases fluid secretion, while inhibition of Na+ transport reduces fluid absorption. Current treatment of diarrhoea includes replacement of fluid and electrolyte losses using oral rehydration solutions, and drugs targeting intestinal motility or fluid secretion. Therapeutics in the development pipeline target intestinal ion channels and transporters, regulatory proteins and cell surface receptors. This Review describes pathogenic mechanisms of secretory diarrhoea, current and emerging therapeutics, and the challenges in developing antidiarrhoeal therapeutics

Thiagarajah J. R. et al. Nat. Rev. Gastroenterol. Hepatol. advance online publication 30 June 2015; doi:10.1038/nrgastro.2015.111

Introduction Diarrhoeal diseases have been a major health problem such as Shigella and Salmonella; and parasites, such as throughout history.1 In the past, diarrhoeal diseases Entamoeba histolytica and Cryptosporidium parvum.5 were often fatal and disease outbreaks spread quickly, The most common cause of severe diarrhoea worldwide affecting large populations. Today, despite the success is rotavirus (28% of diarrhoeal episodes), with V. cholerae of interventions such as oral and intravenous rehydra- producing at least 1–2% of cases.6 Severe diarrhoea out- tion therapy, secretory diarrhoea remains a substantial breaks that rapidly affect large populations are often cause of mortality and morbidity worldwide, particularly associated with complex humanitarian emergencies such in children and the elderly. In 2015, it is estimated that as the displacement of people into refugee camps, natural worldwide 577,000 children aged <5 years and 502,000 disasters such as earthquakes, and armed conflict, Division of 2 Gastroenterology, adults aged >70 years will die from diarrhoeal diseases. leading to the loss of health and sanitation infrastructure. Hepatology and For these vulnerable populations, the mortality risk due Examples include the refugee crisis in Rwanda in 1994, Nutrition, Children’s Hospital Boston, to diarrhoeal disease is often further increased by asso- the conflict in Zimbabwe in 2008, and the earthquake Harvard Medical ciated risk factors such as malnutrition and pre-existing in Haiti in 2010. School, 300 Longwood enteric infections.3 In addition to the mortality risk, Many noninfectious causes of diarrhoea are promi- Avenue, Boston, MA 02115, USA repeated diarrhoeal episodes are associated with long- nent in developed countries. Diarrhoea is associated (J.R.T.). Departments term impairment of physical and mental development, with adverse effects of drugs, particularly certain cancer of Physiology and 7,8 Medicine, Division with an estimated global loss of ~1,400 years of healthy and HIV therapeutics. Up to 28% of patients with 2,4 of Gastroenterology, life due to disability per 100,000 population. HIV treated with protease inhibitors report more than Johns Hopkins The prevalence of diarrhoeal disease, as for other four loose or watery stools per day.7 Intestinal inflam- University School of Medicine, Ross 925, important global causes of childhood mortality such matory and autoimmune conditions, such as ulcerative 720 Rutland Avenue, as pneumonia and malaria, is correlated closely with colitis, Crohn’s disease and coeliac disease, can have a Baltimore, MD 21205, 9,10 USA (M.D.). climate and economic development. The most severely substantial diarrhoeal component. IBS is prevalent in Departments of affected regions include developing countries in sub- developed countries; 10–20% of the adult population Medicine and Saharan Africa and South Asia.2 The major causes of in the US are estimated to have IBS, of which around Physiology, 1246 11 Health Sciences diarrhoeal diseases in developing countries are infec- one-third suffer from chronic diarrhoea. Severe secre- East Tower, University tious, including enterotoxin-producing bacteria, such tory diarrhoea is also caused by rare congenital dis of California, 500 Parnassus Avenue, as Vibrio cholerae and enterotoxigenic Escherichia coli; orders, such as microvillus inclusion disease, familial San Francisco, viruses, such as rotavirus; enteroinvasive bacteria, diarrhoea syndrome and tufting enteropathy, as well CA 94143, USA 12–15 (A.S.V.). as by peptide-secreting neuroendocrine tumours. However, infectious causes of diarrhoea still represent Competing interests Correspondence to: a large proportion of the disease burden in developed A.S.V. A.S.V. is a named inventor on several CFTR and CaCC inhibitor alan.verkman@ patents owned by the University of California, San Francisco. countries. The incidence of diarrhoea caused by rota- ucsf.edu J.R.T. and M.D. declare no competing interests. viruses has fallen dramatically over the past 5 years

Figure 1 | Mechanisms of intestinal fluid absorption Key points ▶ and secretion in secretory diarrhoeas. a | Luminal and ■■ Diarrhoeal disease remains a major global health burden basolateral membrane transporters and intracellular ■■ Secretory diarrhoea results from abnormal fluid and electrolyte absorption signalling mechanisms are involved in intestinal fluid and/or secretion absorption and secretion by enterocytes. b | Some bacteria ■■ Ion channels and transporters such as the cystic fibrosis transmembrane secrete enterotoxins that increase intracellular cyclic conductance regulator and sodium/hydrogen exchanger 3 are targets for nucleotides, resulting in Cl– secretion and inhibition of antisecretory antidiarrhoeal drugs NHE3 and Na+ absorption. Invasive bacteria cause a tissue ■■ New compounds targeting intestinal transporters are in development for inflammatory response involving recruitment of immune antidiarrhoeal therapy, including small molecules, natural compounds and cells and release of cytokines, resulting in intracellular existing drugs Ca2+ signalling. c | The rotaviral protein NSP4 causes ■■ Antidiarrhoeal therapies might be useful as stand-alone therapy or together with elevation of cytoplasmic Ca2+ concentration by binding to oral rehydration solutions integrin-α1β2, galanin and/or by activation of enteric nerves. Rotaviral NSP4 also inhibits NHE3 and SLC5A1. d | Congenital diarrhoeas result from rare inherited with the widespread administration of the rotavirus genetic mutations in several intestinal proteins. vaccine, although the incidence of diarrhoea caused e | Activation of inflammatory signalling pathways such by noroviruses has increased and become the leading as NF‑κB result in Ca2+ or cyclic nucleotide signalling and cause of disease outbreaks from contaminated food in stimulation of Cl– secretion or inhibition of Na+ absorption. the US.16 The main bacterial causes of food-related diar- The release of inflammatory mediators such as TNF and IL‑6 by activated T cells and neutrophils can also stimulate rhoeal disease in the US are Salmonella enterica (19,000 Cl– secretion. Abbreviations: CaCC, calcium-activated hospitalizations per year) and Campylobacter jejuni chloride channel; CaSR, calcium-sensing receptor; CFTR, 16 (8,000 hospitalizations per year), and there have been cystic fibrosis transmembrane conductance regulator; CT, annual enterohaemorrhagic E. coli outbreaks since the cholera toxin; DRA, down regulated in adenoma Cl–/HCO3– early 1990s. exchanger; EC, enterochromaffin; ENaC, epithelial Na+ In this Review, we describe the major pathogenic channel; ENS, enteric nervous system; EPCAM, epithelial mechanisms of secretory diarrhoea, discuss currently cell adhesion molecule; Gal, galanin; GALR1, galanin available pharmacological therapies and therapies that receptor 1; GUCY2C, guanylate cyclase C (heat stable enterotoxin receptor); 5‑HT, 5‑hydroxytryptamine; MYO5B, are being developed, and examine the major challenges unconventional myosin-5b; NHE, sodium/hydrogen in the development of diarrhoeal therapeutics. exchanger; NKCC, Na/K/Cl symporter; NSP4, nonstructural protein 4; RABs, Ras-related proteins; SCFA, Mechanisms of diarrhoeal disease short-chain fatty acid; SLC5A1, sodium/glucose Diarrhoea results from excessive secretion and/or cotransporter; STa, heat-stable toxin; TLR, toll-like impaired absorption of fluid and electrolytes across receptor; VIP, vasoactive intestinal peptide. the intestinal epithelium (Figure 1). The movement of fluid between the intestinal lumen and blood is driven + – – by the active transport of ions, mainly Na , Cl , HCO3 with D-glucose (or D-galactose), with the electro and K+, and solutes, mainly glucose. Fluid absorption or neutral glucose transporter SLC2A2 facilitating glucose secretion involves the coordinated activity of membrane exit across the basolateral membrane.23,24 In the colon, transporters located on the apical (lumen-facing) and in addition to electroneutral Na+ transport by Na+/H+ basolateral (circulation-facing) epithelial membranes.17 exchange (proximal colon), absorption is facilitated by The intestinal epithelium is structurally configured into the epithelial Na+ channel and short-chain fatty acid long, finger-like projections (villi) and glandular, tube- transporters.25,26 Intracellular messengers, including like structures (crypts), with the relative villus-to-crypt Ca2+ and cyclic nucleotides such as cAMP and cGMP, ratio differing along the intestine. Functionally, both inhibit the activity of apical Na+ transporters.27–30 There absorption and secretion can occur in the same epithelial is considerable cross-activation of the intestinal epithelial cells, although secretory processes predominate in crypts second messengers cAMP and Ca2+.31,32 and absorptive processes in villi. Intestinal fluid secretion is driven by transepithelial Fluid absorption is driven by the active transport of Cl– secretion through basolateral and apical Cl– chan- + – – – Na across the epithelium with parallel Cl or HCO3 nels and transporters. Cl is transported into the cell absorption. The electrochemical driving force for this at the basolateral membrane by a Na/K/Cl symporter process is the basolateral Na+/K+-ATPase. In the small (NKCC1, also known as SLC12A2), which is driven by intestine, fluid absorption is facilitated by the sodium/ the Na+ concentration gradient produced by the Na+/ hydrogen exchanger 3 (NHE3, also known as SLC9A3), K+-ATPase.33 Basolateral K+ channels (KCNQ1/KNE3 sodium/glucose cotransporter 1 (SLC5A1), and and KCNN4) provide the electrochemical driving force – – – – 34 Cl /HCO3 exchanger (DRA [SLC26A3] and PAT1 for apical Cl exit across Cl channels, primarily the [SLC26A6]).18–20 Electroneutral fluid absorption is cyclic-nucleotide-activated cystic fibrosis transmem- carried out by the coordinated activity of NHE3 with brane conductance regulator (CFTR) and Ca2+-activated – – – – 33 Cl /HCO3 exchangers (PAT1 for HCO3 absorption and Cl channels (CaCCs). Enteric nerves and cell surface DRA for Cl– absorption in the jejunum and colon).21,22 receptors such as the calcium-sensing receptor (CaSR) Substrate-specific transporters such as SLC5A1 facilitate are thought to modulate intracellular signalling pathways cotransport of Na+ across the apical membrane together and hence electrolyte absorption and secretion.35,36

a Absorption Secretion Na+ SLC5A1 DRA NHE3 H2O Glucose – – Na+ Cl– Na+ Na+ SCFA Cl Cl ENaC CFTR CaCC CaSR – + + HCO3 H Na Tight junction – – –

cGMP cAMP Ca2+ cAMP Ca2+

2Cl– 2K+ 2K+ Na+ K+ K+ CaSR

H2O + Cl– + 3Na 3Na NKCC K+ Na+/K+-ATPase Na+/K+-ATPase channels ACh Gal VIP Circulation His 5-HT Enteric Enk nervous SP system Sst

b Bacterial diarrhoeas c Viral diarrhoeas

Vibrio/E.coli CFTR DRA NHE3 CaCC SLC5A1 – – Cl – + Cl Na+ Glucose Cl– Na+ CT Cl Na ENaC Rotavirus CaCC NHE3 STa Salmonella Shigella – + + + HCO3 H Na H – – – EC NSP4 cGMP 2+ cells Ca 2+ α1β2 Ca cAMP integrin Immune NSP4 cells 5-HT 2Cl– 2Cl– + + VIP Na+ K+ Gal GALR1 Na K

ENS e.g. IL–6, IL-8, NKCC K+ TNF ENS NKCC K+ channels channels

d Congenital diarrhoeas e In ammatory diarrhoeas Congenital Na Congenital Glucose/galactose diarrhoea chloridorrhoea malabsorption Cl– Cl– TLRs Na+ CaCC CFTR NHE3 – NHE3 DRA SLC5A1 Cl Na+ Cl– Na+ Glucose Commensal Guanylin bacteria Microvilli H+ GUCY2C CFTR + – – + H HCO3 RABs cAMP cGMP Ca2+ Cell signalling Microvillus MYO5B pathways EPCAM inclusion disease Defective cell e.g. NF-κB 2Cl– adhesion Na+ K+ Innate immunity signalling 2Cl– + + Macrophages Na K Dendritic cells In ammatory NKCC Neutrophils mediators e.g. TNF, IL-6 Adaptive NKCC immunity T cells

Nature Reviews | Gastroenterology & Hepatology

Bacterial diarrhoeas levels of intracellular cyclic nucleotides, resulting in Bacteria such as V. cholerae and enterotoxigenic E. coli activation of apical CFTR Cl– channels and hence Cl– secrete specific enterotoxins (for example cholera toxin secretion (Figure 1b).37,38 Data from immortalized and and heat-stable enterotoxin, respectively) that increase primary human intestinal cells show that elevation of

cAMP, cGMP and Ca2+ concentrations by bacterial in Ca2+ or cyclic nucleotide signalling and stimulation of enterotoxins also inhibits NHE3.39,40 Bacteria can also Cl– secretion or inhibition of Na+ absorption (Figure 1e). increase various humoral agonists, neurotransmitters or The release of inflammatory mediators such as TNF neuropeptide receptors such as 5-hydroxytryptamine, and IL-6 by activated T cells and neutrophils, which VIP peptides and the galanin receptor type 1,41 thus causes degranulation of mucosal mast cells and release activating Cl– secretion and inhibiting Na+ absorption.42 of histamine and prostaglandins, can also stimulate Cl– Invasive bacteria such as Salmonella and Shigella cause a secretion.63,64 Epithelial Na+ absorption is also probably tissue inflammatory response involving recruitment of affected by inflammation, with studies showing down- immune cells and release of cytokines, resulting in intra- regulation of NHE1 (also known as SLC9A1) and NHE3 cellular Ca2+ signalling.43 Enteropathogenic and invasive in IBD, and defective NHE3 function and regulation by bacteria also result in alterations in transport protein adaptor proteins in an IL-10-deficient mouse model of expression, with several studies showing evidence of colitis.65–67 Predominantly inflammatory diarrhoea is impaired Na+ and Cl– absorption.40,44,45 seen in response to some bacterial pathogens, such as Clostridium difficile, which is the most common cause of Viral diarrhoeas nosocomial antibiotic-associated diarrhoea. C. difficile- Enteric rotavirus infection causes fluid secretion as associated diarrhoea and colitis occurs secondary to well as structural changes in the intestinal epithelium,46 elaborated toxins that activate epithelial inflammatory producing an age-related secretory diarrhoea.47,48 An signalling pathways and recruit immune cells.68 elaborated rotaviral protein (NSP4) is thought to act as an enterotoxin causing elevation of cytoplasmic Current management and treatment Ca2+ concentration by binding to a membrane recep- The acute treatment of infectious diarrhoeal diseases

tor (integrin α1β2), the neuropeptide galanin and/or by centres around prompt and complete amelioration of activation of enteric nerves (Figure 1c).49–51 Rotaviral dehydration. In hospitals, particularly in developed NSP4 also inhibits NHE3 and SLC5A1.52 The molecu- countries, this treatment is often achieved through lar mechanisms by which other enteric viruses such as intravenous fluid replacement.69 In developing coun- norovirus cause diarrhoea is not known at present.53 tries and non-hospital settings oral rehydration solu- Multiple pathogenic mechanisms for drug-induced tion (ORS) is the mainstay of treatment.70 Although diarrhoeas are likely to exist, but there is evidence ORS effectively treats dehydration when administered that certain drugs such as HIV protease inhibitors and appropriately, it does not change fluid losses, diarrhoeal chemotherapy agents induce diarrhoea through intra- output or duration of illness. Most of the current and cellular Ca2+-dependent mechanisms similar to those in historical therapeutics used for symptomatic treat- rotaviral diarrhoea.54,55 ment are classed as antimotility agents or antisecretory agents based on their mechanism of action (Table 1). Congenital diarrhoeas For diarrhoeas caused by enteric infections, various Congenital diarrhoeas can result from rare inher- antibiotics are also used depending on the pathogenic ited genetic mutations in several intestinal proteins organism.71 Antibiotics shorten illness duration and (Figure 1d). The target genes in many of these familial are used particularly for dysentery (bloody diarrhoea), enteropathies have been discovered with the advent of but selection of appropriate antibiotics requires labora- low-cost gene sequencing. Mutations in the Na+ trans- tory diagnosis of the pathogenic organism, which often porter SLC5A1 underlie glucose and galactose mal is not available. Although antibiotics are effective in – – absorption, and mutations in the Cl /HCO3 exchanger reducing symptoms and the duration of infectious diar- DRA result in congenital Cl– diarrhoea.56,57 A mutation rhoeas, their delayed onset of action means they do not in the guanylin receptor GUCY2C, which causes famil- prevent immediate dehydration. Also, concerns about ial diarrhoea syndrome, results in constitutive cGMP antibiotic resistance and the fact that they are contra activation with consequent CFTR-mediated Cl– secre- indicated in specific enteric infections have prevented tion and NHE3 inhibition.14 Alterations in the integrity their recommendation for widespread use.72,73 of intestinal structure caused by mutations in proteins such as MYO5B and the epithelial cell adhesion mol ORS ecule also result in altered signalling with putative acti- ORS is an orally ingested solution that stimulates intesti- vation of electrolyte secretion.58,59 Additional details nal Na+ absorption by SLC5A1 and Na+-coupled amino about congenital diarrhoeas can be found elsewhere.60,61 acid transporters. The current WHO-recommended ORS is hypo-osmolar (245 mOsm/l) to increase water absorp- Inflammatory diarrhoea tion.74 The widespread use of ORS over the past four Intestinal inflammation is seen in autoimmune diseases decades has remarkably reduced the mortality associated such as IBD and coeliac disease. Although the predomi- with diarrhoeal disease by ~70%.75 However, ORS admin- nant feature of these diseases is chronic tissue damage istration in developing countries has stagnated since secondary to inappropriate immune cell activation, 1995, with ORS only used in 30–35% of children with several overlapping signalling pathways affect intesti- diarrhoea aged <5 years.76,77 In the past decade, alterna- nal fluid transport homeostasis.62 Activation of epithelial tive forms of ORS have been developed. In one form, inflammatory signalling pathways such as NF-κB result the substrate linked to Na+ absorption is nonabsorbable

Table 1 | Current antidiarrhoeal therapies Therapy (dose, administration) Mechanism of action Indications Notes and adverse effects References [Brand name]

Alosetron (1–2 mg daily, oral) 5-hydroxytryptamine3 Refractory Contraindicated for patients with Koch et al. [Lotronex®, Prometheus Laboratories, receptor antagonist; diarrhoea- constipation; intestinal ileus, obstruction, (2004)135 USA] antimotility, antisecretory predominant IBS perforation, ischaemic colitis Cholestyramine resin (4–12 g daily, oral) Sequesters bile salts; Bile acid Unlabelled indication; impaired Koch et al. [Prevalite®, Upsher-Smith Laboratories, USA; antisecretory, malabsorption absorption of other drugs; malabsorption (2004)135 Questran®, Bristol–Myers Squibb, USA] proabsorption diarrhoea; ileal of fat-soluble vitamins Colesevelam (4–12 g daily, oral) [Welchol®, resection Daiichi-Sankyo, USA; Cholestagel®, Genzyme, Netherlands] Crofelemer (250 mg daily, oral) CFTR and CaCC channel Diarrhoea associated Not approved for infectious or other Macarthur et [Fulyzaq®, Salix Pharmaceuticals, USA] inhibitor; antisecretory with antiretroviral diarrhoeas al. (2013)89 therapy for HIV/AIDS

Diphenoxylate and atropine (5–20 mg μ-opioid receptor agonist; Acute nonspecific Contraindicated in diarrhoea associated with Karim et al. daily, oral) antimotility diarrhoea enterotoxigenic bacteria, pseudomembranous (1972)83 [Lomotil®, AMCo, UK] colitis and ulcerative colitis; CNS depression, dizziness, paralytic ileus

Loperamide (4–16 mg daily, oral) μ-opioid receptor agonist; Acute nonspecific Contraindicated in bacterial enterocolitis, DuPont et al. [Imodium®, Johnson & Johnson, USA] antimotility diarrhoea, chronic pseudomembranous colitis and ulcerative (1990)85; diarrhoea associated colitis; constipation, nausea Koch et al. with IBD (2004)135; Ericsson et al. (1990)137 Racecadotril (100–300 mg daily, oral) Enkephalinase inhibitor; Acute nonspecific Not approved in USA; headache Hamza et al. [Hidrasec®, Tiorfan®, Bioproject, France] antisecretory diarrhoea (1999)87 Bismuth subsalicylate (0.5–4 g daily, oral) Inhibition of prostaglandin Acute mild Faecal discoloration, tinnitus, CNS DuPont et al. [Pepto-Bismol®, Procter & Gamble, USA; synthesis; antisecretory; nonspecific diarrhoea depression, dizziness, Reyes syndrome (1990)85 Bismatrol] antimicrobial in children

Abbreviations: CaCC, calcium-activated chloride channel; CFTR, cystic fibrosis transmembrane conductance regulator; CNS, central nervous system.

starch, which is relatively resistant to digestion by pancre- the potential serious adverse effects of antimotility atic amylase and is metabolized by bacteria to short-chain drugs together with a narrow therapeutic index has fatty acids in the colon where it stimulates Na+ absorp- limited their recommendation and use, particularly for tion by NHE2 (also known as SLC9A2).74,78 In clinical infectious diarrhoeas. trials, patients with cholera who receive nonabsorbable starch ORS have a shorter duration of diarrhoea and Antisecretory agents require less parenteral fluid support than patients who Reducing intestinal fluid secretion has been a rela- receive conventional ORS.79 Zinc has been shown to tively underexploited area for antidiarrhoeal thera- shorten the duration of acute diarrhoea and is part of peutics. Historically, bismuth subsalicylate was shown the WHO-recommended ORS.80 The mechanism of this to have antidiarrhoeal efficacy and early mechanistic effect of zinc is unclear, and whether zinc deficiency plays studies indicated that salicylates such as aspirin inhibit a role has not been established. In vitro, zinc blocks Cl– enterotoxin-induced Cl– secretion and promote Na+ secretion by inhibiting K+ channels in the basolateral absorption.86 Racecadotril, an encephalinase inhibi- membrane and stimulating NHE3.81,82 tor, or its active metabolite thiorphan, initially showed promise as an antidiarrhoeal. Inhibition of the break- Antimotility agents down of endogenous encephalins could exert anti Drugs that inhibit intestinal motility have been used secretory effects through encephalin-stimulated extensively to treat diarrhoea. The putative mechanism activation of epithelial μ-opioid receptors. Small clini- of action for antimotility drugs is increased Na+ and cal studies initially showed efficacy in cholera-toxin- fluid absorption as a result of slow intestinal transit. mediated fluid secretion; however, a large double-blind, Loperamide and diphenoxylate are μ-opioid agonists placebo-controlled trial showed no improvement in that are widely used for mild, nonspecific diarrhoea. diarrhoeal output or duration.87,88 More recently, a nat- They are not recommended in bacterial diarrhoeas pri- ural-product antisecretory agent, crofelemer, has been marily owing to the risk of paralytic ileus, and diphen approved for use in HIV-related diarrhoeas based on oxylate also has substantial central opioid effects.83 a clinical trial showing efficacy in improving chronic 89 5-hydroxytryptamine3 antagonists such as alosetron diarrhoea in patients with HIV. Crofelemer is a hetero have shown efficacy in chronic diarrhoea related to geneous proanthocyanidin oligomer extracted from the IBS;84 however, their use has been limited by concerns bark latex of the South American tree Croton lechleri. of ileus and ischaemic colitis. Although loperamide is The putative mechanism of action for crofelemer is widely used and effective in mild acute diarrhoea,85 inhibition of Cl– channels in the apical membrane.

Table 2 | Potential antidiarrhoeal therapies in developing countries, although increasing evidence shows that they might be beneficial as adjunctive Name/class Target and mechanism Potential Development indication stage antidiarrhoeal therapies. Antisecretory Inhibition of enteric Acute secretory Phase III factor nervous system; increased diarrhoea New antidiarrhoeal therapies epithelial permeability Most of the currently used antidiarrhoeal therapies are decades old and only in the last 5 years has there CaCCinh‑A01 Absorbable CaCC inhibitor Acute secretory Preclinical diarrhoea been renewed interest in development of antidiarrhoeal 98 iOWH032 Extracellular-acting CFTR Acute secretory Phase I therapeutics, with several novel drug candidates tar- inhibitor diarrhoea geting intestinal fluid transport (Table 2). Some of these new therapeutic candidates have emerged from CFTRinh‑172 Absorbable CFTR inhibitor Acute secretory Preclinical diarrhoea greater understanding of the mechanisms of intestinal Clotrimazole K+ channel inhibitor Acute secretory FDA-approved for fluid secretion and the use of high-throughput screen- diarrhoea other indication ing technology. Figure 2 depicts the molecular targets Eluxadoline μ-opioid receptor agonist Diarrhoea- Phase III for antidiarrhoeal therapeutics at various stages of predominant IBD development. Gallotannins CaCC inhibitors Acute secretory Preclinical diarrhoea Nutritional CFTR inhibitors supplement Activation of CFTR Cl– channels in the small intestine LX1033 Tryptophan hydroxylase Diarrhoea- Phase II and colon occurs in secretory diarrhoeas caused by bac- inhibitor; decreased predominant IBD terial enterotoxins secreted in cholera and Traveller’s 5‑hydroxytryptamine synthesis diarrhoea.99 Intestinal Cl– and fluid secretion are absent Lysophosphatidic Lysophosphatidic acid Acute secretory Preclinical in CFTR-knockout mice and in patients with cystic fibro- acid receptor 2 antagonist diarrhoea Nutritional sis,100,101 and the use of CFTR inhibitors blocks colonic supplement Cl– transport in human tissue.99 High-throughput screen- MalH-lectin Inhibitor of the external Acute secretory Preclinical ing has revealed three chemical classes of small-molecule pore of CFTR diarrhoea CFTR inhibitors. The thiazolidinone CFTRinh-172 NHE3 C‑terminal NHE3 regulatory Acute secretory Preclinical (Figure 3) inhibits CFTR by binding at or near Arg347 peptide complex agonist diarrhoea and stabilizes the channel in its closed state.102 Studies in Obeticholic acid Semi-synthetic bile acid Bile acid Phase II mouse models of cholera and intestinal fluid secretion malabsorption diarrhoea induced by heat-stable toxin have demonstrated efficacy 99,103 PPQ/BPO Absorbable CFTR inhibitors Acute secretory Preclinical of CFTRinh-172. Another class of CFTR inhibitors, diarrhoea which probably interfere with ATP gating, are the PPQ/ 104,105 R‑568 Calcium-sensing receptor Acute secretory Preclinical BPO compounds; the most potent of these com- – agonist diarrhoea pounds, (R)-BPO-27, inhibits CFTR Cl conductance

Abbreviations: BPO, benzopyrimido-pyrrolo-oxazine-dione; CaCC, calcium-activated chloride channel; CFTR, with IC50 ~4 nM and has shown efficacy in models of cystic fibrosis transmembrane conductance regulator; NHE, sodium/hydrogen exchanger; PPQ, pyrimido- polycystic kidney disease,104 but has not yet been tested pyrrolo-quinoxalinedione. in models of diarrhoea. Glycine hydrazides such as GlyH-101 are another However, in vitro studies showed crofelemer to be a class of CFTR inhibitors that bind to the CFTR pore weak and partial antagonist of CFTR, although a rela- on its extracellular surface as demonstrated by patch tively strong inhibitor of CaCCs.90 It is unclear whether clamp, molecular modelling and efficacy of a mem- this inhibition of CaCCs underlies the efficacy of brane-impermeant polyethylene glycol-hydrazide crofelemer in HIV-related diarrhoea. conjugate.106 The GlyH-101 analogue iOWH032 has completed a safety clinical trial,107 although a planned Probiotics phase II trial (NCT02111304) has been terminated Several meta-analyses and clinical studies in developed before patient enrolment. iOWH032 is unlikely to be countries suggest that probiotics prevent or reduce the effective because it is a weak CFTR inhibitor and can duration of diarrhoea.91–93 However, these effects seem undergo rapid convective washout in the intestine.108 to be highly strain-dependent and dose-dependent. To address the potency and washout limitations, non- Studies using specific bacterial strains have shown absorbable macromolecular conjugates have been syn- effects on expression and/or function of DRA, CFTR thesized containing a malonic acid hydrazide (MalH) and NKCC transporters, suggesting that probiotics moiety that inhibits CFTR. One such conjugate, MalH- – affect mechanisms of Cl secretion and electroneutral lectin, inhibited CFTR with an IC50 down to 50 nM, absorption.94–96 A double-blind, placebo-controlled remained bound to CFTR for >6 h, and reduced mor- study in children aged 1–6 years showed that a specific tality in a neonatal mouse model of cholera.109 The high probiotic (Lactobacillus reuteri) significantly reduced potency of the MalH-lectin conjugate and its resistance the incidence of acute diarrhoea, particularly in chil- to washout is possibly due to trapping in the glycocalyx dren with poor nutritional status.97 Currently, probiotics on enterocytes. Multivalent MalH-polyethylene glycol are not routinely recommended in community settings conjugates have also been synthesized and shown to be

Preclinical Phase I–II NHE3 peptide CaCCinh-A01 Gallotannins Phase III LPA LX1033 MalH-lectin Cl– Cl– EC cell Na+ CaCC CFTR iOWH032 Enteric nerves LPAR2 PPQ + Epithelial H BPO cells

CFTRinh-172

Bile salts

2Cl– Na+ K+ K+ AF CaSR Eluxadoline Clotrimazole Obeticholic acid NKCCinh NKCC K+ channels Intestinal villi R-568 Figure 2 | Potential therapies for secretory diarrhoeas at various stages of development and their molecular targets. Nature Reviews | Gastroenterology & Hepatology Various molecular targets including intestinal ion channels and transporters, regulatory proteins and cell surface receptors are found on epithelial cells lining the intestine, enteric nerves and enterocytes. Novel therapeutics targeting these molecules are in the development pipeline. Abbreviations: AF, antisecretory factor; BPO, benzopyrimido-pyrrolo-oxazine- dione; CaCC, calcium-activated chloride channel; CaSR, calcium-sensing receptor; CFTR, cystic fibrosis transmembrane conductance regulator; EC, enterochromaffin; LPA, lysophosphatidic acid; LPAR2, LPA receptor 2; NHE, sodium/hydrogen exchanger; NKCC, Na/K/Cl symporter; PPQ, pyrimido-pyrrolo-quinoxalinedione.

CFTR inhibitiors with nanomolar potency,110 greater in vitro, and was efficacious in mouse models of cholera chemical stability and lower cost than lectin-containing and rotaviral diarrhoea.115 Natural products represent CFTR inhibitor conjugates. inexpensive and readily available potential therapies for serious secretory diarrhoeas. CaCC inhibitors CaCCs provide a second route for Cl– secretion by NKCC1 inhibitors enterocytes and might also be involved in enterotoxin- The electroneutral NKCC1 transporter is the major mediated secretory diarrhoeas by cross-talk in signal- pathway for Cl– entry into enterocytes from the blood ling mechanisms. CaCCs probably represent the primary side and is therefore an attractive target for development pathway for apical membrane Cl– secretion in rotaviral of inhibitors that are predicted to block both CFTR- and perhaps other viral diarrhoeas,48 as well as in secre- mediated and CaCC-mediated secretory diarrhoeas. tory diarrhoeas induced by some HIV protease inhib NKCC inhibitors such as bumetanide are approved for itors54 and chemotherapeutics.55 The molecular identity use as diuretics, as the kidney expresses the NKCC2 of the major CaCC(s) in enterocytes remains unclear. isoform. Although novel NKCC1 inhibitors are emerging The CaCC anoctamin-1 is expressed in interstitial cells from screening efforts, challenges in their development of Cajal and has an important role in intestinal motil- for diarrhoeal therapy include obtaining high selectivity ity,111 but its role in Cl– conductance in enterocytes over NKCC2 (to prevent diuretic action) or confining remains uncertain.112 their action to the intestine, and preventing potential Small-molecule and natural-product CaCC inhibitors off-target effects such as hearing impairment seen in have been identified. A phenotype-based screen in HT-29 NKCC1-knockout mice.116 cells identified several small-molecule CaCC inhibitors, the most potent being the 3-acyl-2-aminothiophene CaSR agonists 113 CaCCinh-A01. CaCCinh-A01 prevented watery diar- The CaSR is a G-protein-coupled receptor expressed rhoea in a neonatal mouse model of rotavirus.114 throughout the small intestine and colon.117 In the small Screening of natural products to find potential CaCC intestine the CaSR is expressed on the basolateral mem- inhibitors identified tannic acid, which motivated the branes of crypt and villus epithelial cells and on the apical discovery that polyphenolic gallotannins strongly inhibit surface of villus cells,117 while in the colon it is present on CaCCs. Remarkably, oral administration of an alcohol- the apical and basolateral membrane of crypt cells and free red wine extract prevented rotaviral diarrhoea in on cells of the enteric nervous system and some entero neonatal mice, without effect on the rotaviral infection.114 endocrine cells. Extracellular signals that activate the The red wine extract did not inhibit CFTR or prevent CaSR include divalent (Ca2+, Mg2+ and Cd2+) and triva- cholera-toxin-induced intestinal fluid secretion. In lent (gadolinium3+ and La3+) cations, and some L-amino another natural-product study, diarrhoea remedies from acids. Activation of the CaSR seems to have multiple sources around the world were screened for inhibition actions on electrolyte transport, including decreasing of Cl– channels. A commonly used Thai herbal remedy enterotoxin-induced Cl– secretion and reversing inhibi- fully inhibited both CFTR and CaCC Cl– conductance tion of Na+ absorption.35 This effect may in part involve

Absorbable CFTR inhibitors External acting CFTR inhibitors

O F F Br Br N OH OH F S O O S H H N N N N N Br N Br H H O OH O NH HO CFTRinh-172 GlyH-101 HN S

O CH3 HN Na+ N O– H3C N O O S O O S O O Br S – O N O N Na+ Lectin HN N OH N N H H H O N O Br PPQ-102 iOWH032 MalH-lectin

CaCC inhibitors O OH

N NH H N N S HO N S O O O O S CH3

CaCCinh-A01 T16Ainh-A01

OH HO OH OH R O HO O OH OH OH HO O HO O HO OH OH HO O R O O O OH O O OH HO O O OH HO OH O OH O O HO OH OH n = 1–28 HO O O O R O OH O OH OH HO O HO O O OH HO O OH OH Procyanidin R = H OH OH Prodelphinidin R = OH OH HO OH OH Tannic acid Crofelemer

Figure 3 | Chemical structures of CFTR and CaCC inhibitors for secretory diarrhoeas. Abbreviations: CaCC, calcium-activated Nature Reviews | Gastroenterology & Hepatology chloride channel; CFTR, cystic fibrosis transmembrane conductance regulator; PPQ, pyrimido-pyrrolo-quinoxalinedione.

2+ + breakdown of cyclic nucleotides by phosphodiesterase Ca -sensitive K channels in enterocytes with IC50 ~5 μM activation35 and regulation of tight junction assembly.118 and has emerged as a potential antidiarrhoeal drug can- CaSR activation by calcimimetic drugs such as R-568 didate.120 Clotrimazole is approved by the FDA for other reduces fluid losses stimulated by bacterial enterotoxins indications, with its primary adverse effects related to and has been proposed as a potential antidiarrhoeal, dose-dependent inhibition of cytochrome P450 enzymes. although to date no clinical studies have investigated Other K+ channel inhibitors, including clotrimazole ana- CaSR activation by calcimimetics or Ca2+ itself. logues121 and more potent new chemical entities, could be potential antisecretory development candidates. K+ channel inhibitors Several studies have shown that inhibition of basolateral NHE3 agonists K+ channels reduces enterotoxin-mediated fluid secre- Studies in cell culture and rodent models suggest that tion.119 The antifungal clotrimazole blocks both cAMP and NHE3 is inhibited in secretory diarrhoeas, which seems

to be an exaggeration of the NHE3 inhibition that occurs μ-opioid and δ-opioid inhibitor with antimotility and physiologically early in the postprandial state.122 NHE3 analgesic properties developed primarily for diarrhoea- activity in enterocytes is inhibited following acute eleva- predominant IBS. Results from phase II trials showed tions in cAMP, cGMP or Ca2+ concentrations, as pro- some clinical improvement in stool consistency and duced by bacterial enterotoxins, ionophores or humoral abdominal pain, and little rebound constipation.132 agonists including 5-hydroxytryptamine and carba- Another compound aimed at diarrhoea-predominant chol.28,123,124 These mechanisms probably apply in human IBS showing efficacy in phase II trials is LX1033, an secretory diarrhoeas. NHE3 activity is reduced in con- inhibitor of tryptophan hydroxylase that affects intes- genital Na+ diarrhoea as seen in intestinal biopsies,125 with tinal motility by inhibition of 5-hydroxytryptamine mutations in the transport domain of NHE3 account- synthesis.133 A semi-synthetic bile acid analogue, obeti ing for this autosomal recessive condition (A. Janecke cholic acid, which activates the nuclear bile acid recep- et al., unpublished data). Human enteroids generated tor (farnesoid X receptor), relieves symptoms in patients from proximal small intestine of healthy individuals with bile acid diarrhoea, which has been suggested to show NHE3 activity under basal conditions, which is contribute to diarrhoea in some patients with IBS.134 The acutely stimulated by dexamethasone and inhibited by action of obeticholic acid is thought to involve a feed cAMP, cGMP, Ca2+, bacterial enterotoxins and rotaviral back mechanism with an increase in the bile acid recep- infection.126 A peptide that mimics part of the NHE3 tor, which results in increased intestinal production of C-terminal domain and prevents NHE3 inhibition by fibroblast growth factor 19 and uptake into the portal cAMP, Ca2+ and cholera toxin has potential utility as a system, thus reducing hepatic bile acid synthesis and proabsorptive therapeutic. intraluminal bile salt concentration.

Antisecretory factor Conclusions and future challenges A secreted, 41 kilodalton protein named antisecretory Although diarrhoeal diseases remain a major global factor released mainly from the pituitary gland 127 has health challenge, there has been a remarkable lack of been shown in rodent models to prevent intestinal fluid effective new therapeutics. Part of the reason for the secretion induced by cholera toxin, heat-stable toxin, lack of progress is that global and regional health policy C. difficile toxin A, Campylobacter toxin and prosta has rightly focused on approaches targeted at preven- glandins.128 The active component of antisecretory factor tion, such as vaccines, improved sanitation, and better consists of seven amino acids in its N-terminus. The health delivery including the use of ORS. As described antisecretory factor-16 peptide, consisting of the active herein, there is now an emerging pipeline of potential N-terminal domain of the antisecretory factor, also blocks antidiarrhoeal agents. In developing countries where intestinal fluid secretion. The exact mechanism of action diarrhoeas are primarily caused by infection, these for antisecretory factor is unclear, but the antisecretory new agents might be useful as adjuncts or additives to effect of antisecretory factor-16 was abolished by vagot- ORS, reducing diarrhoeal output and hence protecting omy, suggesting its action is on nerves and perhaps on against severe dehydration particularly in the early stages capillary permeability. A modified egg-yolk product of disease, and secondarily promoting ORS usage. In enriched with antisecretory factor (Salovum®, AS-Faktor complex medical emergencies, where availability of rehy- AB, Sweden) reduced the duration of acute and prolonged dration fluids might be limited, such therapeutics could (>7 days) diarrhoea in a randomized, placebo-controlled provide life-saving alleviation of fluid loss. To be benefi- trial in children aged 7–60 months.129 Further clinical and cial in developing countries the ideal therapeutic should mechanistic studies are needed for this promising drug. have a wide therapeutic index, be stable in harsh environ- ments and be extremely low-cost. Major challenges in Lysophosphatidic acid moving forward include funding, logistics and design of Lysophosphatidic acid is a naturally occurring phospho- informative clinical trials. The development of drugs that lipid present in many foods. Several studies have sug- can be used safely in children and the elderly, where the gested that interaction of lysophosphatidic acid with its disease burden is greatest, poses additional challenges in intestinal receptor (lysophosphatidic acid receptor 2) terms of drug adverse effects and patient recruitment for results in the formation of a macromolecular complex clinical trials. For indications such as diarrhoeas related that modulates CFTR activity. Lysophosphatidic acid to chronic inflammatory diseases, IBD or drug-induced receptor agonists prevent enterotoxin-mediated fluid diarrhoeas, the development pathway for antisecretory secretion in rodents, suggesting that LPA-based nutri- or proabsorptive compounds is clearer, as in the case of tional supplements could be used as adjunct therapy crofelemer. Progress towards new antisecretory agents in diarrhoea.130 Lysophosphatidic acid also stimulates has been accelerated with the elucidation of new regula- NHE3 in the small intestine through lysophosphatidic tory pathways in intestinal fluid transport, raised aware- acid receptor 5 on Na+-absorptive cells.131 ness of natural-product remedies, and emergence of new tools such as ion-sensitive fluorescent proteins and Other potential therapies human enteroid models for drug screening. The next and Several other potential antidiarrhoeal therapeutics with more challenging process will be translating these dis- specific indications are currently in phase II or phase coveries into safe, low-cost therapies to reduce the global III clinical trials (Figure 2). Eluxadoline is a mixed health and economic burden of diarrhoeal diseases.

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