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Discovery and Development of Antisecretory Drugs for Treating Diarrheal Diseases

2013; Elsevier BV; Volume: 12; Issue: 2 Linguagem: Inglês

10.1016/j.cgh.2013.12.001

1542-7714

Jay R. Thiagarajah, Euna Ko, Lukmanee Tradtrantip, Mark Donowitz, A. S. Verkman,

Eosinophilic Esophagitis

Diarrheal diseases constitute a significant global health burden and are a major cause of childhood mortality and morbidity. Treatment of diarrheal disease has centered on the replacement of fluid and electrolyte losses using oral rehydration solutions. Although oral rehydration solutions have been highly successful, significant mortality and morbidity due to diarrheal disease remains. Secretory diarrheas, such as those caused by bacterial and viral enterotoxins, result from activation of cyclic nucleotide and/or Ca2+ signaling pathways in intestinal epithelial cells, enterocytes, which increase the permeability of Cl- channels at the lumen-facing membrane. Additionally, there is often a parallel reduction in intestinal Na+ absorption. Inhibition of enterocyte Cl- channels, including the cystic fibrosis transmembrane conductance regulator and Ca2+-activated Cl- channels, represents an attractive strategy for antisecretory drug therapy. High-throughput screening of synthetic small-molecule collections has identified several classes of Cl- channel inhibitors that show efficacy in animal models of diarrhea but remain to be tested clinically. In addition, several natural product extracts with Cl- channel inhibition activity have shown efficacy in diarrhea models. However, a number of challenges remain to translate the promising bench science into clinically useful therapeutics, including efficiently targeting orally administered drugs to enterocytes during diarrhea, funding development costs, and carrying out informative clinical trials. Nonetheless, Cl- channel inhibitors may prove to be effective adjunctive therapy in a broad spectrum of clinical diarrheas, including acute infectious and drug-related diarrheas, short bowel syndrome, and congenital enteropathies. Diarrheal diseases constitute a significant global health burden and are a major cause of childhood mortality and morbidity. Treatment of diarrheal disease has centered on the replacement of fluid and electrolyte losses using oral rehydration solutions. Although oral rehydration solutions have been highly successful, significant mortality and morbidity due to diarrheal disease remains. Secretory diarrheas, such as those caused by bacterial and viral enterotoxins, result from activation of cyclic nucleotide and/or Ca2+ signaling pathways in intestinal epithelial cells, enterocytes, which increase the permeability of Cl- channels at the lumen-facing membrane. Additionally, there is often a parallel reduction in intestinal Na+ absorption. Inhibition of enterocyte Cl- channels, including the cystic fibrosis transmembrane conductance regulator and Ca2+-activated Cl- channels, represents an attractive strategy for antisecretory drug therapy. High-throughput screening of synthetic small-molecule collections has identified several classes of Cl- channel inhibitors that show efficacy in animal models of diarrhea but remain to be tested clinically. In addition, several natural product extracts with Cl- channel inhibition activity have shown efficacy in diarrhea models. However, a number of challenges remain to translate the promising bench science into clinically useful therapeutics, including efficiently targeting orally administered drugs to enterocytes during diarrhea, funding development costs, and carrying out informative clinical trials. Nonetheless, Cl- channel inhibitors may prove to be effective adjunctive therapy in a broad spectrum of clinical diarrheas, including acute infectious and drug-related diarrheas, short bowel syndrome, and congenital enteropathies. Diarrheal disease is a major health burden worldwide and represents a leading cause of mortality and morbidity. This burden, as with many major diseases, falls disproportionately on the very young and the elderly. At present, diarrheal disease is the second leading cause of mortality globally in children younger than age 5, and repeated episodes of dehydration from diarrhea are associated with impaired physical and mental development.1Walker C.L. Rudan I. Liu L. et al.Global burden of childhood pneumonia and diarrhoea.Lancet. 2013; 381: 1405-1416Abstract Full Text Full Text PDF PubMed Scopus (1519) Google Scholar, 2Moore S.R. Lima N.L. Soares A.M. et al.Prolonged episodes of acute diarrhea reduce growth and increase risk of persistent diarrhea in children.Gastroenterology. 2010; 139: 1156-1164Abstract Full Text Full Text PDF PubMed Scopus (125) Google Scholar In developing countries, the major causes of diarrheal diseases are primarily infectious, including enterotoxin-producing bacteria, such as Vibrio cholerae and enterotoxigenic Escherichia coli; viruses, such as rotavirus; enteroinvasive bacteria, such as Shigella and Salmonella; and parasites, such as Entamoeba histolytica and Cryptosporidium parvum.1Walker C.L. Rudan I. Liu L. et al.Global burden of childhood pneumonia and diarrhoea.Lancet. 2013; 381: 1405-1416Abstract Full Text Full Text PDF PubMed Scopus (1519) Google Scholar Major noninfectious causes of diarrhea include drug side effects, particularly with certain cancer and human immunodeficiency virus (HIV) therapeutics, and diarrhea secondary to intestinal inflammatory and autoimmune diseases, such as ulcerative colitis, Crohn's disease, and celiac disease.3Binder H.J. Mechanisms of diarrhea in inflammatory bowel diseases.Ann NY Acad Sci. 2009; 1165: 285-293Crossref PubMed Scopus (44) Google Scholar, 4Green P.H. Jabri B. Celiac disease.Annu Rev Med. 2006; 57: 207-221Crossref PubMed Scopus (173) Google Scholar Diarrhea is also a major problem in patients with short bowel syndrome; in rare congenital disorders, such as microvillus inclusion disease and tufting enteropathy; and with peptide-secreting neuroendocrine tumors.5Canani R.B. Terrin G. Recent progress in congenital diarrheal disorders.Curr Gastroenterol Rep. 2011; 13: 257-264Crossref PubMed Scopus (42) Google Scholar Oral rehydration solution (ORS) to replace fluid losses and promote intestinal fluid absorption has been the primary therapy for infectious diarrheas, reducing mortality 4-fold over the last 30 years.6Munos M.K. Walker C.L. Black R.E. et al.The effect of oral rehydration solution and recommended home fluids on diarrhoea mortality.Int J Epidemiol. 2010; 39: i75-i87Crossref PubMed Scopus (189) Google Scholar However, there remains an unmet need for alternative and adjunctive antidiarrheal therapeutics to complement ORS, in part because of limitations in ORS availability, acceptability, and adequate administration, and for symptom relief. Diarrhea results from excessive secretion and/or impaired absorption of fluid and electrolytes across the intestinal epithelium. Movement of fluid across the intestinal epithelium is driven by active transport of ions, mainly Na+, Cl-, and K+, and solutes, mainly glucose. Fluid absorption in the small intestine involves several luminal transporters, including a Na+/H+ exchanger, Na+-glucose cotransporter, and Cl-/HCO3- exchanger.7Lin R. Murtazina R. Cha B. et al.D-glucose acts via sodium/glucose cotransporter 1 to increase NHE3 in mouse jejunal brush border by a Na+/H+ exchange regulatory factor 2-dependent process.Gastroenterology. 2011; 140: 560-571Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar, 8Walker N.M. Simpson J.E. Brazill J.M. et al.Role of down-regulated in adenoma anion exchanger in HCO3- secretion across murine duodenum.Gastroenterology. 2009; 136: 893-901Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar As in epithelia in general, the electrochemical driving force is established by a basolateral Na+K+-ATPase pump. In the colon, fluid absorption is also facilitated by the epithelial Na+ channel and short-chain fatty acid transporters.9Gawenis L.R. Franklin C.L. Simpson J.E. et al.cAMP inhibition of murine intestinal Na/H exchange requires CFTR-mediated cell shrinkage of villus epithelium.Gastroenterology. 2003; 125: 1148-1163Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar, 10Murtazina R. Kovbasnjuk O. Chen T.E. et al.NHERF2 is necessary for basal activity, second messenger inhibition, and LPA stimulation of NHE3 in mouse distal ileum.Am J Physiol Cell Physiol. 2011; 301: C126-C136Crossref PubMed Scopus (34) Google Scholar, 11Binder H.J. Role of colonic short-chain fatty acid transport in diarrhea.Annu Rev Physiol. 2010; 72: 297-313Crossref PubMed Scopus (214) Google Scholar Targeting of fluid-absorptive pathways for diarrheal therapy is the subject of a companion review. Intestinal fluid secretion is driven by active transepithelial Cl- secretion, creating the electrochemical force for paracellular Na+ secretion and the osmotic driving force for transcellular water secretion (Figure 1). Cl- is transported into the cell at the basolateral membrane by a Na+/K+/2Cl- cotransporter, which is driven by Na+ and Cl- concentration gradients produced by the Na+K+-ATPase and basolateral K+ channels. The electrochemical gradient drives Cl- secretion across the luminal membrane Cl- channels, primarily the cAMP-activated channel cystic fibrosis transmembrane conductance regulator (CFTR) and Ca2+-activated Cl- channels (CaCC).12Barrett K.E. Keely S.J. Chloride secretion by the intestinal epithelium: molecular basis and regulatory aspects.Annu Rev Physiol. 2000; 62: 535-572Crossref PubMed Scopus (392) Google Scholar Enterotoxin-producing bacteria, such as V cholerae and enterotoxigenic E coli, produce secretory diarrhea primarily by activation of CFTR-mediated Cl- secretion.13Thiagarajah J.R. Broadbent T. Hsieh E. et al.Prevention of toxin-induced intestinal ion and fluid secretion by a small-molecule CFTR inhibitor.Gastroenterology. 2004; 126: 511-519Abstract Full Text Full Text PDF PubMed Scopus (134) Google Scholar Viral diarrheas, such as caused by rotavirus, are thought to result in secretion by causing elevation in cytoplasmic Ca2+ and consequent activation of luminal CaCCs.14Morris A.P. Scott J.K. Ball J.M. et al.NSP4 elicits age-dependent diarrhea and Ca2+ mediated I– influx into intestinal crypts of CF mice.Am J Physiol. 1999; 277: G431-G444PubMed Google Scholar Drug-related diarrhea caused by HIV protease inhibitors is also thought to involve CaCCs.15Rufo P.A. Lin P.W. Andrade A. et al.Diarrhea-associated HIV-1 APIs potentiate muscarinic activation of Cl- secretion by T84 cells via prolongation of cytosolic Ca2+ signaling.Am J Physiol Cell Physiol. 2004; 286: C998-C1008Crossref PubMed Scopus (31) Google Scholar However, the contribution of Cl- secretion in the pathogenesis of most drug-related diarrheas, congenital pediatric enteropathies, and many bacterial, viral, and parasitic infections remains untested. Despite these limitations in current knowledge, inhibition of luminal CFTR and CaCC Cl- channels represents an attractive target for potential antidiarrheal therapeutics. Our laboratory developed and carried out cell-based high-throughput screens to identify Cl- channel modulators using genetically encoded, cytoplasmic fluorescent halide sensors, including the yellow fluorescent protein YFP-H148Q/I152L, whose fluorescence is strongly reduced by I-.16Jayaraman S. Haggie P. Wachter R. et al.Mechanism and cellular applications of a green fluorescent protein-based halide sensor.J Biol Chem. 2000; 275: 6047-6050Abstract Full Text Full Text PDF PubMed Scopus (259) Google Scholar Target-based assays used epithelial cells expressing YFP-H148Q/I152L and CFTR17Ma T. Thiagarajah J.R. Yang H. et al.Thiazolidinone CFTR inhibitor identified by high-throughput screening blocks cholera toxin-induced intestinal fluid secretion.J Clin Invest. 2002; 110: 1651-1658Crossref PubMed Scopus (593) Google Scholar or the CaCC TMEM16A.18Namkung W. Phuan P. Verkman A.S. TMEM16A inhibitors reveal TMEM16A as a minor component of CaCC conductance in airway and intestinal epithelial cells.J Biol Chem. 2011; 286: 2365-2374Abstract Full Text Full Text PDF PubMed Scopus (289) Google Scholar The high-throughput screens involved addition of test compound and Cl- channel activation (by cAMP agonists for CFTR, Ca2+ agonists for TMEM16A), followed by extracellular I- addition to drive cellular I- influx. Potential inhibitors were identified as compounds reducing I- influx as monitored by the kinetics of YFP-H148Q/I152L fluorescence decrease. Because the identity of the major enterocyte CaCC is not clear, phenotype-based screening was done to identify intestinal CaCC inhibitors, using a human intestinal epithelial cell line (HT-29) stably expressing YFP-H148Q/I152L by lentiviral transfection.19De La Fuente R. Namkung W. Mills S. et al.Small-molecule screen identifies inhibitors of a human intestinal calcium-activated chloride channel.Mol Pharmacol. 2008; 73: 758-768Crossref PubMed Scopus (173) Google Scholar Three chemical classes of nanomolar-potency small-molecule CFTR inhibitors have been identified from screening of synthetic small-molecule collections. The thiazolidinone CFTRinh-172 (Figure 2A) inhibits CFTR Cl- conductance by binding near arginine-347 on the cytoplasmic side of CFTR and stabilizing the channel closed-state.20Caci E. Caputo A. Hinzpeter A. et al.Evidence for direct CFTR inhibition by CFTRinh-172 based on arginine 347 mutagenesis.Biochem J. 2008; 413: 135-142Crossref PubMed Scopus (87) Google Scholar Studies on CFTRinh-172 analogs have identified the chemical structural determinants of CFTR inhibition and have provided analogs with a range of activities and aqueous solubilities.21Sonawane N.D. Verkman A.S. Thiazolidinone CFTR inhibitors with improved water solubility identified by structure-activity analysis.Bioorg Med Chem. 2008; 16: 8187-8195Crossref PubMed Scopus (49) Google Scholar CFTRinh-172 has shown antisecretory efficacy in rodent diarrhea models, including a closed-intestinal loop model in which fluid accumulation is measured in response to luminal cholera toxin (Figure 2A). A more recently identified class of CFTR inhibitors targeting the cytoplasmic surface of CFTR are the PPQ/BPO compounds, with the best compound (R-BPO-27) having IC50 ∼4 nM.22Snyder D.S. Tradtrantip L. Yao C. et al.Potent, metabolically stable benzopyrimido-pyrrolo-oxazine-dione (BPO) CFTR inhibitors for polycystic kidney disease.J Med Chem. 2011; 54: 5468-5477Crossref PubMed Scopus (73) Google Scholar The PPQ/BPO compounds have shown efficacy in models of polycystic kidney disease in which cyst expansion involves CFTR Cl- secretion but have not been tested in diarrhea models.23Tradtrantip L. Sonawane N.D. Namkung W. et al.Nanomolar potency pyrimido-pyrrolo-quinoxalinedione CFTR inhibitor reduces cyst size in a polycystic kidney disease model.J Med Chem. 2009; 52: 6447-6455Crossref PubMed Scopus (78) Google Scholar A third chemical class of small-molecule CFTR inhibitors, the glycine hydrazides, targets the extracellular CFTR surface in the channel pore itself.24Norimatsu Y. Ivetac A. Alexander C. et al.Locating a plausible binding site for an open channel blocker, GlyH-101, in the pore of the cystic fibrosis transmembrane conductance regulator.Mol Pharmacol. 2012; 82: 1042-1055Crossref PubMed Scopus (35) Google Scholar These compounds offer the unique opportunity for development as externally acting, nonabsorbable antisecretory agents; however, by targeting an external site on the intestinal lumen a potential barrier is accessing CFTR in the deep intestinal crypts against a strong convective washout force during secretory diarrheas. Small-molecule glycine hydrazides, such as the original compound GlyH-10125Muanprasat C. Sonawane N.D. Salinas D. et al.Discovery of glycine hydrazide pore-occluding CFTR inhibitors: mechanism, structure-activity analysis, and in vivo efficacy.J Gen Physiol. 2004; 124: 125-137Crossref PubMed Scopus (230) Google Scholar and an analog being studied clinically (iOWH032),26de Hostos E.L. Choy R.K. Nguyen T. Developing novel antisecretory drugs to treat infectious diarrhea.Future Med Chem. 2011; 3: 1317-1325Crossref PubMed Scopus (30) Google Scholar are unlikely to be effective in significant diarrheas because they are washed off within seconds and have poor IC50 (>10 μM) at the interior-negative enterocyte membrane potential. Following structure-activity analysis, we synthesized sticky glycine hydrazide analogs with improved potency down to 50 nM that resist intestinal washout.27Sonawane N.D. Zhao D. Zegarra-Mora O. et al.Lectin conjugates as potent, nonabsorbable CFTR inhibitors for reducing intestinal fluid secretion in cholera.Gastroenterology. 2007; 132: 1234-1244Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar A macromolecular conjugate containing a CFTR inhibitor moiety linked by a polar spacer to a lectin, which bound strongly to the enterocyte surface glycocalyx, produced improved survival in a suckling mouse model of cholera (Figure 2B). The initial phenotype-based screen in HT-29 cells yielded several small-molecule CaCC inhibitors, the most potent being the 3-acyl-2-aminothiophene CaCCinh-A01, which fully inhibited CaCC-dependent halide flux in different intestinal cell lines with IC50 ∼1 μM. CaCCinh-A01 likely targets the CaCC directly based on patch-clamp studies and its lack of effect on cytoplasmic Ca2+ signaling. CaCCinh-A01 was shown recently to prevent watery diarrhea in a neonatal mouse model of rotavirus.28Ko E.A. Jin B.J. Namkung W. et al.Chloride channel inhibition by a red wine extract prevents rotaviral secretory diarrhea in neonatal mice.Gut. 2013 Sep 19; ([Epub ahead of print])Google Scholar Small-molecule inhibitors of the CaCC TMEM16A were identified in a target-based screen.18Namkung W. Phuan P. Verkman A.S. TMEM16A inhibitors reveal TMEM16A as a minor component of CaCC conductance in airway and intestinal epithelial cells.J Biol Chem. 2011; 286: 2365-2374Abstract Full Text Full Text PDF PubMed Scopus (289) Google Scholar However, although TMEM16A is the major CaCC in salivary gland and in interstitial cells of Cajal in the intestine, it probably represents a minor contributor to Cl- conductance in enterocytes. Natural products represent a potentially attractive source of antidiarrheal therapeutics, because they are generally inexpensive and have the potential for rapid translation to the clinic. In addition, there is a long history of anecdotal evidence of efficacy of various antidiarrheal remedies in many parts of the world. A drug/natural product screen done in our laboratory for CaCC inhibitors unexpectedly revealed tannic acid as a general CaCC inhibitor. Subsequent studies showed strong CaCC inhibition by red wines that contain chemically related polyphenolic gallotannins.29Namkung W. Thiagarajah J.R. Phuan P.W. et al.Inhibition of Ca2+-activated Cl- channels by gallotannins as a possible molecular basis of health benefits of green tea and red wine.FASEB J. 2010; 24: 4178-4186Crossref PubMed Scopus (170) Google Scholar A wide range of CaCC inhibition activities was found in different red wines, although white wines and various grape extracts showed no inhibition. Motivated by the likely involvement of CaCC activation in rotaviral diarrhea, we recently showed that an alcohol-free >1 kilodalton red wine extract prevented rotaviral diarrhea in neonatal mice, without effect on the rotaviral infection.28Ko E.A. Jin B.J. Namkung W. et al.Chloride channel inhibition by a red wine extract prevents rotaviral secretory diarrhea in neonatal mice.Gut. 2013 Sep 19; ([Epub ahead of print])Google Scholar As shown in Figure 2C, oral administration of the red wine extract stained the stool red but prevented watery diarrhea. In control studies, a red wine extract with minimal CaCC activity in vitro did not prevent rotaviral diarrhea, and the red wine extract used in Figure 2C did not inhibit CFTR or prevent cholera toxin–induced diarrhea. The use of red wine extracts for CaCC-dependent diarrhea thus warrants potential clinical testing. In a separate study, we recently screened various diarrhea remedies from sources around the world for those showing Cl- channel inhibition. A commonly used Thai herbal remedy was identified that fully inhibited both CFTR and CaCC Cl- conductance in vitro, and was efficacious in mouse models of cholera and rotaviral diarrhea (Tradtrantip and coworkers, unpublished data, 2013). The remedy also inhibited intestinal smooth muscle contraction and motility. Natural products thus represent a potentially inexpensive and readily available therapy for secretory diarrhea with a defined mechanism of action. A natural product extract, crofelemer, was recently approved for treatment of diarrhea associated with HIV drug therapy.30Yeo Q.M. Crutchley R. Cottreau J. et al.Crofelemer, a novel antisecretory agent approved for the treatment of HIV-associated diarrhea.Drugs Today (Barc). 2013; 49: 239-252Crossref PubMed Scopus (12) Google Scholar Crofelemer is a heterogeneous proanthocyanidin oligomer extracted from the bark latex of South American tree Croton lechleri. In vitro studies showed crofelemer as a weak and partial (∼60%) antagonist of CFTR, although a relatively strong inhibitor of CaCC with IC50 100-fold Kd), and sustained high luminal inhibitor concentration or slow inhibitor dissociation.32Jin B.J. Thiagarajah J.R. Verkman A.S. Convective washout reduces the antidiarrheal efficacy of enterocyte surface-targeted antisecretory drugs.J Gen Physiol. 2013; 141: 261-272Crossref PubMed Scopus (20) Google Scholar Washout is a significant concern for small-molecule CFTR glycine hydrazides, such as iOWH032, and potentially for several of the natural product agents. Antisecretory drug therapy has considerable potential in reducing morbidity and mortality associated with infectious, drug-induced, and other diarrhea. The identification of synthetic small molecules and potent natural products, and the repurposing of existing medications, present a promising preclinical pipeline of drug candidates. The overall human and economic cost of diarrheal disease globally demands a comprehensive approach that includes pharmacologic therapies in addition to existing public health priorities, such as improvements in access to ORS, vaccination, and sanitation. Although multiple challenges remain in the development of antisecretory drugs, including the funding of informative clinical trials, the next decade may bring the exciting prospect of new drugs to combat diarrheal disease.