HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Full Text (PDF) All Versions of this Article: 20/1/130 05-4818fjev1    most recent Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Sonawane, N. D. Articles by Verkman, A. S. Search for Related Content PubMed PubMed Citation Articles by Sonawane, N. D. Articles by Verkman, A. S.

Luminally active, nonabsorbable CFTR inhibitors as potential therapy to reduce intestinal fluid loss in cholera

Departments of Medicine and Physiology, Cardiovascular Research Institute, University of California, San Francisco, California, USA

¹Correspondence: 1246 Health Sciences East Tower, University of California, San Francisco, CA 94143-0521, USA. E-mail: verkman{at}itsa.ucsf.edu

SPECIFIC AIMS

Intestinal fluid secretion in diarrhea involves active secretion of chloride into the intestinal lumen by enterocytes, which creates the driving force for sodium and water secretion. Cell culture and animal studies indicate that cystic fibrosis transmembrane conductance regulator (CFTR) provides the principle route for chloride secretion at the luminal membrane in enterotoxin-mediated secretory diarrheas produced by infection with Vibrio cholera and Escherichia coli. Pharmacological CFTR inhibition has thus been proposed as a strategy to reduce fluid losses in cholera and other enterotoxin-mediated diarrheas, which remain a major problem in the developing world.

Our lab has identified by high-throughput screening two classes of potent CFTR inhibitors that blocked cholera toxin-induced intestinal fluid secretion in rodent models. The thiazolidinone CFTR_inh-172 (Fig. 1 a) produces a voltage-independent CFTR chloride channel block with prolongation of mean channel closed time. CFTR_inh-172 is rapidly absorbed across the intestinal wall and undergoes enterohepatic recirculation. In contrast, the glycine hydrazide GlyH-101 appeared by electrophysiological studies to inhibit CFTR by binding to a site at its external pore. GlyH-101 block of CFTR chloride conductance was rapid, and produced inward rectification with reduced mean channel open time. The purpose of this study was to design and characterize membrane-impermeant, nonabsorbable CFTR inhibitors that inhibit CFTR function when added at the apical cell surface. Such compounds could be used as novel probes of external CFTR pore structure and potentially as nontoxic antidiarrheals.

View larger version (26K): [in this window] [in a new window]   Figure 1. Small molecule CFTR inhibitors. a) Structures of the thiazolidinone CFTRinh–172 and the glycine hydrazide GlyH-101. b) Nonabsorbable CFTR inhibitors: malonic acid dihydrazides (MalH-x) and glycine hydrazides [GlyH-(PEG)n].

PRINCIPAL FINDINGS

1. Synthesis of polar, nonabsorbable CFTR inhibitors
We synthesized a series highly polar, nonabsorbable CFTR inhibitors based on the glycine hydrazide scaffold (Fig. 1b ). Structure-activity analysis indicated the glycyl methyl group as the unique site on the glycine hydrazide scaffold where modifications could be tolerated. One strategy for design of water soluble and membrane impermeant CFTR inhibitors was modification of GlyH-101 by linking it with bulky moieties containing polar groups such as sulfonic acid, or carboxy or hydroxy with pK_a < 7. A second strategy was conjugation of GlyH-101 to the water soluble polymer polyethylene glycol (PEG). In initial studies to explore the types of substitutions that could be tolerated without loss of activity, we synthesized a series of derivatives containing substituted phenyl, hydroxyethyl, ethoxycarbonyl, carboxyl, and ethyl at the glycyl methyl position of GlyH-101. A series of malic acid hydrazides had greatest CFTR inhibitory potency, even better than the parent compound, GlyH-101.

MalH-1 is structurally similar to GlyH-101 except for an additional benzaldehyde moiety, making it doubly charged, bulkier, and more hydrophilic, which conferred greater water solubility (5 mM) compared with GlyH-101, and reduced membrane permeability. MalH-2 carries two disulfonic acid groups, whereas MalH-3 contains one sulfonic acid moiety with a hydrophilic thiourea link. MalH-2 and MalH-3 are freely water soluble, exceeding 50% wt/volume at 20°C in saline. Compounds containing the PEG moiety had water solubility of >10 mM.

2. Compounds rapidly inhibited CFTR when added to the apical cell surface
CFTR inhibition was assayed by short-circuit current analysis in FRT cells expressing human wild-type CFTR. Apical membrane chloride current was measured after permeabilization of the cell basolateral membrane in the presence of a transepithelial chloride gradient, and CFTR chloride channel stimulation by the cell-permeant cAMP agonist CPT-cAMP. Under these conditions the measured current is a CFTR-dependent chloride current. The data shown in Fig. 2 a indicate prompt reduction in chloride current by the various compounds when added to the apical bathing solution, with inhibitory potencies (K_i) in the range of 2–8 µM and nearly complete inhibition at higher concentrations. The CFTR inhibition was reversible after washout (Fig. 2b ).

View larger version (23K): [in this window] [in a new window]   Figure 2. Inhibition of apical membrane chloride current in FRT epithelial cells expressing human wild-type CFTR. Chloride current was measured by short circuit current analysis in cells subjected to a chloride ion gradient and after permeabilization of the basolateral membrane. CFTR was stimulated by 100 µM CPT-cAMP. a) Increasing concentrations of MalH compounds were added as indicated. b) Representative wash-out study showing reversible CFTR inhibition by MalH-3.

Transepithelial permeability of the CFTR inhibitors was assessed by the Caco-2 assay in which appearance of compounds in the basal solution was assayed at 4 h after compound addition to the apical solution. For each of the compounds in Fig. 1b , appearance in the basal solution could not be detected at 4 h. In this Caco-2 assay, CFTR_inh-172 was 45% equilibrated at 4 h. Intestinal absorption was measured in living mice from compound disappearance from the lumens of closed mid-jejunal loops over 2 h. In these experiments, raffinose was included in the infusate solutions to prevent fluid absorption. There was < 2% compound absorption per hour, whereas > 90% of the thiazolidinone CFTR_inh–172 was absorbed over 2 h and 65% of GlyH-101.

3. Inhibitors block cholera toxin-induced intestinal fluid secretion without affecting absorption
Antidiarrheal efficacy was assayed in closed mid-jejunal loops in mice. Loops were injected with saline or solutions of cholera toxin containing different inhibitor amounts. Intestinal fluid secretion at 6 h was measured. The data summary in Fig. 3 (left) shows a loop weight-to-length ratio (corresponding to 100% inhibition) of 0.09 in saline-injected loops, and 0.28 (corresponding to 0% inhibition) in cholera toxin-injected loops. Each compound inhibited loop secretion in a dose-dependent manner with essentially complete inhibition at the higher amounts.

View larger version (22K): [in this window] [in a new window]   Figure 3. Antidiarrheal properties of CFTR inhibitors. Left: Inhibition of cholera toxin-induced fluid secretion. Loops were injected with saline or saline containing 1 µg cholera toxin (CT) with indicated compound amounts. Loop weight-to-length ratio was measured at 6 h (SD, n=3–5 mice). Right: Schematic of enterocyte showing CFTR-dependent chloride secretion producing sodium and water secretion. CFTR inhibition by an external pore blocker reduces the volume of secretory diarrhea by inhibiting chloride and consequently net fluid secretion without impairing fluid absorption.

To determine whether the compounds affected intestinal fluid absorption, measurements of remaining fluid were done in closed intestinal loops at 20 and 30 min after injection of 100 µL saline containing 10 mM glucose (without or with compounds). There was no significant effect of the CFTR inhibitors on intestinal fluid absorption. Toxicity studies in cell cultures (50–100 µM for 2 days) and in mice (4–8 mg/Kg, twice daily for 4 days) showed no detectable toxicity. LC/MS analysis indicated that the compounds were highly stable at pH > 4 and in the presence of intestinal contents.

CONCLUSIONS AND SIGNIFICANCE

CFTR is a unique target for antidiarrheal therapy because of its location at the lumen-facing surface of enterocytes and its role as the rate-limiting step in ion secretion caused by several enterotoxins including cholera toxin (Fig. 3 , right). The glycine hydrazide-based CFTR inhibitors synthesized here undergo little intestinal absorption and are effective in preventing cholera toxin-induced fluid secretion in a rodent model of intestinal fluid secretion. The potential advantages of antidiarrheal therapy using a nonabsorbable compound are that high concentrations can be achieved in the gut lumen with minimal concerns about toxicity and off-target effects related to cellular uptake and systemic absorption. The nonabsorbable CFTR inhibitors reported here can be synthesized at relatively low cost, which is an important consideration for antidiarrheal applications for use in developing countries.

The mainstay of current therapy in cholera is oral rehydration solution (ORS) therapy to prevent the consequences of massive volume and electrolyte depletion. Cholera toxin and other enterotoxins produce diarrhea by binding to ganglioside receptors at the enterocyte lumen, resulting in elevated cyclic nucleotide concentrations, protein kinase activation, and CFTR phosphorylation. Vaccines and antibiotic therapy aimed at reducing bacterial load are under clinical evaluation. Alternative strategies to reduce intestinal fluid losses in cholera have been proposed, such as enkephalinase inhibition to reduce cyclic nucleotide concentration, and inhibition of toxin binding to cell surface receptors. CFTR inhibition is predicted to reduce intestinal fluid losses in cholera and other enterotoxin-mediated diarrheas, and may be of particular benefit in young and elderly subjects where morbidity and mortality remain high despite ORS therapy, as well as where ORS therapy is not available or practical. Also, since ORS therapy does not reduce stool volume or the duration of diarrhea, CFTR inhibitors may have a role in reducing the duration and clinical severity of cholera. Large animal testing and clinical studies will be needed to determine the utility of the nonabsorbable CFTR inhibitors developed here.

FOOTNOTES

To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.05-4818fje;

This article has been cited by other articles:

				B. Yang, N. D. Sonawane, D. Zhao, S. Somlo, and A. S. Verkman Small-Molecule CFTR Inhibitors Slow Cyst Growth in Polycystic Kidney Disease J. Am. Soc. Nephrol., July 1, 2008; 19(7): 1300 - 1310. [Abstract] [Full Text] [PDF]

				N. D. Sonawane, O. Zegarra-Moran, W. Namkung, L. J. V. Galietta, and A. S. Verkman {alpha}-Aminoazaheterocyclic-Methylglyoxal Adducts Do Not Inhibit Cystic Fibrosis Transmembrane Conductance Regulator Chloride Channel Activity J. Pharmacol. Exp. Ther., May 1, 2008; 325(2): 529 - 535. [Abstract] [Full Text] [PDF]

				C. Routaboul, C. Norez, P. Melin, M.-C. Molina, B. Boucherle, F. Bossard, S. Noel, R. Robert, C. Gauthier, F. Becq, et al. Discovery of {alpha}-Aminoazaheterocycle-Methylglyoxal Adducts as a New Class of High-Affinity Inhibitors of Cystic Fibrosis Transmembrane Conductance Regulator Chloride Channels J. Pharmacol. Exp. Ther., September 1, 2007; 322(3): 1023 - 1035. [Abstract] [Full Text] [PDF]

				A. Reynolds, A. Parris, L. A. Evans, S. Lindqvist, P. Sharp, M. Lewis, R. Tighe, and M. R. Williams Dynamic and differential regulation of NKCC1 by calcium and cAMP in the native human colonic epithelium J. Physiol., July 15, 2007; 582(2): 507 - 524. [Abstract] [Full Text] [PDF]

This Article Full Text (PDF) All Versions of this Article: 20/1/130 05-4818fjev1    most recent Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Sonawane, N. D. Articles by Verkman, A. S. Search for Related Content PubMed PubMed Citation Articles by Sonawane, N. D. Articles by Verkman, A. S.