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GASTROINTESTINAL, HEPATIC, PULMONARY, AND RENAL

Talniflumate Increases Survival in a Cystic Fibrosis Mouse Model of Distal Intestinal Obstructive Syndrome

Dalton Cardiovascular Research Center (N.M.W., J.E.S., K.T.B., L.L.C.) and Department of Biomedical Sciences (J.E.S., L.L.C.), University of Missouri, Columbia, Missouri; and Genaera Corporation (R.C.L.), Plymouth Meeting, Pennsylvania

Received for publication August 30, 2005
Accepted December 13, 2005.

	Abstract

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Intestinal disease in cystic fibrosis (CF) mice closely mirrors aspects of obstructive syndromes in CF patients. The pathogenesis involves accumulation of mucoid debris in the crypts that fuse with intestinal content to form obstructing mucofeculant impactions. Treatment involves modalities that increase the fluidity of the luminal content, such as osmotic laxatives and liquid diets. We investigated the effects of talniflumate (Lomucin, Genaera Corporation, Plymouth Meeting, PA), a compound that may be beneficial to treatment of CF intestinal disease based on three mechanisms of action: mucus synthesis inhibition by blockade of the murine calcium-activated chloride channel 3 (mCLCA3), nonsteroidal anti-inflammatory effects, and inhibition of Cl^–/HCO ^–₃ exchanger(s) involved in intestinal NaCl absorption. Cohorts of CF mice were fed control diet or diets containing either talniflumate (0.4 mg/g chow) or ibuprofen (0.4 mg/g chow) for 21 days to assess survival. Talniflumate significantly increased CF mouse survival from 26 to 77%, whereas ibuprofen had no effect (22% survival). Oral talniflumate did not alter crypt goblet cell numbers or change intestinal expression of mCLCA3 but tended to decrease crypt mucoid impaction. Ussing chamber studies indicated that talniflumate slightly increased the basal short-circuit current of CF intestine, but the change was not sensitive to secretagogue stimulation or bumetanide inhibition. In contrast, intracellular pH measurements of intact intestinal villous epithelium indicated that talniflumate significantly inhibited apical membrane Cl^–/HCO ^–₃ exchange by >50%. We conclude that oral talniflumate increases the survival of CF mice, possibly by the beneficial effects of decreasing small intestinal NaCl absorption through the inhibition of apical membrane Cl^–/HCO ^–₃ exchanger(s).

Talniflumate (Lomucin, Genaera Corporation, Plymouth Meeting, PA) is a phthalidyl ester of niflumic acid. Previous studies have shown that niflumic acid is an inhibitor of the calcium-activated chloride channel hCLCA1 [and its murine homolog, murine calcium-activated chloride channel 3 (mCLCA3), alias gob-5] (Pauli et al., 2000; Zhou et al., 2002). Because the expression of hCLCA1 has been closely associated with increased mucin production by epithelia of CF and asthmatic patients (Toda et al., 2002; Hauber et al., 2003), the proposed mechanism of action for talniflumate or niflumic acid is inhibition of hCLCA function in mucus overproduction (Melton, 2002). This hypothesis has been strengthened by demonstrations that these compounds inhibit mucin synthesis and release in cell culture and animal model systems (Zhou et al., 2002; Bertrand et al., 2004) (M. McLane, K. J. Holroyd, and R. C. Levitt, unpublished data). However, drugs of this class possess other pharmacological properties, including nonsteroidal anti-inflammatory activity through an inhibitory action on cyclooxygenases (Insel, 1996) and inhibition of Slc26a Cl^–/HCO ^–₃ exchanger activity (i.e., Slc26a3, alias down-regulated in adenoma, and Slc26a6, alias putative anion transporter-1 or chloride formate exchanger), which play a major role in NaCl absorption across the intestine (Jacob et al., 2002; Wang et al., 2005).

The combination of these properties (mucus synthesis inhibitor, nonsteroidal anti-inflammatory drug, Cl^–/HCO ^–₃ exchange inhibitor) in an orally tolerated drug may be useful in the treatment of CF intestinal disease. Therefore, we tested whether oral talniflumate treatment would reduce mortality resulting from obstructive disease in CF mice after acute withdrawal of PEG laxative treatment. These results were compared with the effect of oral ibuprofen, a cyclooxygenase inhibitor of the same class that does not inhibit mucin production (Zhou et al., 2002). In additional studies of CF mice maintained on PEG laxative, we evaluated the effect of talniflumate on goblet cell number, crypt lumen distension, mCLCA3 expression, transepithelial bioelectric properties, and Cl^–/HCO ^–₃ exchange in intact intestinal preparations.

	Materials and Methods

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Animals. Mice (23–45 days old) homozygous for gene-targeted disruption of the murine homolog of cftr (S489X mutation, cftr^tm1UNC mice) or homozygous for the cftr F508 mutation (cftr^tm1Kth mice) and maintained on a C57BL/6J background were used (Snouwaert et al., 1992; Zeiher et al., 1995). In some experiments, litter mate cftr+/+ mice were used as wild-type (WT) controls. The mutant mice were identified by using a PCR-based analysis of tail snip DNA, as previously described (Clarke and Harline, 1996). Differences in survival parameters (see below) between the two CF mouse models were not found; therefore, data from the two groups of mice were combined using a ratio of approximately 25% cftr^tm1UNC/75% cftr^tm1Kth mice (CF mice). All mice were maintained ad libitum on standard laboratory chow (Formulab 5008 Rodent Chow; Purina, St. Louis, MO). Drinking water contained an oral osmotic laxative (Colyte; Schwartz Pharma, Seymour, IN) with the following composition: 60.00 g/l PEG, 1.46 g/l NaCl, 0.75 g/l KCl, 1.68 g/l NaHCO₃, and 5.68 g/l Na₂SO₄. The PEG-treated drinking water was provided ad libitum until the experimental period. The mice were housed singly in temperature (22–26°C) and light (12-/12-h light/dark cycle)-controlled rooms in the Association for Assessment and Accreditation of Laboratory Animal Care-accredited animal facility at the Dalton Cardiovascular Research Center. Mice were sacrificed by CO₂ asphyxiation followed by a surgically induced pneumothorax. All experiments involving animals were approved by the University of Missouri Animal Care and Use Committee.

Survival Studies. Survival studies were conducted by replacement of the PEG laxative drinking water with tap water, a regimen that has been shown previously to result in high mortality (>90%) from obstructive intestinal impactions for CF mice maintained on a solid diet (Clarke et al., 2004). Four days before PEG withdrawal (i.e., the "lead-in" period), the standard mouse chow was replaced with pulverized mouse chow containing 0.4 mg/g talniflumate (i.e., 0.4 mg of talniflumate/g chow), 0.4 mg/g ibuprofen, or untreated (control). To minimize wastage of the pulverized diet, the food was placed in a weighted glass dish covered by 1.3-cm galvanized wire mesh that allowed "head-only" access. After PEG withdrawal but with continuance of the experimental diets, the mice were weighed daily. The mean body weight for all groups was similar after the lead-in period (control, 16.9 ± 0.8 g, n = 19; talniflumate, 17.3 ± 1.0 g, n = 13; ibuprofen, 16.1 ± 0.9 g, n = 9; N.S.). Daily water consumption was also measured and was similar among all groups (control, 0.34 ± 0.02; talniflumate, 0.35 ± 0.02; ibuprofen, 0.29 ± 0.03 ml/g body weight/day; N.S.). The mice were sacrificed 21 days after PEG laxative withdrawal or when body weight declined by >2 g in 1 day or >1 g for 2 consecutive days. Upon necropsy, all CF mice that died spontaneously or that were sacrificed prior to 21 days were found to have an obstructing impaction in either the proximal ileum or at the ileal-cecal junction of the intestine. In a separate survival study, CF mice were provided a reduced concentration of PEG laxative (70%) in the drinking water and consumed either the talniflumate or control diet.

Ussing Chamber Studies. CF mice were maintained with 70% PEG laxative in the drinking water and fed either the talniflumate or control diet for 7 to 10 days. At the conclusion of this period, the mice were sacrificed, and sections of proximal jejunum and cecum were immediately removed for bioelectric measurements, histology, or mRNA expression studies (see below). For bioelectric measurements, intestinal sections were mounted full thickness in standard Ussing chambers (0.238-cm² exposed surface area) as previously described (Clarke and Harline, 1996). Briefly, the intestinal preparations were bathed on the mucosal and serosal surfaces with warmed (37°C) Krebs bicarbonate Ringers containing 115 mM NaCl, 2.4 mM K₂HPO₄, 0.4 mM KH₂PO₄, 25 mM NaHCO₃, 1.2 mM CaCl₂, and 1.2 mM MgCl₂ and gassed with 95% O₂/5% CO₂, pH 7.4. Glucose (10 mM) was added to the serosal bath; mannitol (10 mM) was substituted for glucose in the mucosal bath to avoid an inward current because of Na⁺-coupled glucose cotransport (Clarke et al., 1992). Transepithelial short-circuit current (I_sc, in microamperes per centimeter squared) was measured using an automatic voltage clamp (VCC-600; Physiologic Instruments, San Diego, CA) and total tissue conductance (G_t, millisiemens per centimeter squared tissue surface area) was determined every 5 min by measuring the current deflections resulting from a 5-mV transepithelial pulse and applying Ohm's law. The serosal bath served as ground in all experiments. For experiments measuring the I_sc response to stimulation of intracellular cAMP or Ca²⁺, the jejunal sections were treated with either 10 µM forskolin (added to mucosal and serosal baths) or 100 µM carbachol (added to the serosal bath), respectively. For experiments measuring Na⁺-coupled glucose current, the I_sc was recorded before and after the addition of 10 mM glucose to the mucosal bath. For pH stat method to measure net HCO ^–₃ secretion, the luminal bath did not contain HCO ^–₃ and was vigorously gassed with 100% O₂. The bath pH was clamped at 7.4 by neutralizing the appearance of base with 5 mM HCl using an automatic titrator (Radiometer Analytical, Lyon, France). The serosal-to-mucosal flux of bicarbonate (J_smHCO₃) was measured as the steady-state rate of H⁺ equivalents required for neutralization per hour and normalized to tissue surface area (microequivalents per centimeter squared per hour).

Northern Blot Analysis. Northern blot analysis of total mRNA was performed as previously described (Clarke et al., 2004). Total RNA from murine duodenum was extracted using TRI-Reagent (Molecular Research Center, Cincinnati, OH), according to manufacturer's instructions. RNA was mixed with Glyoxal sample buffer (Bio-Whittaker Molecular Applications, Rockland, ME), separated by 1% agarose gel electrophoresis, and transferred to a Hybond-N⁺ nylon membrane (Amersham Biosciences, Piscataway, NJ). The blot was probed using -dCTP ³²P-labeled cDNA PCR products for mCLCA3 and the L32 ribosomal protein. For the mCLCA3 probe, the RT-PCR product was obtained using the sense and antisense oligonucleotide primers with the following sequences: 5'-GAAAGCTGCAGGATGGAATC-3' and 5'-GACTGGTTGATTTCTTGCCTG-3'. For the L32 probe, the RT-PCR product was obtained using the sense and antisense oligonucleotide primers with the following sequences: 5'-CATCTGTTTTACGGCATCATG-3' and 5'-AGCTCCCATAACCGATGTTGG-3'. Radiographic density of bands was measured using a Kodak Imaging Station 2000R (Eastman Kodak, Rochester, NY), and the expression level of mCLCA3 was normalized to L32.

Histology. Jejunal or cecal sections were fixed in buffered 2.5% glutaraldehyde/2.0% paraformaldehyde, embedded in paraffin for sectioning (5-µm thickness), and stained with either H&E or Alcian Blue, pH 2.5, with a periodic acid Schiff counterstain. Using an upright microscope (BX50WI; Olympus, Tokyo, Japan), histological sections were scanned at low power for longitudinal crypt crosssections extending from the base to crypt mouth (three crypts/section/mouse). Crypts were photographed using a SensiCam digital camera (Cooke, Auburn Heights, MI) at 100x magnification (40x objective plus 2.5x photo eyepiece), and morphological measurements of the crypt lumen diameter, crypt diameter, or number of goblet cells/crypt were obtained using ImagePro Plus (Media Cybernetics, Carlsbad, CA).

Intracellular pH Measurement of Villous Epithelium. The method used for imaging villous epithelial cells in intact murine intestine has been previously described (Simpson et al., 2005). Briefly, WT litter mates of CF mice, i.e., cftr+/+, were sacrificed, and proximal duodenum was removed, opened longitudinally, and the mucosa was stripped of the underlying muscle layers. The intestinal segment was mounted apical side up on a horizontal perfusion chamber in which mucosal and serosal surfaces were independently bathed. The intestinal preparations were treated with indomethacin (1 µM, mucosal and serosal baths) and tetrodotoxin (0.1 µM, serosal bath) to minimize the effect of endogenous prostaglandins and neural tone, respectively (Bukhave and Rask-Madsen, 1980; Sheldon et al., 1988). Villi were immobilized under a fine nylon mesh and washed for 5 min with a solution containing 100 µM DL-dithiothreitol. The duodenum was incubated in the presence of 16 µM 2',7'-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein acetoxymethyl ester (BCECF-AM) for 10 min on the apical side in an isethionatebicarbonate Ringer solution (IBR) containing 140.0 mM Na⁺, 55.0 mM Cl^–, 55.0 mM isethionate^–, 25.0 mM HCO ^–₃, 5.2 mM K⁺, 5.0 mM N-Tris methyl-2-aminoethanesulfonic acid, 4.8 mM gluconate^–, 2.8 mM PO₄^2–, 1.2 mM Ca²⁺, 1.2 mM Mg²⁺, 10.0 mM glucose, and 6.8 mM mannitol that was gassed with 95% O₂/5% CO₂ at 37°C, pH 7.4. The basolateral superfusate consisted of a Cl^–-free IBR (Cl^– replaced with isethionate^–) gassed with 95% O₂/5% CO₂ at 37°C, pH 7.4, and contained 1 µM 5-(N-ethyl-n-isopropyl)-amiloride to block the activity of Na⁺/H⁺ exchanger isoform 1. During BCECF-AM incubation, duodenal villi were viewed at 100x magnification (40x water immersion objective plus 2.5x photo eyepiece), and 10 epithelial cells from the midregion of a single villus were selected for ratiometric analysis. Changes in intracellular pH (pH_i) were measured by the dual-excitation wavelength technique (440 and 495 nm), and the villi were imaged at a 535-nm emission wavelength. Ratiometric images were acquired at 20-s intervals with a Sensi-Cam digital camera (Cooke), and images were processed using Axon Imaging Workbench 2.2 (Axon Instruments, Union City, CA). The 495-:440-nm ratios were converted to pH_i using a standard curve generated by the K⁺/nigericin technique (Thomas et al., 1979; Boyarsky et al., 1998). Experiments to measure Cl^–/HCO ^–₃ exchange consisted of pH_i alkalization induced by replacement of apical Cl^– with isethionate^– on an equimolar basis. After a stable pH_i was obtained (approximately 2 min), pH_i recovery was initiated by replacing isethionate^– with Cl^–, as previously described (Simpson et al., 2005). Rates of anion exchange during alkalization and recovery (pH_i/t) were calculated from a linear regression of the values from the first 90 s of the initial pH_i changes during Cl^– removal and replacement, respectively. For studies of talniflumate treatment, the intestinal preparation was exposed to either 100 µM talniflumate or vehicle [0.4% dimethyl sulfoxide (DMSO)] in the apical bath for 5 min prior to the measurements of Cl^–/HCO ^–₃ exchange activity.

Materials. Talniflumate (Lomucin) and mouse chow diets containing talniflumate, ibuprofen, or vehicle were generously provided by Genaera Corporation. The fluorescent dye BCECF-AM was obtained from Molecular Probes (Eugene, OR). Tetrodotoxin was obtained from Biomol International L.P. (Plymouth Meeting, PA). All other materials were obtained from either Sigma Aldrich (St. Louis, MO) or Fisher Scientific (Springfield, NJ). For drug treatment of the in vitro preparations, talniflumate was added from a 100 mM stock solution in DMSO, forskolin was added from a 10 mM stock in DMSO, and carbachol was added from a 100 mM stock in IBR.

Statistics. A Kaplan-Meier log-rank analysis with a post hoc Holm-Sidak test for all pair-wise comparisons was used to compare survival curves between treatment groups. A Student's t test assuming equal variances was used to compare data for two treatment groups, and a one-way analysis of variance with a post hoc Bonferroni's test was used to compare data from more than two treatment groups. p < 0.05 was considered statistically significant. All data are expressed as mean ± S.E.M.

	Results

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Talniflumate But Not Ibuprofen Increases the Survival of CF Mice. Mucus and debris accumulating within the intestinal crypts can fuse with luminal contents, resulting in an obstructing impaction and the fatal sequela of peritonitis in untreated CF mice (Snouwaert et al., 1992). To test the efficacy of talniflumate in preventing fatal intestinal disease, CF mice initially treated with a PEG laxative in the drinking water were divided into three groups with different diets (control, talniflumate, and ibuprofen). The ibuprofen group was included because compounds of this class have known nonsteroidal anti-inflammatory activity (Insel, 1996), and ibuprofen has been shown to have beneficial effects in the treatment of CF patients (Konstan et al., 1995). The CF mice in the three groups consumed similar amounts of the diets (control, 0.22 ± 0.01; talniflumate, 0.22 ± 0.04; and ibuprofen, 0.15 ± 0.06 g/g body weight/day, N.S.), resulting in an average daily dose of 88 mg talniflumate/kg body weight or 60 mg ibuprofen/kg body weight. As shown in Fig. 1A, 14 of 19 (73.7%) CF mice on the control diet died during the 21-day study period, with greatest loss occurring between days 4 and 10. Of the CF mice consuming the ibuprofen diet, seven of nine (77.8%) died during the survival study period. Most CF mice on the ibuprofen diet died within 4 days (66%), suggesting an additional untoward effect of the ibuprofen; however, the greatest loss in the control group also occurred early in the study (42.1% loss within 6 days), and differences between the two survival curves were not statistically significant. In contrast to the control and ibuprofen groups, only 3 of 13 (23.1%) CF mice consuming the talniflumate diet died during the 21-day period. The survival for CF mice consuming the talniflumate diet was significantly increased compared with that for CF mice consuming either the control or ibuprofen diets.

View larger version (18K): [in this window] [in a new window]   Fig. 1. Survival curves. A, survival after withdrawal of a PEG osmotic laxative for CF mice consuming talniflumate (88 mg/kg body weight/day, n = 13), ibuprofen (60 mg/kg body weight/day, n = 9), or control diet (n = 19). a and b, curves with different letters are significantly different. B, survival of mice maintained on 70% polyethylene glycol osmotic laxative (PEG concentration that yields 50% survival) in the drinking water and consuming either control (n = 8) or talniflumate (88 mg/kg body weight/day, n = 7) diets (log-rank test, p < 0.082).

Withdrawal of the PEG laxative from the CF mice results in the highest mortality rates during the first few days (<10 days) after the switch to untreated drinking water. We questioned whether the beneficial effects of talniflumate would also be observed in CF mice that were subjected to a less severe disease insult. Preliminary studies indicated that dilution of PEG laxative to 70% of full-strength yields an approximately 50% mortality rate in untreated CF mice (Walker et al., 2004). Because the greatest mortality occurred within the first 2 weeks after PEG withdrawal (see Fig. 1A), the 14-day survival of CF mice maintained on 70% PEG laxative in the drinking water was compared between mice consuming either the control or talniflumate diets. As shown in Fig. 1B, all CF mice on the talniflumate diet survived, whereas 37.5% of the CF mice consuming the control diet died during the 14-day study period.

Talniflumate Treatment Does Not Significantly Affect Goblet Cell Numbers or Mucus Impaction in the Intestinal Crypts of CF Mice. It is well documented that the CF mouse intestine recapitulates the histopathological appearance of intestinal disease in CF patients, which is hallmarked by goblet cell hyperplasia and mucus impaction of the crypts (Grubb and Gabriel, 1997). Previous investigations in cell culture systems and animal disease models have shown that talniflumate treatment can significantly decrease rates of mucus production (Melton, 2002; Zhou et al., 2002). Therefore, we evaluated goblet cell numbers and the ratio of crypt lumen diameter to crypt diameter (as a measure of mucus impaction of the intestinal crypts) in CF mice treated with either the talniflumate or control diets. Because water consumption often decreases for 1 to 2 days after switching from PEG-containing drinking water to tap water (data not shown), the CF mice were maintained on 70% PEG laxative in the drinking water and consumed the test diets for a 7- to 10-day period. At the conclusion of the study, the CF mice were sacrificed, and intestinal samples were taken for histological examination. As shown by the cumulative data in Fig. 2A, goblet cell numbers in the crypts of either the small intestine (jejunum, left panel) or large intestine (cecum, right panel) were not significantly different between the CF mice consuming either the talniflumate or control diets. As a measure of small bowel crypt impaction, the ratio of jejunal crypt lumen diameter/crypt diameter in the two groups of CF mice was measured as shown diagrammatically in Fig. 2B, right panel. The cumulative data from these measurements (Fig. 2B, left panel) indicated that the mean crypt lumen/crypt diameter ratio for the talniflumate group was slightly, although nonsignificantly, reduced compared with that in CF mice on the control diet. Thus, we cannot rule out the possibility that talniflumate has a beneficial effect on reducing crypt impaction in CF mice.

View larger version (42K): [in this window] [in a new window]   Fig. 2. A, comparison of goblet cell number per crypt cross-section in the jejunum (left panel) and the cecum (right panel) for CF mice maintained on 70% PEG osmotic laxative and consuming either control or talniflumate (88 mg/kg body weight/day) diet. Bars, mean data from six mice (two to three crypts/mouse) consuming the control diet (jejunum, total of 17 crypts; cecum, total of 18 crypts), and six mice (two to three crypts/mouse) consuming the talniflumate diet (jejunum, total of 18 crypts; cecum, total of 21 crypts). B, left panel, ratio of crypt lumen diameter to total crypt diameter of jejunal crypt cross-sections for CF mice maintained on 70% PEG osmotic laxative and consuming either control or talniflumate diet. Bars, mean data from six mice (three crypts/mouse for total of 18 crypts) consuming the control diet, and seven mice (three crypts/mouse for total of 18 crypts) consuming the talniflumate diet. Right panel, photomicrograph depicting measurements of crypt lumen diameter (short lines) and crypt diameter (long lines) for lumen/crypt diameter ratio of the jejunum.

View larger version (36K): [in this window] [in a new window]   Fig. 3. Top panel, Northern blot of mCLCA3 and L32 mRNA expression in the small intestine of CF mice maintained on 70% PEG osmotic laxative and consuming either control or talniflumate (88 mg/kg body weight/day) diet. Bottom panel, densitometric measurements of mCLCA3 expression normalized to L32 expression for control or talniflumate-treated intestine (n = 3).

View larger version (15K): [in this window] [in a new window]   Fig. 4. Isc of jejuna from CF mice maintained on 70% PEG in the drinking water and consuming either control or talniflumate (88 mg/kg body weight/day) diet for 7 days (n = 6). Left panel, Isc under basal conditions and following treatment with forskolin (10 µM, mucosal and serosal bath treatment) or carbachol (100 µM, serosal bath treatment). Dashed line, average basal Isc for WT (Gawenis et al., 2003). Right panel, Isc following addition of 10 mM glucose to the mucosal bath. Transepithelial resistances were not different between control and talniflumate intestine (data not shown). *, significantly different from control.

Talniflumate Significantly Inhibits Cl^–/HCO ^–₃ Activity in Murine Intestinal Epithelium in Vitro. At least two members of the Slc26a family of sulfate transporters (Slc26a3 and Slc26a6) provide Cl^–/HCO ^–₃ exchange function at the apical membrane of intestinal epithelia (Jacob et al., 2002; Wang et al., 2005). These anion exchangers provide important functions of bicarbonate secretion and Cl^– absorption across the intestine. For example, loss of function mutations in SLC26A3 (alias down-regulated in adenoma) cause the human disease congenital chloride diarrhea (CLD), which is characterized by copious acidic diarrhea resulting from reduced NaCl absorption across the intestine (Mount and Romero, 2004). Studies of recombinant proteins have shown that the activity of the Slc26a exchangers is significantly inhibited by niflumic acid (Chernova et al., 2003, 2005), and, recently, we have shown that niflumic acid inhibits 60% of the basal Cl^–/HCO ^–₃ exchange activity in native intestinal epithelium from both WT and CF mice (Simpson et al., 2005). To determine whether this effect of niflumic acid is physiologically relevant, we performed ex vivo pH stat measurements of net HCO ^–₃ secretion across native murine small intestine and found that 100 µM niflumic acid decreased net HCO ^–₃ secretion by 62.6% [net HCO ^–₃ secretory flux (in microequivalents per centimeter squared per hour): control = 2.3 ± 0.2; niflumic acid = 0.9 ± 0.2, n = 11, p < 0.05].

Because niflumic acid significantly inhibits apical membrane Cl^–/HCO ^–₃ exchange in both WT and CF mouse intestine (Simpson et al., 2005), we asked whether its phthalate derivative talniflumate also inhibits Cl^–/HCO ^–₃ exchange activity in the murine small intestine. Using WT villous epithelium, microfluorometry studies show that robust Cl^–/ HCO ^–₃ exchange activity is present at the apical membrane of the villous epithelium. As demonstrated in Fig. 5A, the epithelial cells alkalize when Cl^– is removed from the luminal bath because the Cl^–/HCO ^–₃ exchanger(s) operate in the "reverse" mode, i.e., Cl^–_out/HCO ^–_3in exchange. After readdition of luminal Cl^–, the cells acidify as the exchanger(s) operate in the "forward" mode, i.e., Cl^–_in/HCO ^–_3out, and the rate of Cl^–/HCO ^–₃ exchange is measured during the initial linear phase of pH_i changes. To evaluate the effect of talniflumate on this transport process, WT intestinal epithelium was treated with either 100 µM talniflumate or vehicle (0.4% DMSO) for approximately 5 min before measuring Cl^–/ HCO ^–₃ exchange activity by Cl^– removal and replacement. As shown in Fig. 5B, talniflumate significantly inhibited the rate of Cl^–/HCO ^–₃ exchange by 55% compared with vehicle control. Thus, talniflumate, like niflumic acid, inhibits the complement of apical membrane anion exchangers in the murine intestinal epithelium.

View larger version (19K): [in this window] [in a new window]   Fig. 5. A, representative experiment showing measurement of Cl–/ HCO –3 exchange in intact WT intestinal villous epithelial cells (n = 6). Following Cl– removal from the luminal perfusate, Cl– exits the cell in exchange for HCO –3 and increases the pHi. Return of Cl– to luminal perfusate results in the Cl– entry in exchange for HCO –3, which decreases pHi. B, rates of Cl–/HCO –3 exchange activity in the villous epithelial cells of WT murine small intestine treated with either talniflumate (100 µM, mucosal bath treatment) or vehicle (DMSO) control. *, significantly different from vehicle control (n = 4–5).

	Discussion

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Survival studies of CF mice consuming either the talniflumate or control diets while simultaneously being treated with 70% PEG laxative in the drinking water were used to control for confounding factors associated with abrupt PEG withdrawal. For example, we had noted that water consumption often decreased for 1 to 2 days immediately after switching from PEG-containing drinking water to tap water (data not shown). Preliminary studies indicated that dilution of the PEG laxative to 70% of full strength resulted in an approximately 50% effective concentration with respect to CF mouse survival. Using 70% PEG laxative in the drinking water, survival of CF mice consuming the control diet was 62.5%, whereas 100% of CF mice consuming the talniflumate diet survived. Thus, talniflumate treatment appeared to have a beneficial effect even when disease severity was lessened by simultaneous laxative treatment.

Goblet cell metaplasia and mucus overproduction are fundamental to the pathogenesis of cystic fibrosis (Welsh et al., 1995). When tested in cell culture systems and animal models of airway disease, talniflumate and its parent compound niflumic acid have been shown to inhibit mucus production (Melton, 2002; Zhou et al., 2002; Bertrand et al., 2004). Investigations into the mechanism of action have largely focused on the ability of these compounds to block activity of the calcium-activated Cl^– channels, hCLCA1 and mCLCA3 (Gandhi et al., 1998; Gruber et al., 1998). These proteins show a strong positive correlation with mucus overproduction in the lungs of interleukin (IL)-9 transgenic mice and in human primary lung cultures treated with IL-4, IL-9, and IL-13 (Toda et al., 2002; Hauber et al., 2003; M. McLane, K. J. Holroyd, and R. C. Levitt, unpublished data). Moreover, expression of hCLCA1 in NCI-H292 cells induces soluble gel-forming mucin production, which is inhibited by niflumic acid treatment (Zhou et al., 2002). Despite the beneficial effects of niflumic acid on these models of mucus production, the role of hCLCA1 Cl^– channel activity in the process of mucus production remains unexplained. Recently, studies of calcium-stimulated granule exocytosis in mucin-secreting cells indicate that niflumic acid interferes with Ca²⁺ influx across the plasma membrane, a process that initiates mucin granule release (Bertrand et al., 2004). Based on evidence that talniflumate may reduce mucus production in the intestine, we examined goblet cell numbers, mCLCA3 expression, and the ratio of crypt lumen diameter/crypt diameter in mice consuming the talniflumate diet and treated with 70% PEG laxative in the drinking water (to avoid spurious effects of impending impaction). However, talniflumate treatment did not result in any additional effects beyond that provided by the 70% PEG laxative treatment. The only positive indicator was a trend toward a reduction of crypt lumen diameter, which is an indirect measure of mucus impaction of the crypt. Thus, the evidence from these basic measurements of intestinal mucus production was not consistent with a major effect of talniflumate on mucus overproduction in the CF intestine, but a beneficial effect of talniflumate cannot be ruled out because changes in mucus production may not be detectable by the methods used or the effect of talniflumate may overlap with the effects of simultaneous PEG laxative treatment.

The transepithelial bioelectric parameters of the intestine from CF mice consuming the talniflumate or control diets, in the presence of 70% PEG laxative, were compared ex vivo in Ussing chamber studies. Talniflumate treatment did not have an appreciable effect on the integrity of the intestine or the paracellular shunt as indicated by the lack of changes in transepithelial conductance (data not shown) and I_sc response to glucose, i.e., Na⁺-coupled glucose absorption. Interestingly, the talniflumate diet caused a slight increase in the basal I_sc, although the change did not normalize the I_sc relative to that of unstimulated intestine from wild-type mice and was not responsive to secretagogue stimulation by forskolin or carbachol. The increased I_sc indicates an inward current that either results from electrogenic anion secretion or cation absorption. However, the I_sc was not sensitive to inhibition of Cl^– secretion with bumetanide, and very little activity of the epithelial Na channel can be detected in the midjejunum of the mouse (Clarke and Harline, 1996). Thus, elucidation of the ionic basis of increased basal I_sc in the intestine of mice consuming the talniflumate diet will require additional studies.

The recent discovery that at least two members of the SLC26A family of anion transporters can function as Cl^–/ HCO ^–₃ exchangers in cell systems and have been immunolocalized to the apical membrane of the intestinal epithelium has given identity to the major proteins involved in intestinal Cl^– absorption (Jacob et al., 2002; Wang et al., 2005). SLC26A6 is localized to the surface epithelium of the small intestine, whereas SLC26A3 is expressed in the surface epithelium throughout the intestinal tract with greatest amounts in duodenum and the large intestine (Melvin et al., 1999; Jacob et al., 2002). Although a genetic disease entity has not been identified with mutations of SLC26A6, loss-of-function mutations in SLC26A3 are known to cause CLD (Mount and Romero, 2004). In individuals affected with CLD, loss of intestinal Cl^– absorption can result in severe diarrheal fluid loss (Kere et al., 1999). It has been shown in heterologous expression studies that SLC26A3 and SLC26A6 are inhibited by the action of niflumic acid (Chernova et al., 2003, 2005). Furthermore, it has been proposed that pharmacological inhibition of the SLC26A transporters in the intestine is a potential therapy for the clinical impaction states that characterize CF intestinal disease (Chernova et al., 2003). Using microspectrofluorometry, we have recently shown that niflumic acid inhibits Cl^–/HCO ^–₃ exchange by 60 to 65% in intact intestinal epithelia from both WT and CFTR-null mice (Simpson et al., 2005). Therefore, using the same technique, we investigated the effect of luminally applied talniflumate on Cl^–/HCO ^–₃ exchange across the apical membrane of villous epithelium in the WT murine duodenum, a site where both Slc26a3 and Slc26a6 are expressed (Jacob et al., 2002; Wang et al., 2002). Talniflumate resulted in >50% reduction in the rate of Cl^–/HCO ^–₃ exchange across the apical membrane of the intestine. These results are consistent with the proposal that talniflumate has a beneficial effect on the survival of CF mice by reducing the incidence of intestinal impactions through pharmacological inhibition of intestinal Cl^– absorption. A diagram depicting the beneficial effect of Cl^–/HCO ^–₃ exchange inhibition in the CF intestine is shown in Fig. 6. In the absence of anion secretion in the CF intestine, NaCl absorptive processes dominate electrolyte transport and contribute to dehydration of the luminal content. Inhibition of apical membrane Cl^–/HCO ^–₃ exchanger(s), similar to the effect of SLC26A3 mutations in congenital chloride-losing diarrhea patients (Kere et al., 1999), reduces transcellular Cl^– absorption, resulting in the retention of (Na⁺)Cl^– and water that increases the fluidity of the luminal content. Thus, therapies directed at the inhibition of the SLC26A Cl^–/HCO ^–₃ exchangers of the intestine may be useful in the treatment of distal intestinal obstructive syndrome in cystic fibrosis patients.

View larger version (21K): [in this window] [in a new window]   Fig. 6. Schematic diagram depicting the beneficial effects of Cl–/HCO –3 exchange inhibition in the treatment of distal intestinal obstructive syndrome in CF patients. A, in CF patients, net NaCl and water secretion are deficient due to the absence of the CFTR anion channel activity and the process of NaCl absorption via coupled Na+/H+, Cl–/HCO –3 exchangers (Na+/H+ exchanger isoform 3 and AE, respectively) dominates in much of the intestine, thereby contributing to dehydration of the luminal content. B, inhibition of apical membrane Cl–/HCO –3 exchanger(s) reduces intestinal Cl– absorption and Na+ absorption (Kere et al., 1999), resulting in the retention of NaCl and water in the lumen that increases the fluidity of the luminal content.

	Acknowledgements

 
We acknowledge the support and technical expertise of Mike McLane and K. J. Holroyd at Genaera Corporation (Plymouth Meeting, PA).

	Footnotes

 
This work was supported by Cystic Fibrosis Foundation Therapeutics, Inc. Grant CLARKE01W0 and by the National Institutes of Health Grant DK44816 (to L.L.C.).

doi:10.1124/jpet.105.094847.

ABBREVIATIONS: CF, cystic fibrosis; CFTR, cystic fibrosis transmembrane conductance regulator; PEG, polyethylene glycol; hCLCA1, human calcium-activated chloride channel 1; mCLCA3, murine calcium-activated chloride channel 3; WT, wild type; PCR, polymerase chain reaction; I_sc, short-circuit current; BCECF-AM, 2',7'-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein acetoxymethyl ester; IBR, isethionate-bicarbonate Ringers; pH_i, intracellular pH; DMSO, dimethyl sulfoxide; CLD, congenital chloride diarrhea; IL, interleukin.

Address correspondence to: Dr. Lane L. Clarke, 324D Dalton Cardiovascular Research Center, 134 Research Park Drive, University of Missouri, Columbia, MO 65211. E-mail: clarkel{at}missouri.edu

	References

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