Prevention of toxin-induced intestinal ion and fluid secretion by a small-molecule CFTR inhibitor  Jay R. Thiagarajah, Talmage Broadbent, Emily Hsieh, Alan S. Verkman  Gastroenterology  Volume 126, Issue 2, Pages 511-519 (February 2004) DOI: 10.1053/j.gastro.2003.11.005

Figure 1 Characterization of mouse closed-intestinal loop model. (A) Intestinal loops were injected with 200 μL buffer and loop weight measured at indicated times (mean ± SEM, 4 mice per time point). Inset (top): Chemical structure of CFTRinh-172. Inset (lower): Percentage fluid absorption at 30 minutes with and without CFTRinh-172 (20 μg IP, n = 4) in wild-type mice and with and without forskolin (20 μmol/L)/IBMX (100 μmol/L) in CF mice (n = 3). (B) Time course of cholera and STa toxin-induced fluid secretion in mouse closed-loop model. Dashed line shows control (saline-injected) loops of same mice. Data for injected loops (1 μg cholera toxin/loop, 0.1 μg STa toxin/loop) as mean ± SEM (4–6 mice). Gastroenterology 2004 126, 511-519DOI: (10.1053/j.gastro.2003.11.005)

Figure 2 CFTRinh-172 inhibition of intestinal fluid secretion after cholera toxin in mice. (A) Dose response for inhibition of fluid accumulation in mouse loop model. Mice were given single doses of CFTRinh-172 by intraperitoneal injection, and loop weight (mean ± SEM, 4–6 mice per dose) was measured at 6 hours. Dashed line indicates average weight in saline-injected control loops of same mice. (B) Persistence of CFTRinh-172 inhibition. Mice were injected with 20 μg CFTRinh-172 (IP) at indicated times before or after cholera toxin administration (4–6 mice per time point). (C) Time course of plasma 14C-CFTRinh-172 radioactivity after IV injection (tail vein, left ordinate) and oral administration (CFTRinh-172 in TPGS, right ordinate). Data shown as counts per minutes per μCi injected (4 mice). (D) 14C-CFTRinh-172 accumulation in gastrointestinal organs at 6 hours after IV and oral 14C-CFTRinh-172 administration (4 mice). (E) Inhibition of cholera toxin-induced fluid secretion by orally administered CFTRinh-172 (200 μg in TPGS) in mouse open-loop model. Data shown as ratio of weight of entire small intestine 6 hours after oral gavage before vs. after luminal fluid removal (mean ± SEM, 4 mice per group, ∗P < 0.01). (F) CFTRinh-172 permeability across Caco-2 monolayers (mean ± SEM, 18 inserts) with Papp = 16 × 10−6 cm/s. Gastroenterology 2004 126, 511-519DOI: (10.1053/j.gastro.2003.11.005)

Figure 3 CFTRinh-172 inhibition of STa and cholera toxin-induced fluid secretion in mouse and rat closed-loop models. (A) Dose response for inhibition of STa toxin-induced fluid accumulation in mouse loop model. Mice were given single doses of CFTRinh-172 by intraperitoneal injection, and loop weight (mean ± SEM, 4–6 mice per dose) was measured at 5 hours. Dashed line indicates average weight in saline-injected control loops of same mice. (B) Intestinal fluid accumulation in CF mice. Loops were injected with cholera (1 μg) or STa toxin (0.1 μg) (4–6 mice per group), ∗P < 0.05. Inhibition of cholera toxin (C) and STa toxin (D) induced fluid secretion in rat intestinal loops (SEM, 4 rats per group), ∗P < 0.01. Gastroenterology 2004 126, 511-519DOI: (10.1053/j.gastro.2003.11.005)

Figure 4 CFTRinh-172 inhibition of forskolin- and STa toxin-stimulated short-circuit current in mouse ileum (A) and human ileum (B). STa toxin shown as upper inset; no-inhibitor time control as insets. Data representative of studies on 5 mice and 2 sets of human tissues. CFTRinh-172 was added to both sides of tissue. Glucose (10 mmol/L) was present in all solutions. Half-maximal inhibition was 9 ± 3 μmol/L for forskolin-stimulated short-circuit current in mouse ileum. Amiloride (10 μmol/L) was present in the apical solutions. Gastroenterology 2004 126, 511-519DOI: (10.1053/j.gastro.2003.11.005)

Figure 5 Short-circuit analysis of CFTRinh-172 inhibition of Cl− secretion in T84 colonic epithelial cells. (A) Data shown as representative traces from experiments on 5–12 inserts per condition. CFTRinh-172 was added to both sides of cell layers. CFTR agonists include forskolin (left), 8-Br-cGMP (middle), and CFTRact-16 (right). Insets show no-inhibitor time controls for each agonist. (B) Left: CFTRinh-172 inhibition of forskolin-stimulated short-circuit current after basolateral permeabilization with amphotericin B (250 μg/mL). Representative of 6 experiments. Middle: Average dose response for CFTRinh-172 inhibition of forskolin-stimulated (circles) and 8-Br-cGMP-stimulated (triangles) short-circuit current in permeabilized vs. nonpermeabilized T84 cells (mean ± SEM, 6–12 inserts). Right: CFTRinh-172 inhibition of forskolin-stimulated short-circuit current in the presence of high K+ (68 mmol/L) in the basolateral solution with low Cl− in the apical solution. Representative of 4 experiments. Gastroenterology 2004 126, 511-519DOI: (10.1053/j.gastro.2003.11.005)