Sildenafil normalizes bowel transit in preclinical models of constipation

Abstract

Guanylyl cyclase-C (GC-C) agonists increase cGMP levels in the intestinal epithelium to promote secretion. This process underlies the utility of exogenous GC-C agonists such as linaclotide for the treatment of chronic idiopathic constipation (CIC) and irritable bowel syndrome with constipation (IBS-C). Because GC-C agonists have limited use in pediatric patients, there is a need for alternative cGMP-elevating agents that are effective in the intestine. The present study aimed to determine whether the PDE-5 inhibitor sildenafil has similar effects as linaclotide on preclinical models of constipation. Oral administration of sildenafil caused increased cGMP levels in mouse intestinal epithelium demonstrating that blocking cGMP-breakdown is an alternative approach to increase cGMP in the gut. Both linaclotide and sildenafil reduced proliferation and increased differentiation in colon mucosa, indicating common target pathways. The homeostatic effects of cGMP required gut turnover since maximal effects were observed after 3 days of treatment. Neither linaclotide nor sildenafil treatment affected intestinal transit or water content of fecal pellets in healthy mice. To test the effectiveness of cGMP elevation in a functional motility disorder model, mice were treated with dextran sulfate sodium (DSS) to induce colitis and were allowed to recover for several weeks. The recovered animals exhibited slower transit, but increased fecal water content. An acute dose of sildenafil was able to normalize transit and fecal water content in the DSS-recovery animal model, and also in loperamide-induced constipation. The higher fecal water content in the recovered animals was due to a compromised epithelial barrier, which was normalized by sildenafil treatment. Taken together our results show that sildenafil can have similar effects as linaclotide on the intestine, and may have therapeutic benefit to patients with CIC, IBS-C, and post-infectious IBS.

Fig 1. PDE-5 inhibitors increase cGMP and regulate homeostasis in the colon epithelium.

(A) cGMP levels in the intestinal mucosa of untreated mice (Ctrl), or 5 h following gavage (Gav) or intraperitoneal injection (IP) of vardenafil or sildenafil, or mice provided sildenafil in water ad libitum (Sild Water) for 5 days. (B) Daily intake by mice provided water alone (Control) or water containing 35μg/ml sildenafil. (C, D) Quantification of goblet cells (ABPAS) and (E, F) Ki-67- positive cells in colon mucosa of CD-1 mice treated with vardenafil IP (vard) or sildenafil in drinking water (sild) for 5 days. Data are shown as means, error bars represent SEM, n = 6 (A, B), n = 3 (D, F). *P<0.05, ***P<0.001 by two-tailed Student’s t-test.

Fig 2. Sildenafil increases EE cell density in colon mucosa of healthy mice but has no effect on transit time or fecal water content.

(A) Quantification of enteroendocrine cells (CgA) from stained tissue sections from colons of mice treated for five days with sildenafil, linaclotide, or untreated (Ctrl). (B) Quantification of enteroendocrine cells in colons from mice treated with various doses of sildenafil ad libitum in drinking water for five days. (C) CgA-positive cells over time following ad libitum sildenafil treatment. C57/BL6 mice were treated with sildenafil or linaclotide for five days prior to intestinal transit assay of (D) total transit or (E) upper intestinal transit. (F) Fecal pellets were collected from transit assays and measured for fecal water content. Data are shown as means, error bars represent SEM, n = 3 (A-C), n = 12 (D-E). *P<0.05, **P<0.005 by two-tailed Student’s t-test, each treatment group is compared to control (A, B) or time 0 (C).

Fig 3. Mice recovering from DSS-induced inflammation have increased intestinal transit time and fecal water content.

(A) 6–8 week old C57/BL6 mice were administered 3% DSS in drinking water for five days then allowed to recover for 3 weeks. (B) H&E and AB/PAS staining of a section of distal colon from a healthy mouse and a DSS-recovery mouse shows residual inflammation in recovery mice. Arrows show edema (upper panel) and crypt loss (lower panel) in the recovery animals. (C) Intestinal transit time for DSS-recovery mice for different recovery times. (D) Distance that a gavaged bolus of charcoal traveled in 10 minutes in mice recovered from DSS for three-weeks, compared to healthy mice. (E) Fecal water content of healthy and DSS-recovery mice. Data are shown as means, error bars represent SEM, n = 6 (C-E). *P<0.05, ***P<0.001 by two-tailed Student’s t-test.

Fig 4. Sildenafil decreases intestinal transit time in a DSS-recovery model of IBS.

(A,B) Quantification of 5-HT-positive cells in stained sections of colon from healthy mice, or those recovered from DSS treatment (DSS recovery). Mice were either untreated controls (Ctrl) or treated with sildenafil for five days (Sild). (C) Intestinal transit time and (D) distance traveled by a gavaged bolus of charcoal were measured in healthy and DSS-recovery mice. Mice were either untreated (Ctrl), or treated with sildenafil or linaclotide acutely (1 hr; Acute Sild, Acute Lin respectively) and 5-days or (Sild, Lin respectively). Data are shown as means, error bars represent SEM, n = 6 (B), n = 12 (C-D). *P<0.05, ** P<0.005, ***P<0.001 by one-way ANOVA and Tukey’s post hoc analysis.

Fig 5. Sildenafil regulates fecal water content in DSS-recovery mice.

Fecal pellets from healthy mice, or those recovered from DSS treatment (DSS recovery) were collected to measure (A) total wet and dry mass of pellets and (B) fecal water content. Mice were either untreated (Ctrl), or treated with sildenafil or linaclotide acutely (1 hr; Acute Sild, Acute Lin respectively) or 5-days (Sild, Lin respectively). (C) Mice were either provided water (healthy) or 3% DSS for 5 days (DSS). Barrier function was assessed using the FITC-dextran approach as described in the Materials and Methods. Mice exposed to DSS were either untreated (Ctrl) or treated with an acute dose of sildenafil (Sild). (D) Levels of tight junctional proteins claudin 4 and occludin were measured by western blot in mucosa collected from untreated mice (Ctrl) and mice treated sildenafil for 5 days (Sild). Data are shown as means, error bars represent SEM, n = 12 (A, B), n = 6 (C, D). *P<0.05, ***P<0.001 by one-way ANOVA and Tukey’s post hoc analysis.

Fig 6. Sildenafil normalizes transit in loperamide-induced constipation model.

Mice were either provided vehicle (healthy) or 10 mg/kg loperamide (constipated). (A) Total intestinal transit, (B) distance traveled by a gavaged bolus of charcoal, and (C) fecal water content were measured in untreated (Ctrl) or sildenafil treated (Sild) mice as indicated. Data are shown as means, error bars represent SEM, n = 12 (A-C). *P<0.05, ***P<0.001 by one-way ANOVA and Tukey’s post hoc analysis.