Bioactivity of Oral Linaclotide in Human Colorectum for Cancer Chemoprevention

Abstract

Guanylate cyclase C (GUCY2C) is a tumor-suppressing receptor silenced by loss of expression of its luminocrine hormones guanylin and uroguanylin early in colorectal carcinogenesis. This observation suggests oral replacement with a GUCY2C agonist may be an effective targeted chemoprevention agent. Linaclotide is an FDA-approved oral GUCY2C agonist formulated for gastric release, inducing fluid secretion into the small bowel to treat chronic idiopathic constipation. The ability of oral linaclotide to induce a pharmacodynamic response in epithelial cells of the colorectum in humans remains undefined. Here, we demonstrate that administration of 0.87 mg of oral linaclotide daily for 7 days to healthy volunteers, after oral colon preparation with polyethylene glycol solution (MoviPrep), activates GUCY2C, resulting in accumulation of its product cyclic (c)GMP in epithelial cells of the cecum, transverse colon, and distal rectum. GUCY2C activation by oral linaclotide was associated with homeostatic signaling, including phosphorylation of vasodilator-stimulated phosphoprotein and inhibition of proliferation quantified by reduced Ki67-positive epithelial cells. In the absence of the complete oral colonoscopy preparation, linaclotide did not alter cGMP production in epithelial cells of the colorectum, demonstrating that there was an effect related to the laxative preparation. These data show that the current FDA-approved formulation of oral linaclotide developed for small-bowel delivery to treat chronic idiopathic constipation is inadequate for reliably regulating GUCY2C in the colorectum to prevent tumorigenesis. The study results highlight the importance of developing a novel GUCY2C agonist formulated for release and activity targeted to the large intestine for colorectal cancer prevention. Cancer Prev Res; 10(6); 345-54. ©2017 AACR.

Figure 1

CONSORT flow diagram of subject progress through the phases of the clinical trial.

Figure 2. Cyclic GMP pharmacodynamic response to linaclotide or placebo in healthy volunteers in Stage I

Cyclic GMP pharmacodynamic response was calculated as described in Methods. C, cecum; TC, transverse colon, R, rectum.

Figure 3. VASP phosphorylation in mucosal biopsies from healthy subjects treated with linaclotide or placebo in Stage I

Phosphorylated VASP was quantified by densitometry following immunoblot analysis of biopsies from the cecum, transverse colon, and rectum. The amount of phosphorylated VASP was normalized to the epithelial marker villin. For each intestinal segment, the ratio of normalized phosphorylated villin on day 1 (pre-dose) and 7 (post-dose) were calculated. *, p<0.05.

Figure 4. Cell proliferation in crypts in mucosal biopsies from healthy subjects treated with linaclotide or placebo in Stage I

Proliferation was quantified by enumerating cells expressing Ki67 by immunofluorescence. Ki67 was enumerated in 10–20 crypts in each biopsy and means were calculated. For each intestinal segment, the ratio of mean Ki67 expression on day 1 (pre-dose) and 7 (post-dose) were calculated. C, cecum; TC, transverse colon, R, rectum. ***, p<0.001.

Figure 5. Cyclic GMP pharmacodynamic response to linaclotide or placebo in healthy volunteers in Stage II and III

Cyclic GMP pharmacodynamic response was calculated as described in Methods in rectal biopsies of healthy subjects in (A) Stage II, (B) Stage III following tap water enemas, and (C) Stage III following PEG enemas.