Published OnlineFirst April 10, 2017; DOI: 10.1158/1940-6207.CAPR-16-0286 Research Article Cancer Prevention Research Bioactivity of Oral Linaclotide in Human Colorectum for Cancer Chemoprevention David S. Weinberg1, Jieru E. Lin2, Nathan R. Foster3, Gary Della'Zanna4, Asad Umar4, Drew Seisler3, Walter K. Kraft2, David M. Kastenberg5, Leo C. Katz5, Paul J. Limburg6, and Scott A. Waldman2 Abstract Guanylate cyclase C (GUCY2C) is a tumor-suppressing recep- GUCY2C activation by oral linaclotide was associated with tor silenced by loss of expression of its luminocrine hormones homeostatic signaling, including phosphorylation of vasodila- guanylin and uroguanylin early in colorectal carcinogenesis. This tor-stimulated phosphoprotein and inhibition of proliferation observation suggests oral replacement with a GUCY2C agonist quantified by reduced Ki67-positive epithelial cells. In the may be an effective targeted chemoprevention agent. Linaclotide absence of the complete oral colonoscopy preparation, linaclo- is an FDA-approved oral GUCY2C agonist formulated for gastric tide did not alter cGMP production in epithelial cells of the release, inducing fluid secretion into the small bowel to treat colorectum, demonstrating that there was an effect related to the chronic idiopathic constipation. The ability of oral linaclotide to laxative preparation. These data show that the current FDA- induce a pharmacodynamic response in epithelial cells of the approved formulation of oral linaclotide developed for small- colorectum in humans remains undefined. Here, we demon- bowel delivery to treat chronic idiopathic constipation is inad- strate that administration of 0.87 mg of oral linaclotide daily for equate for reliably regulating GUCY2C in the colorectum to 7 days to healthy volunteers, after oral colon preparation with prevent tumorigenesis. The study results highlight the impor- polyethylene glycol solution (MoviPrep), activates GUCY2C, tance of developing a novel GUCY2C agonist formulated for resulting in accumulation of its product cyclic (c)GMP in epi- release and activity targeted to the large intestine for colorectal thelial cells of the cecum, transverse colon, and distal rectum. cancer prevention. Cancer Prev Res; 10(6); 345–54. Ó2017 AACR. Introduction aspirin (acetylsalicylic acid) and other NSAIDs represent the most thoroughly investigated class of colorectal cancer chemo- Colorectal cancer is the fourth most commonly diagnosed prevention agents. However, given the established risk/benefit cancer in the United States, with approximately 150,000 new profile of these agents, the widespread use of acetylsalicylic acid cases recorded each year (1). Over the course of a lifetime, about or other NSAIDs strictly for colorectal cancer chemoprevention 1 in 20 U.S. residents will be diagnosed with this disease. seems unlikely in the average-risk population. Despite advances in early detection and treatment, the mortality Guanylate cyclase C (GUCY2C) is the intestinal epithelial rate for colorectal cancer remains nearly 50%. Although screen- cell receptor (2) for the endogenous hormones guanylin and ing and surveillance continue to be the cornerstone of colorectal uroguanylin. Hormone–receptor interaction activates the intra- cancer prevention, chemoprevention has emerged as a comple- cellular catalytic domain, which converts guanosine triphos- mentary approach among higher risk participants. To date, phate to cyclic guanosine monophosphate (cGMP). This cyclic nucleotide activates signaling intermediates, including cGMP- dependent protein kinase, which phosphorylates downstream 1Department of Medicine, Fox Chase Cancer Center, Philadelphia, Pennsylvania. 2Department of Pharmacology and Experimental Therapeutics, Thomas Jeffer- effectors, including vasodilator-stimulated phosphoprotein son University, Philadelphia, Pennsylvania. 3Mayo Foundation for Medical Edu- (VASP) and cystic fibrosis transmembrane conductance regu- cation and Research, Mayo Clinic, Rochester, Minnesota. 4Division of Cancer lator (CFTR). Phosphorylation of CFTR opens this chloride Prevention, NCI, NIH, Bethesda, Maryland. 5Division of Gastroenterology, channel, resulting in fluid and electrolyte secretion. This mech- Department of Medicine, Thomas Jefferson University, Philadelphia, Pennsyl- anism has been coopted by bacteria that secrete heat-stable vania. 6Division of Gastroenterology and Hepatology, Department of Internal enterotoxins, which are structural and functional homologs Medicine, Mayo Clinic, Rochester, Minnesota. of guanylin and uroguanylin, to induce GUCY2C-dependent Note: Supplementary data for this article are available at Cancer Prevention diarrhea (3–5). Beyond secretion, GUCY2C and its ligands also Research Online (http://cancerprevres.aacrjournals.org/). regulate intestinal homeostasis along the crypt–villus axis ClinicalTrials.gov identifier: NCT01950403 by restricting proliferative dynamics and coordinating cell Corresponding Author: Scott A. Waldman, Department of Pharmacology and cycle, differentiation, and metabolic circuits (6–8). In that Experimental Therapeutics, Thomas Jefferson University, 1020 Locust Street, context, guanylin and uroguanylin are the most commonly Room 368, Philadelphia, PA 19107. Phone: 215-955-6086; Fax: 215-955-5681; lost gene products in colorectal cancer in animals and humans E-mail: [email protected] (9–11). Of significance, epithelial cells undergoing transfor- doi: 10.1158/1940-6207.CAPR-16-0286 mation continue to express GUCY2C. Indeed, colon cancer cells Ó2017 American Association for Cancer Research. overexpress GUCY2C compared with normal adjacent mucosa www.aacrjournals.org 345 Downloaded from cancerpreventionresearch.aacrjournals.org on September 25, 2021. © 2017 American Association for Cancer Research. Published OnlineFirst April 10, 2017; DOI: 10.1158/1940-6207.CAPR-16-0286 Weinberg et al. Preregistered n = 46 Screen failure reasons (n = 22) Parcipant decision 4 Alternates* 5 Lost to follow-up** 4 Polyps found at baseline 2 Registered Procs 2 n = 24 Invesgator decision 1 Other reasons 4 Stage I Stage II Stage III Stage III (MoviPrep followed by (MoviPrep followed by (tap water enema followed (PEG enema followed by colonoscopy) colonoscopy) by sigmoidoscopy) sigmoidoscopy) Linaclode/placebo Linaclode/placebo Linaclode/placebo Linaclode/placebo 0.87 mg days 1–7 0.87 mg days 1–7 0.87 mg days 1–7 0.87 mg days 1–7 n = 6 n = 6 n = 6 n = 6 * Subjects not needed due to full stage accrual by the me of eligibility assessment compleon. ** Subjects did not present for sigmoidoscopy Figure 1. CONSORT flow diagram of subject progress through the phases of the clinical trial. (12, 13). Moreover, we have previously demonstrated that the stomach's acidic environment, which stimulates fluid secre- pharmacologic or genetic delivery of GUCY2C ligands opposes tion in the proximal small bowel. In its current formulation, intestinal tumorigenesis in mice (14, 15). only approximately 1% to 3% of orally administered linaclo- Taken together, these data support that GUCY2C is a tumor- tide or its active metabolite is recovered in stool (16). This suppressing receptor and when silenced, due to the loss of study examined whether sufficient concentrations of the orally expression of guanylin and uroguanylin, universally contributes administered peptide can successfully engage GUCY2C in epi- to early development of colorectal cancer. These properties high- thelial cells of the colon and rectum, key targets for chemo- light the potential value of oral replacement with GUCY2C prevention. The study comprised three stages (Fig. 1). In stage I, agonists as targeted prevention for colorectal cancer. Oral we evaluated the ability of a single oral daily dose of 0.87 mg of GUCY2C agonists have impressive safety profiles in preclinical linaclotide administered for 7 days to activate cGMP produc- through late-stage clinical trials for the treatment of chronic tion in the colon and rectum sampled by colonoscopic biopsy constipation syndromes. Given the paucity of compounds proven following oral bowel preparation. Stage II explored the ability safe and effective for colorectal cancer chemoprevention, this class of that same dose to activate rectal GUCY2C (the most distant of agent warrants further investigation. Linaclotide is an FDA- site for chemoprevention) by sigmoidoscopy sampling follow- approved GUCY2C agonist formulated for immediate gastric ing oral bowel preparation. In stage III, we explored the release, with bioactivity in the small intestine. It is approved for ability of linaclotide 0.87 mg to activate GUCY2C in rectal the treatment of irritable bowel syndrome with constipation mucosa. Biopsies were obtained by sigmoidoscopy following and for chronic idiopathic constipation. The chemoprevention- rectal preparation by tap water or PEG enema. Stage III was relevant pharmacodynamic response of linaclotide in the human designed to determine whether the orally administered colonic colon was not assessed during the agent's development. Here, bowel preparation affected the colonic distribution of linaclo- we evaluated the effects of linaclotide in epithelial cells of the tide. We anticipated that successful completion of these colorectum in healthy volunteers. three stages would offer a dose reduction employing sigmoid- oscopy and distal bowel cleansing by enema to identify the optimal dose of linaclotide for a subsequent chemoprevention trial. Materials and Methods The study was approved by the IRBs of the Mayo Clinic Study design (Rochester,MN),ThomasJefferson