Linaclotide is a potent and selective guanylate cyclase C agonist that elicits pharmacological effects locally in the gastrointestinal tract

doi:10.1016/j.lfs.2010.03.015

Life Sciences

Volume 86, Issues 19–20, 8 May 2010, Pages 760-765

https://doi.org/10.1016/j.lfs.2010.03.015 Get rights and content

Abstract

Aims

Linaclotide is an orally administered 14-amino acid peptide being developed for the treatment of constipation-predominant irritable bowel syndrome (IBS-C) and chronic constipation. We determined the stability of linaclotide in the intestine, measured the oral bioavailability, and investigated whether the pharmacodynamic effects elicited in rodent models of gastrointestinal function are mechanistically linked to the activation of intestinal guanylate cyclase C (GC-C).

Main methods

Linaclotide binding to intestinal mucosal membranes was assessed in competitive binding assays. Stability and oral bioavailability of linaclotide were measured in small intestinal fluid and serum, respectively, and models of gastrointestinal function were conducted using wild type (wt) and GC-C null mice.

Key findings

Linaclotide inhibited in vitro [¹²⁵I]-STa binding to intestinal mucosal membranes from wt mice in a concentration-dependent manner. In contrast, [¹²⁵I]-STa binding to these membranes from GC-C null mice was significantly decreased. After incubation in vitro in jejunal fluid for 30 min, linaclotide was completely degraded. Pharmacokinetic analysis showed very low oral bioavailability (0.10%). In intestinal secretion and transit models, linaclotide exhibited significant pharmacological effects in wt, but not in GC-C null mice: induction of increased fluid secretion into surgically ligated jejunal loops was accompanied by the secretion of elevated levels of cyclic guanosine-3′,5′-monophosphate and accelerated gastrointestinal transit.

Significance

Linaclotide is a potent and selective GC-C agonist that elicits pharmacological effects locally in the gastrointestinal tract. This pharmacological profile suggests that orally administered linaclotide may be capable of improving the abdominal symptoms and bowel habits of patients suffering from IBS-C and chronic constipation.

Introduction

Guanylate cyclase C (GC-C), which was originally identified as the receptor for bacterial STa enterotoxin, is a transmembrane receptor with intrinsic guanylate cyclase activity that is predominantly expressed on the luminal surface of intestinal epithelial cells (Schulz et al. 1990). The endogenous peptide hormones guanylin and uroguanylin are GC-C agonists that regulate the guanylate cyclase activity of this receptor. Guanylin and uroguanylin are expressed along the longitudinal axis of the gastrointestinal tract and released into the intestinal lumen, but their expression pattern is distinctly different in major segments of the intestine and in different cell types within the mucosa due to their altered activity in the variable pH microenvironments found at the mucosal surface of the intestine (Wiegand et al., 1992, Li & Goy, 1993, Fan et al., 1996, Hamra et al., 1996, Hamra et al., 1997).

Stimulation of GC-C significantly elevates the intracellular concentration of the second messenger cyclic guanosine-3′, 5′-monophosphate (cGMP), which is involved in the regulation of a broad range of physiological processes, including the activation of cGMP-dependent protein kinase II (PKG-II) (Pfeiffer et al., 1996, Schlossmann et al., 2005). PKG-II phosphorylation regulates the activity of the cystic fibrosis transmembrane conductance regulator (CFTR), an ion channel protein colocalized with PKG-II at the apical surface of intestinal epithelial cells (Seidler et al., 1997, Vaandrager et al., 1998). Thus, the activation of GC-C by guanylin and uroguanylin provides an intrinsic mechanism for the control of intestinal fluid homeostasis. This balance is achieved through the stimulation of transepithelial secretion of chloride (Cl⁻) and bicarbonate (HCO₃⁻) ions and concomitant inhibition of sodium (Na⁺) absorption through a blockade of Na⁺/H⁺ exchange, which results in decreased Na⁺ absorption and increased secretion of water into the intestinal lumen (Forte, 1999, Vaandrager, 2002, Sindic & Schlatter, 2006). Studies in GC-C null mice have confirmed the critical role of this receptor in intestinal fluid homeostasis (Mann et al., 1997, Schulz et al., 1997). Thus, the GC-C receptor has the potential as a therapeutic target for the treatment of functional gastrointestinal disorders such as constipation-predominant irritable bowel syndrome (IBS-C) and chronic constipation.

IBS-C and chronic constipation are highly prevalent functional gastrointestinal disorders, which occur more frequently in women than men. IBS-C is characterized by symptoms of chronically recurring abdominal pain, discomfort and bloating in association with symptoms of constipation (Drossman, 2006, Videlock & Chang, 2007). Chronic constipation symptoms include infrequent bowel movements, hard stool, straining during defecation, and a feeling of incomplete evacuation. Abdominal discomfort and bloating may also be present (Sandler & Drossman, 1987, Koch et al., 1997). For both disorders, current therapeutic options are limited. Hence, there is a continued medical need for more effective and safer therapeutic agents.

Linaclotide is an orally active 14-amino acid peptide of the guanylin family of cGMP-regulating peptides that also includes the heat-stable microbial ST peptides. In this study, we have investigated whether the pharmacological properties of linaclotide in both in vitro and in vivo models of gastrointestinal function are mechanistically linked to the activation of GC-C.

Section snippets

Animals

Male and female wild type (wt) and GC-C null (Gucy2c⁻/⁻) mice (C57/BL6) were a gift from Mitchell Cohen (Cincinnati Children's Hospital Medical Center, Cincinnati, OH, USA). Female CD-1 mice were obtained from Charles River Laboratories (Wilmington, MA). All animal studies were performed in the laboratories of Ironwood Pharmaceuticals and approved by the Institutional Animal Care and Use Committee.

Reagents

Lyophilized linaclotide was obtained from Polypeptide Laboratories, Torrance, CA, USA. Activated

Molecular structure and amino acid sequence of linaclotide

Linaclotide includes several distinct molecular features that provide improved pharmacological properties over those of the endogenous peptide hormones guanylin and uroguanylin. First, the molecular structure of linaclotide is significantly different from those of guanylin and uroguanylin. Specifically, the pharmacologically active conformation of linaclotide is stabilized by three intramolecular disulfide bonds, compared to two such bonds in the endogenous peptides (Fig. 1). Second, the amino

Discussion

Linaclotide is an orally administered 14-amino acid peptide of the guanylin family of cGMP-regulating peptides. Although the pharmacological responses of linaclotide elicited locally in the gastrointestinal tract are related to those of guanylin and uroguanylin, this peptide possesses important structural and pharmacological properties that clearly distinguish it from the endogenous peptide hormones.

The molecular structure and amino acid sequence of linaclotide differ from those of guanylin and

Conflict of interest statement

The authors declare that there are no conflicts of interest.

Acknowledgements

We wish to thank Samuel Rivers for his expert technical assistance in the secretion studies and Carolyn Higgins for her expert support in the analysis of pharmacokinetic studies.

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View full text

Linaclotide is a potent and selective guanylate cyclase C agonist that elicits pharmacological effects locally in the gastrointestinal tract

Abstract

Aims

Main methods

Key findings

Significance

Introduction

Section snippets

Animals

Reagents

Molecular structure and amino acid sequence of linaclotide

Discussion

Conflict of interest statement

Acknowledgements

Gastroenterology

Biochemical and Biophysical Research Communications

Regulatory Peptides

Biochemical and Biophysical Research Communications

Cell

Gastroenterology

Analytical Biochemistry

Gastroenterology Clinics of North America

Biochemical and Biophysical Research Communications

Different segmental transit times in patients with irritable bowel syndrome and “normal” colonic transit time: is there a correlation with symptoms?

Techniques in Coloproctology

The molecular target of MD-1100 is guanylate cyclase C (GC-C), an apical receptor on intestinal epithelial cells

Gastroenterology

Identification of two affinity states for low affinity receptors for Escherichia coli heat-stable enterotoxin: correlation of occupation of lower affinity state with guanylate cyclase activation

Molecular Pharmacology

Guanylate cyclase-C-mediated anti-nociceptive effects of linaclotide in rodent models of visceral pain

Neurogastroenterology and Motility

Regulation of intestinal uroguanylin/guanylin receptor-mediated responses by mucosal acidity

Proceedings of the National Academy of Sciences USA