Pulmonary, Gastrointestinal and Urogenital PharmacologyLinaclotide, through activation of guanylate cyclase C, acts locally in the gastrointestinal tract to elicit enhanced intestinal secretion and transit
Introduction
Linaclotide is a first-in-class, orally administered 14 amino acid peptide of the guanylin peptide family, a family of cyclic guanosine-3′, 5′-monophosphate (cGMP) regulating peptide hormones that also includes the heat-stable microbial ST peptides (Currie et al., 1992, Forte, 2004, Hamra et al., 1993). Guanylin and uroguanylin are primarily expressed along the longitudinal axis of the gastrointestinal tract and released into the intestinal lumen, but their expression pattern and intestinal sites of activity are distinctively different (Fan et al., 1996, Hamra et al., 1993). While uroguanylin exerts its activity primarily in the acidic microenvironment of the proximal duodenum, guanylin is more active in intestinal regions with a more neutral pH microenvironment. Guanylate cyclase C, which is predominantly expressed on the luminal surface of intestinal epithelial cells, is an important regulator of intestinal function and is the molecular target of guanylin and uroguanylin (Schulz et al., 1990). Stimulation of guanylate cyclase C receptors by these peptides leads to increased intracellular levels of the second messenger cGMP. Cyclic GMP is involved in the regulation of a broad range of physiological processes, including the control of intestinal fluid homeostasis (Schlossman et al., 2005, Seidler et al., 1997). Studies in mice genetically deficient in the guanylate cyclase C gene have confirmed the critical role of this receptor in the regulation of intestinal fluid homeostasis (Mann et al., 1997, Schulz et al., 1997). Thus, the guanylate cyclase C receptor appears to be a novel therapeutic target for the treatment of functional gastrointestinal disorders such as irritable bowel syndrome with constipation and chronic constipation.
Irritable bowel syndrome is a highly prevalent, chronic condition that affects 20 to 64 million individuals in the US (Borum, 2001). Since there is no known structural or biochemical etiology, irritable bowel syndrome is characterized as a functional gastrointestinal disorder (Ringel et al., 2001). Principal symptoms of irritable bowel syndrome include abdominal pain or discomfort that is associated with a change in stool frequency or stool form (Drossman et al., 1999, Drossman, 2006). Chronic constipation, a functional gastrointestinal disorder affecting approximately 15% of the US population, also results in patients' reduced quality of life both physically and psychologically (McCallum et al., 2009). Principal symptoms of chronic constipation include infrequent stools, straining, a feeling of incomplete evacuation, rectal and perianal fullness, abdominal discomfort and bloating (McCallum et al., 2009). Despite significant efforts over the past two decades to develop effective drugs for the treatment of these functional gastrointestinal disorders, therapeutic options are limited. Hence, there is a continued medical need for more effective and safer therapeutic agents.
In this study, we report the solution structure of linaclotide and the in vitro binding and agonist activity to guanylate cyclase C receptors. Furthermore, we have determined the stability of linaclotide under conditions mimicking the gastric environment, the rat oral bioavailability of linaclotide, and whether the pharmacodynamic effects elicited locally in the gastrointestinal tract in rat models of gastrointestinal transit and intestinal secretion are linked to the activation of guanylate cyclase C receptors.
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Animals
Male and female CD rats were obtained from Charles River Laboratories (Wilmington, MA). The animals were housed in a temperature (21 °C ± 1 °C) and relative humidity (35% ± 5%) controlled room with 12-h light/dark cycle, with ad libitum access to filtered tap water and standard irradiated pelleted laboratory chow. The rats were provided with at least a 3-day acclimatization period prior to the start of any experiment. At the end of the studies, rats were euthanized by CO2 asphyxiation. All animal
Determination of the solution structure of linaclotide by nuclear magnetic resonance
The accurate mass of linaclotide, determined using LC-TOF/MS is 1525.4, consistent with all six cysteines in the molecule involved in disulfide bonds. This observation was confirmed using 2D 1H-13C-HSQC NMR spectrum of linaclotide. The 13C chemical shifts of the Cβ from the six cysteines of linaclotide were between 34 and 43 ppm, which is consistent with the cysteines being in the oxidized form. The analysis of a database of measured oxidized cysteine 13C Cβ chemical shifts indicates that most
Discussion
Linaclotide is a first-in-class, orally administered 14-amino acid peptide that is in development for the treatment of IBS-C and chronic constipation. Although linaclotide has disulfide linkages and sequence motifs similar to guanylin and uroguanylin and thus should therefore be classified as a member of the guanylin family of guanylate cyclase C agonist peptides, it possesses a number of important pharmacological properties that clearly distinguishes it from the endogenous peptide hormones.
Acknowledgment
The authors thank Sam Rivers for excellent technical support.
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2022, Trends in Pharmacological SciencesCitation Excerpt :Importantly, linaclotide and plecanatide have limited bioavailability (Box 2) and hence have clinical safety as well as being bereft of central adverse effects, unlike traditional analgesics such as μ-opioids or centrally acting neuromodulators and most available cannabinoid agents [6]. Upon binding by GC-C agonists, the intrinsic guanylate cyclase activity of GC-C rapidly elevates the intracellular concentration of the second messenger cyclic guanosine-3′,5′-monophosphate (cGMP) via enzymatic conversion of cytoplasmic guanosine triphosphate (GTP) to cGMP [7–9]. Cyclic GMP is involved in the regulation of a broad range of physiological processes, including activation of cGMP-dependent protein kinase II (PKG-II), and a direct interaction with cyclic nucleotide-gated ion channels and cGMP-regulated phosphodiesterases [9,10].
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