Guanylate cyclase C

Linaclotide inhibits in vitro [¹²⁵I]-STa binding to intestinal mucosal membranes from wt mice in a concentration-dependent manner. On the other hand, there is a significant decrease in [¹²⁵I]-STa binding to these membranes from GC-C null mice. Linacelotide is totally broken down after 30 minutes of in vitro incubation in jejunal fluid[1]. Linaclotide improves defecation by increasing the secretion of water into the lumen and inhibiting the absorption of sodium ions through its action on guanylate cyclase-C receptors on the luminal membrane. The drug is only slightly absorbed into the systemic circulation[2].

Oral bioavailability is extremely low, according to pharmacokinetic analysis (0.10%). In models of intestinal secretion and transit, linaclotide shows notable pharmacological effects in wt mice, but not in GC-C null mice: elevated levels of cyclic guanosine-3',5-monophosphate are secreted along with accelerated gastrointestinal transit when increased fluid secretion is induced into surgically ligated jejunal loops[1]. In patients with chronic constipation, linaclotide reduces abdominal pain and significantly increases weekly spontaneous bowel movements and complete spontaneous bowel movements (CSBMs)[2].

Intestinal mucosal membrane binding assays [1]
STa(p) was radioiodinated (2.200 Ci/mmol) and purified as described by Thompson et al. 1985. Of the two monoiodinated forms of STa generated, the one labeled at the fourth tyrosine was isolated, purified and used as the tracer in this study. The binding reactions were carried out in 50 µl reactions containing 0.1 M sodium acetate pH 5.0, 0.2% BSA, intestinal mucosal membrane protein (50 µg), 53,000 cpm [125I]-STa (11 fmol, 217 pM), and 0.3 nM to 1.0 µM Linaclotide competitor. After incubation at 37 °C for 30 min, the reactions were applied to Whatman GF/C glass-fiber filters (pretreated with 1% polyvinylpyrrolidone) by vacuum filtration. The filters were then rinsed with ice-cold PBS buffer and the trapped [125I]-STa radioligand measured in a scintillation counter. Specific binding was determined by subtracting the [125I]-STa bound in the presence of excess unlabeled Linaclotide from the total binding. Competitive radioligand-binding curves were generated using GraphPad Prism. Nonlinear regression analysis of the binding data was used to calculate the concentration of competitor that resulted in 50% radioligand bound (IC50). Because the [125I]-STa concentration of 217 pM used in these assays was very small compared to its dissociation constant, the calculated IC50 and Ki values are in effect identical. The results are expressed as mean ± standard error of the mean (SEM).

---

Linaclotide metabolism in intestinal fluid [1]
To collect small intestinal fluid samples, two mice were fasted overnight but had access ad libitum to filtered tap water. Laparotomy was performed on anesthetized (isofluorane) mice and their small intestines were exteriorized. The areas of the jejunum selected for ligation were rinsed out with 3 ml of 20 mM Tris–HCl buffer, pH 7.5 prior to the creation of loops 1 to 3 cm in length, which were then injected with 0.2 ml vehicle (Krebs–Ringer solution containing 10 mM glucose, 10 mM HEPES, pH 7.0) (KRGH). The abdominal wall and skin were sutured and the animals were allowed to recover for 30 min. Following recovery, the mice were sacrificed, the loops excised, and the fluid removed and stored at − 20 °C. Linaclotide (0.1 mg/ml) was incubated in either 0.010 ml of intestinal fluid or 0.010 ml phosphate-buffered saline (PBS) (control) at 37 °C for varying amounts of time. The reactions were stopped by addition of 0.015 ml PBS and one volume of ice-cold 12% trichloroacetic acid (TCA), vortexed and centrifuged at 16,000 × g for 5 min at 4 °C. Linaclotide degradation was analyzed by LC–MS/MS using the MassLynx version 4.0 SP4 software for molecular weight prediction and data analysis.

Mice: Three groups (n=3) of female CD-1 mice receive linaclotide (8 mg/kg) intravenously (i.v.), and two groups (n=3) receive linaclotide (8 mg/kg) by gavage (p.o.) in order to assess oral bioavailability. After allowing blood to clot for five minutes, serum is collected and kept at -80°C until sample preparation and LC-MS/MS analysis[1]. The blood is then centrifuged at 13,000×g for three minutes.

---

Oral bioavailability of Linaclotide in mice [1]
To determine oral bioavailability, three groups (n = 3) of female CD-1 mice received Linaclotide (8 mg/kg) intravenously (i.v.), while two groups (n = 3) received Linaclotide (8 mg/kg) by gavage (p.o.). Blood was allowed to clot for 5 min, centrifuged at 13,000 × g for 3 min, and the serum was collected and stored at − 80 °C until sample preparation and analysis by LC–MS/MS. The concentration of linaclotide was determined based on a standard concentration curve of linaclotide generated using a set of standards prepared in mouse serum (lower limit of quantitation was 1.0 ng/ml). Data were collected using Waters MassLynx version 4.0 software. Linaclotide serum concentrations were plotted as a function of time using GraphPad Prism 5.0 software. Pharmacokinetic parameters for oral and intravenous administration were calculated using WinNonlin version 5.2. If no analyte was detected, the concentration was set to zero for calculations of the AUC and oral bioavailability.

---

Intestinal fluid secretion assay [1]
Intestinal loops in wt and GC-C null mice (n = 5–7/group) were surgically ligated after the mice had been placed under isofluorane anesthesia and laparotomy was performed to exteriorize the small intestine. The small intestines were flushed with Krebs–Ringer buffer (K–R, 10 mM glucose, 10 mM HEPES) pH 7.0 (KRGH) and a loop of approximately 3 cm in length was created halfway between the stomach and the cecum. Loops were injected with either 100 µl Linaclotide (5 µg) or 100 µl vehicle (KRGH), and the animals were allowed to recover for 90 min prior to euthanasia. The loops were then excised and the length and weight of each loop was recorded both prior to and after collection of the intestinal fluid content. Fluid secretion was calculated and expressed as the weight to length ratio (W/L), a commonly used surrogate to measure intestinal secretion.

---

Gastrointestinal transit in mice [1]
Male and female wt and GC-C null mice (n = 9/group) were fasted overnight, having access to water ad libitum. The mice received oral doses (200 µl) of either Linaclotide (100 µg/kg) or vehicle (20 mM Tris–HCl, pH 7.5) 10 min prior to a dose of 10% activated carbon/10% gum arabic powder suspension (200 µl in water) administered by gavage. After 5 min, the animals were euthanized and their intestines ranging from the stomach to the cecum were removed. Gastrointestinal transit is expressed as the percentage of the total length of the small intestine traveled by the charcoal front.

Absorption, Distribution and Excretion
Linaclotide is minimally absorbed after oral administration, and its systemic bioavailability is negligible. However, since the ligand-binding domain of the GC-C target is located on the luminal surface of intestinal epithelial cells, systemic exposure is not important for the maximum therapeutic effect of linaclotide. Because plasma concentrations of linaclotide and its active metabolites are below the limit of quantitation, information regarding the area under the curve (AUC) and peak plasma concentration (Cmax) is currently unavailable. In healthy subjects, after a 7-day course of 290 μmol linaclotide once daily, the mean recovery rates of the active peptide in fasting and fed fecal samples were 3% and 5%, respectively. The recovered active peptide constitutes the active metabolite. Given that plasma concentrations of linaclotide cannot be measured after the recommended oral dose, it is expected that linaclotide will not distribute to tissues to any clinically significant extent. No relevant information is available. Given that plasma concentrations of linaclotide cannot be measured after oral administration of therapeutic doses, minimal tissue distribution of linaclotide is expected.
After seven consecutive days of daily administration of 290 μg Linzess, the average recovery rate of the bioactive peptide in fecal samples from fasting and satiety subjects was approximately 5% (fasting) and 3% (satiety), almost entirely in the form of bioactive metabolites.
Absorption of Linzess after oral administration is minimal, resulting in low systemic bioavailability. Following oral administration of 145 μg or 290 μg, plasma concentrations of linaclotin and its bioactive metabolites were below the limits of quantitation. Therefore, standard pharmacokinetic parameters such as area under the curve (AUC), maximum concentration (Cmax), and half-life could not be calculated.
It is currently unknown whether linaclotin is distributed into human milk.
For more complete data on the absorption, distribution, and excretion of linaclotin (8 items in total), please visit the HSDB records page.
Metabolism/Metabolites

Linaclotin is metabolized in the small intestine, where its C-terminal tyrosine residue is shed to form the major bioactive metabolite MM-419447. The disulfide bonds of linaclotide and MM-419447 are reduced in the intestinal lumen, followed by proteolysis and degradation to form smaller peptides and native amino acids, which are absorbed through the intestine. In in vitro rat experiments, linaclotide is resistant to enzymatic hydrolysis by pepsin, trypsin, aminopeptidase, or chymotrypsin. A series of experiments (mainly in rodents) have investigated the metabolism of linaclotide. Linaclotide is metabolized in the intestine, where its disulfide bonds are rapidly broken, making it more readily digestible by enzymes in the gastrointestinal environment. Several metabolites containing 3–13 amino acids have been identified. Only one metabolite, MM-419447, has been shown to have pharmacodynamic activity. Linaclotide is metabolized in the gastrointestinal tract to become the predominantly active metabolite via the loss of terminal tyrosine residues. Both linaclotide and its metabolites are degraded into smaller peptides and native amino acids by proteolytic enzymes in the intestinal lumen. We investigated the metabolic stability of linaclotide under simulated gastrointestinal conditions and characterized the metabolite MM-419447 (CCEYCCNPACTGC), which contributes to the pharmacological action of linaclotide. Systemic exposure to these bioactive peptides was low in both rats and humans. Low systemic and portal vein concentrations of linaclotide and MM-419447 were observed in rats, confirming minimal absorption of both peptides after oral administration. Linaclotide is stable in the acidic environment of the stomach and is converted to MM-419447 in the small intestine. The disulfide bonds of both peptides are reduced in the small intestine and subsequently degraded by proteolytic hydrolysis. Following oral administration of linaclotide, less than 1% of the bioactive peptide was excreted in rat feces, compared to an average excretion of 3–5% in human feces; in both cases, MM-419447 was the major recovered peptide. MM-419447 binds with high affinity to T84 cells in vitro, leading to a significant concentration-dependent accumulation of intracellular cyclic guanosine monophosphate (cGMP). In a rat gastrointestinal model, oral administration of MM-419447 significantly increased fluid secretion in the small intestinal loops, increased intraluminal cGMP levels, and resulted in a dose-dependent acceleration of gastrointestinal transit. These results indicate that the active metabolite is crucial for the pharmacological effects of linaclotide.
Biological Half-Life
Because the concentrations of linaclotide and its active metabolite in plasma are below the limit of quantitation, information regarding the half-life is currently unavailable.
Two male and two female monkeys were intravenously injected with linaclotide at a dose of 15 mg/kg/day for seven consecutive days. …On days 1 and 7, the mean half-life in both sexes was approximately 1.5 hours.
Linaclotide is minimally absorbed in systemic circulation. In a study by Busby et al., 11 patients received 290 μg of linaclotide once daily for seven days, after which plasma samples were collected. No quantitative concentration of linaclotide or its major metabolite MM-419447 was detected. This finding is consistent with absorption data from other clinical trials. When a single dose of 2897 μg of linaclotide was administered, quantifiable concentrations of linaclotide were detected in 2 out of 18 subjects, but its major metabolite was not detected. In a phase III clinical trial for patients with constipation-predominant irritable bowel syndrome (IBS-C) or chronic constipation-predominant irritable bowel syndrome (CC), 465 patients were treated with 290 μg or 145 μg of linaclotide, and plasma linaclotide concentrations were detectable in only 2 patients; linaclotide concentrations were below 0.5 ng/mL, and no quantifiable major metabolite was detected in any of the patients. Because linaclotide acts primarily on the intestine and is minimally absorbed through systemic circulation, its distribution is very small. [2] Studies have shown that linaclotide is relatively stable in gastric juice. After incubation for 3 hours in vitro with trypsin, pepsin, aminopeptidase, or chymotrypsin, linaclotide was not metabolized. Its 13-amino acid active metabolite MM-419447 is produced by incubating linaclotide with carboxypeptidase A. After 6 hours of digestion with carboxypeptidase A, linaclotide is almost completely converted into MM-419447. Animal studies have shown that linaclotide is metabolized faster in the duodenum and jejunal loop than in the ileal loop. In order for linaclotide and its metabolites to be completely degraded (and thus lose their pharmacological activity), these peptides undergo disulfide bond reduction in the intestine, making them more readily digestible by proteolytic enzymes, which break them down into smaller peptides and amino acids, and recycle them by the body. [2] In a phase I open-label, two-period, crossover food effects study in healthy volunteers, fecal samples were collected after subjects were given 290 μg of linaclotide once daily for 7 days, and after a single oral dose of 2897 μg of linaclotide. Almost all of the active peptides recovered in the feces were in the form of the major metabolite MM-419447. The recovery rate of the active peptide ranged from 0% to 20% (mean 3% to 5%) of the administered linaclotide dose. The parent compound was only detected after a 2897 μg dose was taken with food (detected in 3 out of 9 subjects, with a median of 0.4% of the administered linaclotide dose). [2]

Toxicity Summary
Identification and Uses: Linaclotide is a white to off-white powder. Linaclotide is used to treat constipation-predominant irritable bowel syndrome in adults and also to treat chronic idiopathic constipation in adults. Human Exposure and Toxicity: Experience with linaclotide overdose is limited. In the clinical development program of linaclotide, 22 healthy volunteers received a single dose of 2897 micrograms of linaclotide; the safety profile in these subjects was consistent with the overall population receiving linaclotide, with diarrhea being the most common adverse reaction. Linaclotide is contraindicated in infants under 6 years of age, and should be avoided in children and adolescents aged 6–17 years. While the safety and efficacy of linaclotide in patients under 18 years of age have not been established, linaclotide caused death in young rats when administered at a single clinically relevant adult oral dose. Linaclotide did not show genotoxicity in an in vitro assay of chromosomal aberrations in cultured human peripheral blood lymphocytes. Animal Studies: In rats, no systemic exposure to linaclotide was detected at a single oral dose up to 5.0 mg/kg. No effects of linaclotide on survival, body weight, food consumption, clinical observation, or macroscopic assessment were observed. Cynomolgus monkeys were administered linaclotide via single oral doses of 0.5, 1.5, 3.0, and 5.0 mg/kg. Monkeys receiving a single oral dose of linaclotide (1.5 mg/kg or higher) experienced changes in fecal characteristics (unformed and/or loose stools), significantly reduced food intake, and/or abdominal distension. No significant changes in individual body weight were observed in these animals. One monkey was administered linaclotide orally for five consecutive days at a dose of 1.5 mg/kg/day, experiencing unformed and loose stools throughout the administration period and mild abdominal distension on the fourth day of administration. These results indicate that linaclotide is well tolerated in cynomolgus monkeys at single oral doses up to 5.0 mg/kg. However, when linaclotide was administered to juvenile mice at clinically relevant adult doses, mortality was observed. In newborn mice, mortality occurred after one or two oral doses of linaclotide (10 μg/kg/day) on day 7 after birth. These deaths were due to rapid and severe dehydration. Subcutaneous fluid resuscitation prevented death in newborn mice after administration of linaclotide. In studies without fluid resuscitation, tolerance to linaclotide in young mice increased with age. In 2-week-old mice, linaclotide was well tolerated at a dose of 50 μg/kg/day, but death occurred after a single oral dose of 100 μg/kg. In 3-week-old mice, linaclotide was well tolerated at a dose of 100 μg/kg/day, but death occurred after a single oral dose of 600 μg/kg. Linaclotide was well tolerated; in 4-week-old mice, no death was observed after continuous administration at a dose of 1000 μg/kg/day for 7 days; and in 6-week-old mice, no death was observed after continuous administration at a dose of 20000 μg/kg/day for 28 days. The potential risk of teratogenicity of linaclotide was investigated in rats, rabbits, and mice. No maternal toxicity or effects on embryo-fetal development were observed in rats at oral doses up to 100 mg/kg/day and in rabbits at oral doses up to 40 mg/kg/day. Severe maternal toxicity, including death, reduced uterine and fetal weight, and fetal morphological abnormalities, was observed in mice at oral doses of at least 40 mg/kg/day. No maternal toxicity or adverse effects on embryo-fetal development were observed in mice at oral doses of 5 mg/kg/day. Linaclotide had no effect on fertility or reproductive function in male and female rats at oral doses up to 100,000 μg/kg/day. In vitro bacterial reverse mutation (Ames) assays demonstrated that linaclotide is not genotoxic.
Hepatotoxicity
In clinical trials, linaclotide treatment was not associated with significant changes in serum enzyme levels or clinically significant liver injury events. Mild, transient increases in ALT were observed on a probability score of E (unlikely to be a cause of clinically significant liver injury).

---

Effects during pregnancy and lactation
◉ Overview of use during lactation
Linaclotide is minimally absorbed from the gastrointestinal tract. After daily administration of doses up to 290 micrograms, the drug and its active metabolites are undetectable in breast milk. Linaclotide appears to be acceptable in lactating women without requiring special attention.
◉ Effects on breastfed infants
No published information found as of the revision date.
◉ Effects on lactation and breast milk
No published information found as of the revision date.

---

Protein binding
No relevant information available.

---

Multiple clinical studies have confirmed the safety of linaclotide. The most common adverse event was mild to moderate diarrhea (Table 3); other common adverse events included flatulence and abdominal pain. In a 12-week trial, the type and incidence of adverse events in patients treated with linaclotide were similar to those in the control group. The discontinuation rate in the linaclotide group (4.7% in the 145 μg group and 3.8% in the 290 μg group) was higher than that in the placebo group (0.5%), which was attributed to treatment-related diarrhea. The proportion of patients with severe diarrhea as assessed by investigators was higher in the linaclotide group (1.5% vs. 0.2% in the placebo group). No clinically significant differences were found between the two groups in hematological or blood chemistry parameters, urinalysis or electrocardiogram (ECG) results, or vital signs. Safety results were similar to those in the previously summarized 26-week trial. The incidence of diarrhea was higher in the linaclotide group than in the placebo group, and the discontinuation rate was also higher. Among patients who reported diarrhea during treatment with linaclotide, 7.7% had mild diarrhea, 10.0% had moderate diarrhea, and 2.0% had severe diarrhea. Most patients reported diarrhea during treatment within the first 4 weeks. No differences were reported in vital signs, hematological or blood chemistry values, or ECG parameters. [2]

[1]. Linaclotide is a potent and selective guanylate cyclase C agonist that elicits pharmacological effects locally in the gastrointestinal tract. Life Sci. 2010 May 8;86(19-20):760-5.

[2]. Linaclotide: a novel agent for chronic constipation and irritable bowel syndrome. Am J Health Syst Pharm. 2014 Jul 1;71(13):1081-91.

Linaclotide acetate is the acetate form of linaclotide. Linaclotide is a synthetic tetradecapeptide drug and an agonist of intestinal guanylate cyclase type C (GC-C). Its structure is related to the guanosine peptide family and it has secretory, analgesic, and laxative effects. After oral administration, linaclotide binds to and activates GC-C receptors located on the luminal surface of intestinal epithelial cells. This increases the concentration of intracellular cyclic guanosine monophosphate (cGMP), which is derived from guanosine triphosphate (GTP). cGMP activates cystic fibrosis transmembrane conduction regulator (CFTR), stimulating the secretion of chloride and bicarbonate ions into the intestinal lumen. This promotes the excretion of sodium ions into the intestinal lumen, thereby increasing intestinal fluid secretion. This ultimately accelerates the gastrointestinal transit of intestinal contents, improves defecation, and relieves constipation. Elevated extracellular cGMP levels may also exert analgesic effects through a mechanism that is not fully elucidated, which may involve the modulation of nociceptors on pain-inducing fibers in the colon. Linaclotide is minimally absorbed in the gastrointestinal tract.

---

Therapeutic Use
Linzess (linaclotide) is indicated for the treatment of constipation-predominant irritable bowel syndrome (IBS-C) in adults. /US product label includes/
Linzess is indicated for the treatment of chronic idiopathic constipation (CIC) in adults. /US product label includes/

---

Drug Warnings
/Black Box Warning/ Warning: Risk in children. Linzess is contraindicated in children under 6 years of age; in non-clinical studies, a single oral dose of a clinically relevant adult dose of linaclotide resulted in death from dehydration in young mice. Linzess should be avoided in children aged 6 to 17 years. The safety and efficacy of Linzess in children under 18 years of age have not been established.
Linaclotide is contraindicated in infants under 6 years of age and should be avoided in children and adolescents aged 6 to 17 years. The safety and efficacy of linaclotide in pediatric patients have not been established, and the drug resulted in death in toxicology studies in young mice aged 1 to 3 weeks (approximately equivalent to infants under 2 years of age). In young mice, mortality began on day 7 after birth with administration of 10 μg/kg linaclotide once daily, or a single oral dose of 100 μg/kg on day 14, and a single oral dose of 600 μg/kg on day 21. Although no deaths occurred in 6-week-old mice (approximately equivalent to 12-17 years of age) after receiving 20,000 μg/kg linaclotide once daily for 28 days, this drug should be avoided in children and adolescents aged 6-17 years due to reported cases of infant mortality and a lack of safety and efficacy data in pediatric patients. Data are currently unavailable for mice aged 3-6 weeks. In clinical trials, 2% of patients receiving linaclotide for irritable bowel syndrome reported severe diarrhea. Linaclotide is indicated for constipation-predominant irritable bowel syndrome (IBS) or chronic idiopathic constipation. If severe diarrhea occurs, treatment should be interrupted or discontinued. It is currently unknown whether linaclotide is excreted into human breast milk. Although linaclotide and its active metabolites are undetectable in plasma after oral administration at the recommended dose, caution should still be exercised when breastfeeding women take linaclotide. For more drug warnings (full version) (10 in total) on linaclotide, please visit the HSDB record page.

---

Pharmacodynamics
Linaclotide is a laxative with visceral analgesic and secretory effects. In animal studies and clinical trials, linaclotide improved constipation and gastrointestinal symptoms in patients with constipation-predominant irritable bowel syndrome and chronic idiopathic constipation. In animal models, linaclotide has been shown to accelerate gastrointestinal motility and reduce intestinal pain. In animal models of visceral pain, linaclotide reduced abdominal muscle contractions and decreased the activity of pain-sensory nerves. Compared with taking it on an empty stomach, taking linaclotide with a high-fat meal resulted in loose stools and increased bowel frequency. Linaclotide binds to its target, guanylate cyclase C (GC-C), with high affinity and selectivity. Linaclotide and its active metabolites exert their effects locally on the luminal surface of the intestinal epithelium. Because linaclotide is stable in strongly acidic pH environments, its effects are not affected by pH.

---

Objectives
Linaclotide is an orally administered 14-amino acid peptide currently 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 its oral bioavailability, and investigated whether the pharmacodynamic effects produced in a rodent gastrointestinal model are mechanistically related to the activation of intestinal guanylate cyclase C (GC-C). Main Methods:
A competitive binding assay was used to assess the binding of linaclotide to the intestinal mucosa. The stability and oral bioavailability of linaclotide were determined in small intestinal fluid and serum, respectively, and a gastrointestinal model was established using wild-type (wt) and GC-C gene knockout mice. Key Findings:
Linaclotide inhibited the in vitro binding of [125I]-STa to the intestinal mucosa of wt mice in a concentration-dependent manner. Conversely, the binding of [125I]-STa to the intestinal mucosa of GC-C knockout mice was significantly reduced. Linaclotide was completely degraded after in vitro incubation in jejunal fluid for 30 minutes. Pharmacokinetic analysis showed extremely low oral bioavailability (0.10%). In intestinal secretion and transport models, linaclotide exhibited significant pharmacological effects in wild-type mice, but these were not observed in GC-C knockout mice: increased fluid secretion in surgically ligated jejunal loops, accompanied by elevated cyclic guanosine monophosphate (cGMP) levels and accelerated gastrointestinal transport. Significance: Linaclotide is a potent and selective GC-C agonist that exerts its pharmacological effects locally in the gastrointestinal tract. These pharmacological characteristics suggest that oral administration of linaclotide may improve abdominal symptoms and bowel habits in patients with constipation-predominant irritable bowel syndrome (IBS-C) and chronic constipation. [1] Objective: This article reviews the pharmacology, pharmacokinetics, clinical efficacy, and safety of linaclotide in the treatment of chronic constipation (CC) and constipation-predominant irritable bowel syndrome (IBS-C). Abstract: Linaclotide (Linzess, Forest Pharmaceuticals) is a 14-amino acid peptide indicated for the treatment of chronic constipation and constipation-predominant irritable bowel syndrome in adults. Linaclotide acts on guanylate cyclase C receptors on the intestinal luminal membrane, increasing the secretion of chloride and bicarbonate ions into the intestine and inhibiting sodium ion absorption, thereby increasing water secretion in the intestinal lumen and improving defecation; the drug is minimally absorbed in the systemic circulation. Linaclotide has been approved by the U.S. Food and Drug Administration (FDA) for once-daily oral administration for the treatment of chronic constipation (CC) at a dose of 145 μg and for the treatment of constipation-predominant irritable bowel syndrome (IBS-C) at a dose of 290 μg. In a placebo-controlled Phase III clinical trial, linaclotide significantly increased weekly spontaneous bowel movement frequency and total spontaneous bowel movement (CSBM) frequency in patients with chronic obstructive pulmonary disease (CC), while also reducing abdominal pain. In patients with IBS-C, linaclotide was shown to meet FDA-recommended endpoints, such as a reduction of at least 30% in abdominal pain score and CSBM frequency from baseline. The most common adverse reaction to linaclotide was diarrhea, which was reported in 16–20% of clinical trial participants. Conclusion: Linaclotide represents a significant advancement in the treatment of CC and IBS-C, with its unique mechanism of action accelerating intestinal motility. Clinical trials have demonstrated that linaclotide is superior to placebo in the treatment of both CC and IBS-C. Adverse reactions to linaclotide are generally mild and limited to the gastrointestinal tract.

C61H83N15O23S6

1586.77

1585.411

851199-60-5

851199-59-2 (free);

16158207

Cys-Cys-Glu-Tyr-Cys-Cys-Asn-Pro-Ala-Cys-Thr-Gly-Cys-Tyr (disulfide bridge: Cys1-Cys6; Cys2-Cys10; Cys5-Cys13)
H-Cys(1)-Cys(2)-Glu-Tyr-Cys(3)-Cys(1)-Asn-Pro-Ala-Cys(2)-Thr-Gly-Cys(3)-Tyr-OH.CH3CO2H
L-cysteinyl-L-cysteinyl-L-alpha-glutamyl-L-tyrosyl-L-cysteinyl-L-cysteinyl-L-asparagyl-L-prolyl-L-alanyl-L-cysteinyl-L-threonyl-glycyl-L-cysteinyl-L-tyrosine (1->6),(2->10),(5->13)-tris(disulfide) acetic acid

CCEYCCNPACTGCY
CCEYCCNPACTGCY (disulfide bridge: Cys1-Cys6; Cys2-Cys10; Cys5-Cys13)

Typically exists as solid at room temperature

20

30

13

105

3060

14

C(=O)(O)C.O=C1[C@H](CC(=O)N)NC(=O)[C@@H]2CSSC[C@@H](C(N[C@@H]3C(N[C@H](C(N[C@H](C(N[C@H](C(N2)=O)CSSC[C@@H](C(=O)N[C@H](C(=O)O)CC2C=CC(O)=CC=2)NC(=O)CNC(=O)[C@H]([C@H](O)C)NC(=O)[C@H](CSSC3)NC(=O)[C@H](C)NC(=O)[C@@H]2CCCN12)=O)CC1C=CC(O)=CC=1)=O)CCC(=O)O)=O)=O)N

KWFNVZFWXXEJKL-YZDVLOIKSA-N

InChI=1S/C59H79N15O21S6.C2H4O2/c1-26-47(82)69-41-25-101-99-22-38-52(87)65-33(13-14-45(80)81)49(84)66-34(16-28-5-9-30(76)10-6-28)50(85)71-40(54(89)72-39(23-97-96-20-32(60)48(83)70-38)53(88)67-35(18-43(61)78)58(93)74-15-3-4-42(74)56(91)63-26)24-100-98-21-37(64-44(79)19-62-57(92)46(27(2)75)73-55(41)90)51(86)68-36(59(94)95)17-29-7-11-31(77)12-8-291-2(3)4/h5-12,26-27,32-42,46,75-77H,3-4,13-25,60H2,1-2H3,(H2,61,78)(H,62,92)(H,63,91)(H,64,79)(H,65,87)(H,66,84)(H,67,88)(H,68,86)(H,69,82)(H,70,83)(H,71,85)(H,72,89)(H,73,90)(H,80,81)(H,94,95)1H3,(H,3,4)/t26-,27+,32-,33-,34-,35-,36-,37-,38-,39-,40-,41-,42-,46-/m0./s1

((3S,6R,9S,15R,20R,23S,26S,29R,32R,37R,40S,45aS)-32-amino-40-(2-amino-2-oxoethyl)-26-(2-carboxyethyl)-23-(4-hydroxybenzyl)-9-((R)-1-hydroxyethyl)-3-methyl-1,4,7,10,13,22,25,28,31,38,41,47-dodecaoxotetracontahydro-19H-37,20-(epiminomethano)-6,29-(methanodithiomethano)pyrrolo[2,1-s][1,2,27,28]tetrathia[5,8,11,14,17,20,23,32,35,38,41]undecaazacyclotritetracontine-15-carbonyl)-L-tyrosine compound with acetic acid (1

MM416775 acetate; MM 416775; 851199-60-5; MD-1100 acetate; MM-416775; UNII-NSF067KU1M; Linaclotide acetate [USAN]; Linaclotide acetate (USAN); MD-1100; MM-416775; Linaclotide acetate; Linzess; Constella

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

May dissolve in DMSO (in most cases), if not, try other solvents such as H2O, Ethanol, or DMF with a minute amount of products to avoid loss of samples

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

---

Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

View More

Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

---

Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

View More

Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

---

Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

---

 (Please use freshly prepared in vivo formulations for optimal results.)