Linaclotide

Female CD1 mice (age, 8–10 wk) were administered loperamide (0.3 mg/kg, IP; Sigma-Aldrich) to produce constipation. Various amounts of CFTR_act-J027 (0.1, 0.3, 1, 3, and 10 mg/kg) were given at the same time (for IP administration) or 1 hour before (for oral administration) loperamide. Control mice were treated with vehicle only. Some mice were treated orally with lubiprostone (0.5 mg/kg; Sigma-Aldrich) or linaclotide (0.5 mg/kg; Toronto Research Chemicals, Inc, Toronto, Ontario, Canada). After loperamide injection, mice were placed individually in metabolic cages with food and water provided ad libitum. Stool samples were collected for 3 hours, and total stool weight and number of fecal pellets were quantified. To measure stool water content the stool samples were dried at 80°C for 24 hours and water content was calculated as follows: (wet weight - dry weight)/wet weight. Similar studies were performed in cystic fibrosis mice (ΔF508 homozygous) lacking functional CFTR. Some studies were performed using the chemically similar but inactive analog of CFTR_act-J027: 3-(2-amino-5-nitrophenyl)-1-(methyl)-2(1H)-quinoxalinone.

Indomethacin, AMG 9810, HC-030031, carbamazepine, amiloride HCl and morphine were obtained from Sigma-Aldrich (St. Louis, MO, United States). Linaclotide, and asimadoline HCl were obtained from Toronto Research Chemicals (Toronto, ON, Canada). AMG 9810, HC-030031, asimadoline and Linaclotide were administered p.o. as suspensions in 0.5% methylcellulose. amiloride HCl was formulated in 25% hydroxypropyl-β-cyclodextran and administered intraperitoneally while morphine was dissolved in saline and given subcutaneously. Also, with the exception of morphine, all compounds were administered one hour prior to behavioral testing (three hours post-Indomethacin dosing). morphine was administered two hours before testing (or two hours after Indomethacin). Route, vehicle and pre-treatment time for all compounds were chosen based on preliminary dosing experiments, pharmacokinetic (PK) data and/or previously published work. All doses are expressed as the free base and were administered in a dose volume of 10 mL/kg.