Wiretrol

Final dosing and processing conditions were as follows. Male 5–6 week old CF-1 mice were housed in cages with wire mesh bottoms to prevent coprophagia and food was withheld for 16–24 h to reduce the number of pellets present in the colon. Mice were anesthetized with isoflurane and given a 200 μL normal saline (Quality Biological) pre-cleansing enema administered with a plastic flexible feeding tube (Instech Laboratories; 22 G X 25 mm), which was optimized to be sufficient volume to clear the distal colon of pellets prior to dosing. Mice were allowed to regain consciousness for 10 min ambulatory time. Mice were then anesthetized to administer 50 μL of drug solution using a 100 μL WireTrol (Drummond) inserted at a depth of 2 cm into the mouse colon. At designated times, blood was collected into BD Microtainer tubes with K₂EDTA, and plasma was obtained by centrifugation (1300 rcf for 10 min). The distal 4 cm of colon was excised, bisected open to remove pellets as necessary, and cut in half longitudinally with a scalpel. The tissue was stored in cryogenic tubes and flash frozen in liquid nitrogen. All samples were stored at −80˚C after being flash frozen in liquid nitrogen.

Rodents are commonly used as animal models for delivery to the GI tract. However, rodents defecate much more frequently (every 5-10 min for mice) than is typical for humans, and the hard, desiccated pellets do not resemble human feces. To simulate the softer stool consistency and less frequent defecation rate of humans, female 6-8 week old CF-1 mice (Harlan) were starved for 24 h. Mice were housed in cages with wire bottoms to prevent coprophagia. Mice were anesthetized with the drop-method via isoflurane for shorter procedures, or injected with 300 mg/kg Avertin (2, 2, 2-Tribromoethanol), using a 20 mg/mL working solution in phosphate buffered saline (PBS), when more extended anesthesia times were required. The various test solutions were administered intrarectally to mice with a Wiretrol (Drummond, Inc.) at volumes of 20 μL. All experimental procedures were approved by the Johns Hopkins Animal Care and Use Committee.