*2.5. In Vivo Pharmacokinetic (PK) Studies*

All in vivo animal studies were approved by the North Carolina (NC) State IACUC. Yorkshire-cross pigs (~25–35 kg bodyweight to allow an optimal esophagus size approximating a human esophagus size) were obtained from campus-maintained breeding herds at NC State University's Swine Educational Unit, and then transferred to the care of Laboratory Animal Resources (LAR) at the College of Veterinary Medicine (CVM). CVM facilities are AAALAC- and USDA-approved, and are managed by the University Attending Veterinarian and a team of board-certified Laboratory Animal Veterinarians (LAVs), Registered Veterinary Technicians (RVTs), and animal care workers. Endoscopy was performed with the pigs under general anesthesia with assistance from RVTs for anesthesia, using standard of care protocols. Pigs were sedated with a combination of xylazine (0.25 mg/kg) and ketamine (11 mg/kg) administered IM, followed by mask induction of anesthesia with isoflurane vaporized in 100% O2. Pigs were then orotracheally intubated and maintained on isoflurane throughout the procedure. All post-endoscopic care of the animals was performed by LAVs, RVTs, and animal caretakers within LAR, in accordance with protocols established in the IACUC application. Any evidence of postoperative pain was managed with administration of buprenorphine (0.05 mg/kg, IM, q8h as needed).

For endoscopic ring implantation, three sets of 5 pigs were used. In the first set, 3D-printed rings were used without fluticasone (placebo rings) to confirm that the rings could be placed. In order to deploy the rings, the rings were back-loaded onto the inner section of an esophageal overtube (Gardus, Steris Healthcare, Mentor, OH, USA), through which the endoscope was inserted and the esophagus was intubated with the tube and ring in place. Then, the outer section of the overtube was gently advanced over the inner tube to push the ring into position in the proximal esophagus. As this technique was successful, fluticasone-eluting rings were deployed in the two additional sets of pigs. Rings were

allowed to indwell for seven days during which time the pigs were provided with an ad lib diet, and then repeat endoscopy was performed to determine if the ring was still seated at day seven, serum, and plasma specimens were obtained for PK analysis and the animals were euthanized per the standard CVM/LAR protocol consistent with 2009 American Veterinary Medical Association (AVMA) Guidelines on Euthanasia. Pigs were initially sedated as before with xylazine/ ketamine, after which they were administered an overdose of pentobarbital (60 mg/kg, IV). The esophagus was immediately explanted once euthanasia had been confirmed, divided into proximal, mid, and distal segments, and flash-frozen for subsequent PK analysis.

For the endoscopic string implantation, an additional three sets of 5 pigs were used. For the first set, the thicker fluticasone-coated string was advanced through a previously placed esophageal overtube, and then held in place as the overtube was removed. The string was then brought through the posterior aspect of the pig's cheek (caudal to the last molar) after making a small incision and retrieving the string with forceps. Strings were externally fixed in place with 2-0 nylon suture and covered with a section of iodine-impregnated self-adhering drape material. The string was allowed to dwell for 24 h while solid feed was withheld, and buprenorphine analgesia was provided. At 24 h, the animals were euthanized, and serum, plasma, and esophageal samples were obtained as above. The procedure for the second set of pigs was identical to the first, with the exception of a 72 h string dwell time, and after the first 24 h the pigs were allowed to eat a liquid diet. For the last set of pigs, the FTS-impregnated fabric string was endoscopically placed. For this, biopsy forceps were used to grasp a fabric tape tag at the end of the string, and gently advanced this into position using direct visualization. After external fixation as above, this string had a 24 h dwell time, with the same sample procurement protocol as for the first set of pigs.
