3.1.2. In Vivo Pharmacokinetic (PK) Studies

In vivo studies were carried out in a porcine model to assess the local and systemic pharmacokinetics of FTS from drug-eluting strings (Supplementary Figure S6). FTS-loaded strings (25 cm L) were prepared using the dip-coating method described above. The strings were placed along the entire length of the pig's esophagus as described above. FTS level was measured in both esophagus tissue and plasma using LC-MS/MS analysis. The serum samples collected at 1 and 3 days post string administration showed minimum FTS concentrations (FTS < 30 pg/mL). There was only one exception of FTS concentration (57.6 pg/mL) detected in serum sample of one pig at day 3 post string administration. In contrast, FTS was detected in all in vivo esophageal tissue explants with higher FTS concentrations detected in day 1 (105.89 ±10.87 µg) compared to day 3 (14.42 ± 9.9 µg) (Supplementary Figure S6B) for FTS-loaded PCL strings. Ex vivo studies with fluticasone coated PCL strings (2 cm L) on a fresh porcine esophageal section incubated for 1 or 3 days in PBS showed an opposite trend to in vivo studies with an increase in FTS tissue accumulation after 3 days incubation in PBS (102.2 ± 27.6 µg) compared to a 24 h incubation (31.78 ± 13.19 µg) (Supplementary Figure S2B). These results demonstrate that FTS was released from the strings at a continuous rate and was highly accumulated in the esophageal tissue both ex vivo and in vivo with minimal or no accumulation in the blood in vivo.

To evaluate a more clinically relevant string material, additional in vivo PK studies were carried out with FTS-loaded fabric strings in the porcine model (Supplementary Figure S6A). An average of 22.08 ± 0.31 (*n* = 5) mg fluticasone was loaded on 25 cm long (L) strings. Strings were then implanted in the esophagus as described above for 1 day of dwell time. Blood (plasma) samples collected at 1 day showed no absorption of FTS, with an exception of 106 pg/mL detected in one pig. FTS was detected in 4 out of 5 pigs' blood (serum) samples with an average of 83 ± 21.6 pg/mL (*n* = 4). On the other hand, FTS was detected in all in vivo esophageal tissue explants at significantly higher concentrations of 23 ± 12.9 µg, which is orders of magnitude higher compared to serum levels. These results

showed that FTS accumulated in the porcine esophageal tissue explants at high levels (~2 <sup>×</sup> <sup>10</sup><sup>5</sup> serum levels) detected at each collection time point, with minimum systemic absorption of FTS detected in blood (serum/plasma) samples. To corroborate the in vitro release studies and in vivo PK studies, ex vivo studies were performed with FTS-loaded fabric strings (2 cm L, *n* = 3) to determine FTS accumulation in porcine esophagus tissue explants over 24 h incubation in PBS at 4 ◦C (Supplementary Figure S2). An average of 103.9 ± 35.4 µg FTS was accumulated in the esophageal tissue explants, which corroborates the in vivo results demonstrating the absorption of FTS in the esophagus.
