(M1130-10-68) Influence of Bovine Serum Albumin on Colonic Mucus Permeation and Solubility of Celecoxib and Naproxen in Fasted State Simulated Colonic Fluids

Purpose: Proteins present in human colonic fluids can alter apparent drug solubility and thus have an impact on drug behavior in the colon, such as mucus permeation, and consequently, absorption. In this study, bovine serum albumin (BSA) was used as a model protein in fasted state simulated colonic fluids (FaSSCoF), to investigate possible effects on apparent drug solubility (P_app) and colonic mucus permeation.

Methods: Solubility studies: Celecoxib and Naproxen were added in excess to 10 ml FaSSCoF with 0-15 mg/mL BSA and incubated at 37°C under magnetic stirring. Samples were taken for up to 48 h, centrifuged and the supernatant diluted with acetonitrile before quantification by HPLC. The mean apparent solubility was calculated based on the two last time points, 24 h and 48 h. Mucus permeation at equilibrium drug solubility: Celecoxib and Naproxen dissolved at equilibrium solubility (S_eq) in FaSSCoF containing 0-15 mg/mL BSA, was added to the donor side on top of a layer of porcine colonic mucus, harvested from freshly slaughtered pigs (Danish Crown, Ringsted, DK), in a T12 Transwell setup. The receiver side contained FaSSCoF with BSA at the same concentration as on the donor side. The setup was placed on a microplate shaker while in a heating cabinet, where temperature was maintained at 37°C. Samples from the receiver compartment were taken for up to four hours. Four different mucus thicknesses (0, 2, 3 and 4 mm) were evaluated from drug flux and apparent permeability (P_app).

Results: The apparent S_eq for celecoxib and naproxen are presented in Figure 1. Increasing BSA concentration linearly increases the apparent S_eq of celecoxib and naproxen, however, the relative increase is larger for celecoxib, factor of 20 at 15 mg/mL BSA, while for naproxen it was 1.2 at 15 mg/mL BSA, when compared to FaSSCoF to no BSA. The flux of celecoxib and naproxen across porcine colonic mucus, when dissolved at S_eq in FaSSCoF containing 0-15 mg/mL BSA, are presented in Figure 2. When applying celecoxib or naproxen at S_eq on the donor side, the flux increases with increasing BSA concentration. This effect is more prominent for celecoxib than for naproxen, presumably due to the vastly larger relative increase in apparent S_eq. An increase in the mucus layer thickness leads to a decrease in flux irrespective of drug and BSA concentration. The P<sub>app of celecoxib and naproxen are presented in Figure 3. For naproxen, the P<sub>app is independent of BSA concentration. For celecoxib, the P<sub>app of celecoxib is decreasing with increasing BSA concentrations, when there is a mucus layer present.

Conclusion: Our results confirm that proteins in colonic fluids will have an impact on drug behavior in the colon, such as mucus permeation. BSA has solubilizing capacity for both celecoxib and naproxen. Increasing the apparent S_eq by addition of BSA will help facilitate permeation of celecoxib and naproxen across mucus layers, irrespective of thickness. Precisely how BSA helps achieve this increase in flux is not known, however, we can rationalize that the higher available bound drug concentration can act as a reservoir for available monomeric drug molecules that can quickly partition into solution, as free drug permeates across the mucus layer. Another possibility could be that the celecoxib-BSA complex itself permeates across the mucus layer, albeit at a slower rate than free drug. Further studies to understand the mechanism of action BSA works with needs to be conducted.

Acknowledgements: This project has received funding from the European Union´s Horizon 2020 research and innovation programme under the Marie Sklodowska-Curie grant agreement No 956851

Figure 1. Apparent solubility of celecoxib and naproxen in FaSSCoF as a function of BSA concentration. Mean ± SD (n=6)

Figure 2. Flux of celecoxib and naproxen through porcine colonic mucus, when dissolved in FaSSCoF with 0-15 mg/mL BSA at Seq. Mean ± SD (n=3).

Figure 3. Papp of celecoxib and naproxen through porcine colonic mucus, when dissolved in FaSSCoF with 0-15 mg/mL BSA at Seq. Mean ± SD (n=3).