Abstract by Venkatasubramanian Ramakrishnan

Administering therapeutic peptides orally for systemic absorption poses challenges due to physical and chemical degradation of this class of drugs in the gastrointestinal tract (GIT) and poor permeability across intestinal membranes because of their large size and hydrophilicity. To overcome these barriers, formulation strategies that protect the peptide from proteolysis in the GIT and improve permeability across the intestinal membranes are required. Self-nanoemulsifying drug delivery systems (SNEDDS), which are oral lipid-based drug delivery systems, can potentially be used for this purpose, due to their ease of preparation, ability to protect the peptide from proteolytic degradation and innate permeation enhancing capabilities. Exenatide, a GLP-1 analogue, was used as a model drug in this study. To enable loading of exenatide into SNEDDS, its lipophilicity was improved by complexing the peptide with lipids or phospholipids (LPs, Ex:LP) using freeze-drying. The overall aim of this PhD thesis was to improve the bioavailability of exenatide by utilizing phospholipids or lipids (LP) as excipients to design lipophilic complexes (Ex:LP) to be loaded into rationally designed SNEDDS. Furthermore, the ability of the developed in vitro models to predict the in vivo performance was also evaluated.

The aim of the first part of the project was to develop and evaluate the lipophilicity of Ex:LP complexes. Different LPs were selected based on their head group and charge, including anionic LPs (dipalmitoyl phosphatidic acid (DPPA), dipalmitoyl phosphatidylglycerol (DPPG), dimyristoyl phosphatidylglycerol (DMPG)), zwitterionic LPs (soybean phosphatidylcholine (SPC) and monoacylphosphatidylcholine (MAPC)), and one cationic LP (dioleoyl trimethylammonium propane (DOTAP)). The developed Ex:LP improved the miscibility and retention of exenatide in medium chain (C₈-C₁₀) mono-diglycerides (MGDG) during the miscibility and partition studies, respectively. This increase in miscibility and retention of exenatide was not observed for exenatide (alone) or for a physical mixture of exenatide and the respective LP.

The aim of the second part of the project was to develop SNEDDS using design of experiments and characterize for their in vitro properties (droplet size distribution, extent of lipolysis, proteolytic protection of exenatide upon loading Ex:LP complex). Additionally, correlations between different in vitro properties of SNEDDS were evaluated in order to make informed choices regarding SNEDDS compositions for further optimization (such as using enzyme inhibitors and in vivo pharmacokinetic (PK) studies). Fifteen SNEDDS were generated, consisting of medium chain (C₈-C₁₀) triglycerides (MCT), MGDG, polyoxyl-40 hydrogenated castor oil (Kolliphor®RH40), and MAPC. Within this design space, SNEDDS with a small droplet size and a lower extent of lipid digestion resulted in an improved proteolytic protection of exenatide (loaded as Ex.SPC complex) during proteolysis. Three SNEDDS representing the three corners of the response contour plots for droplet size, extent of lipolysis and proteolytic protection, namely N1, N4 and N7 SNEDDS, in the design space were selected for further in vitro assessments. In vitro permeability studies on Caco-2 cells showed that SNEDDS with a higher MGDG content (N4 and N7) significantly increased the permeation of fluorescein isothiocynate dextran-4kDa (FD4), which was used as an easily detectable surrogate for exenatide. Regardless of the type and nature of the Ex:LP complex, the in vitro proteolytic protection of exenatide was highest when loaded into N7 SNEDDS, indicating that the composition of the SNEDDS has a strong influence on the proteolytic stability of exenatide. Finally, the inclusion of enzyme inhibitors such as soybean trypsin inhibitor (STI; protease inhibitors) and Orlistat (lipase inhibitor) in N7 SNEDDS resulted in improved protection of exenatide (loaded as Ex:LP) in the in vitro proteolysis model.

The third phase of the project involved pharmacokinetic (PK) studies in fasted rats after oral gavage, to evaluate the predictability of in vitro techniques utilized in this project along with improving the systemic absorption of exenatide. The initial PK-study revealed that SNEDDS with smaller droplet size, less extent of lipolysis, along with greater protection of exenatide, loaded as Ex:SPC, during proteolysis and enhanced permeability of FD4 across Caco-2 monolayers resulted in improved absorption of exenatide (as Ex:SPC) in fasted rats (N7 SNEDDS). To increase the absorption of exenatide, the dosed lipid volume of N7 was increased from 80 to 240 µL, which resulted in a significant increase in exenatide, as Ex.SPC, absorption. However, no significant increase in bioavailability was found when dosing beyond 160 µL, therefore 160 µL of N7 SNEDDS were chosen for further studies. Upon switching to the other Ex:LP complexes, Ex:DMPG-N7 achieved almost 2.0- and 1.7- fold higher absorption of exenatide compared to Ex:MAPC-N7 and Ex:DOTAP-N7, respectively. This indicates that even though N7 provides high proteolytic protection to exenatide during in vitro proteolysis, the absorption is determined by the type and nature of Ex:LP complex loaded in the optimized N7 SNEDDS. Lastly, on dosing STI and/ or Orlistat loaded Ex:DMPG-N7, inhibition of both proteolysis and lipolysis significantly improved the bioavailability of exenatide, loaded as Ex:DMPG, compared to using only Orlistat or STI loaded into Ex:DMPG-N7.

The final part of this thesis involved a comparative study of two distinct anionic complexing agents, docusate (DOC) and DMPG, in order to evaluate the difference in loading, proteolytic protection and absorption of exenatide loaded into two set of SNEDDS, with high (namely N7) or low (namely M1) MC-glycerides. These SNEDDS displayed improved protection against enzymatic degradation (lipase and protease). By subjecting the resulting formulations to in vitro proteolysis, it was shown that Ex:DOC loaded into SNEDDS with either N7 or M1 SNEDDS, provided a significantly greater protection to exenatide compared to Ex:DMPG loaded N7 or M1 SNEDDS. This suggests that Ex:DOC is better suited for encapsulating exenatide within the lipid droplets. Furthermore, oral administration of Ex:DOC in N7 and M1 SNEDDS to fasted rats resulted in a 1.4- to 1.8-fold increase in absorption of exenatide compared to Ex:DMPG loaded N7 and M1 SNEDDS. These results demonstrate the superiority of DOC as a complexing agent over DMPG for improving the systemic absorption of exenatide loaded into SNEDDS.

Ultimately, the results obtain in the PhD thesis strengthen the potential for utilizing phospholipids or lipids as effective complexing agents to enhance oral delivery of peptides loaded into SNEDDS. This project emphasizes the significance of optimizing the composition of SNEDDS, by taking into account parameters such as droplet size, lipolysis, proteolytic protection, and permeability. Moreover, the developed in vitro techniques employed to predict exenatide absorption in SNEDDS were found to be effective in evaluating SNEDDS performance in rats. The results of this PhD project can provide guidance for developing effective SNEDDS formulations for oral peptide delivery.