(W1130-02-08) Oral Absorption of Semaglutide: Pharmacokinetic Modeling and Molecular Dynamics Simulations

Purpose: Obtaining substantial absorption of peptides into the systemic circulation from the gut is challenging. Semaglutide is a GLP-1 agonist that is used as an anti-diabetes agent and also has demonstrated weight loss effects. A once-daily oral dose of semaglutide can be used clinically, despite the drug being a large lipid-modified peptide. The mechanism of absorption of this drug from the stomach is of considerable interest and the details remain unclear. The goal of this study was to use in silico methods to formulate a hypothesis for this process.

Methods: A pharmacokinetic (PK) model for semaglutide was built using GastroPlus software ver. 9.8.3. and parameters taken from the literature. Physicochemical and pharmacokinetic properties (2) used were introduced into the model in a step-by-step process, and changes in the simulated pharmacokinetic profile were noted to understand key properties required to reproduce the observed data. Semaglutide was represented in GastroPlus using the chemical formula (C187H291N45O59), molecular weight (4114 g/mol) and Log P (-5.8) (3). Each pKa was input individually and the dissolution profile (4) and pH-solubility profile (5) were also included. Model building focused on absorption, while clearance and other pharmacokinetic data were estimated or used as defaults.Molecular dynamics (MD) simulations were conducted using YASARA ver. 22.9.24.W.64. The simulations were run in 0.9% NaCl over 280 ns using the AMBER14 force field. The semaglutide structure was obtained from the RCSB protein databank- PDB ID: 7KI0 (6) and the C-18 fatty acid chain was introduced into the structure through model building. Two simulations were run with different orientations of the lipid chain, essentially in the opposite direction to each other. The same parameters were used in both runs.

Results: A baseline PK model for 10 mg oral semaglutide using simple physicochemical parameters resulted in no absorption. Further exploration of parameters in the software identified the gastric pore radius, molecular radius, paracellular permeability, and gastrointestinal transit time as key model parameters that could be altered to reproduce the observed pharmacokinetic data. Absorption exclusively from the stomach (as shown experimentally) with fitting to the observed PK data was reproduced (Figure 1) with a paracellular P<sub>eff of 0.0045 x 10^-4 cm/s, a molecular radius of 5.82 Å, a gastric pore radius of 10.25 Å, and reduction in the transit time in the intestine to 0.01 h. Variation in the molecular radius from 5.7 to 6.0 Å did not alter the simulated C_max and AUC_0-inf. Similar sensitivity analyses were performed for other parameters. The derived model suggests that for semaglutide to be absorbed from the stomach, the helicity of the peptide has to be maintained and the lipid has to be arranged in such a way that the helical radius is maintained at about 5.8 Å. To examine this requirement, we performed MD simulations on a single semaglutide molecule in a water box. The MD simulations showed that semaglutide can retain a stable helical conformation that is supported by interactions between adjacent amino acids. The most important interaction seems to be between Arg34 and Glu27, which formed an electrostatic interaction with a distance of about 7 Å. Most importantly, we observed that the C-18 lipid chain can wrap tightly around the alpha-helix, such that the chain is unlikely to hinder the paracellular movement of the molecule. Viewed along the helical axis, the lipid made hydrophobic contacts with the peptide and the oxygen atoms in the lipid chain pointed away from the peptide into the solvent (Figure 2).

Conclusion: The results of this study provide a basis for understanding the molecular events leading to absorption of a large peptide from the stomach. The PK model can be used to examine the absorption of different doses under different stomach conditions, while the MD simulation provides insights into the possible conformation of semaglutide, including the association of the lipid with the peptide. At higher concentrations, semaglutide molecules has been shown to self-assemble into higher order structures, but absorption may require single free molecules. We note that the SNAC molecule that is co-administered with semaglutide may have an important role in maintaining monomeric semaglutide, as well as increasing stomach pH and opening the pore radius of the stomach lining (7).

References: 1. Overgaard, R.V., Navarria, A., Ingwersen, S.H. et al. Clinical Pharmacokinetics of Oral Semaglutide: Analyses of Data from Clinical Pharmacology Trials. Clin Pharmacokinet 60, 1335–1348 (2021). https://doi.org/10.1007/s40262-021-01025-x
2. National Center for Biotechnology Information. PubChem Compound Summary for CID 56843331, Semaglutide. https://pubchem.ncbi.nlm.nih.gov/compound/Semaglutide. Accessed Jan. 23, 2024.
3. Stability Indicating RP-UPLC Method Development Validation and Dissolution Testing of Semaglutide . Journal of Pharmaceutical Negative Results. 2022;13(4):1459-1464. doi:10.47750/pnr.2022.13.04.205
4. Bækdal, T.A., Donsmark, M., Hartoft-Nielsen, M.-L., Søndergaard, F.L. and Connor, A. (2021), Relationship Between Oral Semaglutide Tablet Erosion and Pharmacokinetics: A Pharmacoscintigraphic Study. Clinical Pharmacology in Drug Development, 10: 453-462. https://doi.org/10.1002/cpdd.938
5. https://doi.org/10.2210/pdb7ki0/pdb
6. Venanzi, M., Savioli, M., Cimino, R., Gatto, E., Palleschi, A., Ripani, G., Cicero, D., Placidi, E., Orvieto, F., & Bianchi, E. (2020). A spectroscopic and molecular dynamics study on the aggregation process of a long-acting lipidated therapeutic peptide: the case of semaglutide. Soft matter.

Acknowledgements: We thank Simulations Plus for the licenses to the software under University Plus.

Figure 2: Semaglutide viewed along the helical axis showing wrapping of the lipid around the peptide. Oxygen atoms in the lipid are shown in red.

Figure 1: Simulated (line) and observed (squares) plasma concentration- time curve of 10mg oral semaglutide.