In-Depth Comparison of Fasted State Simulated Intestinal Fluid (FaSSIF) Versions via NMR Spectroscopy and Complementary Methods

Efficient screening methods in the early drug development stage are key to quickly finding promising drug candidates. Solutions simulating conditions in the small intestine, the main absorption region for orally administered drugs, such as FaSSIF-V2 and V3, are essential. Updated versions aim to better mimic the natural environment with modified concentrations of the main components, taurocholate and lecithin, or inclusion of oxidation products. Here, we use NMR spectroscopy to investigate the aggregation behavior and interactions between drugs and/or polymers with the components in FaSSIF-V2 and -V3 on a molecular level. BCS-class II compounds carbamazepine, ketoconazole, and efavirenz served as model drugs. We found that while the individual FaSSIF versions alone showed a different apparent diffusion, addition of either drugs or polymers evened out these differences. The more complex composition of FaSSIF-V3 results in a higher signal overlap in the NMR spectra. Furthermore, the simultaneous presence of similar chemical entities with different solubilities is challenging for the DOSY analysis, as one NMR signal contains differently diffusing species. NMR spectroscopy complemented by solubility and DLS measurements as well as MD simulations showed that the different diffusion behavior of FaSSIF-V2 and -V3 is indeed a result of the solubility variations of the lecithin-like molecules. Furthermore, it was observed that all model drugs behaved differently in the FaSSIF media, spanning behavior from barely interacting (carbamazepine) to drug molecules in fast exchange between bile colloids and free solution (ketoconazole) to molecules fully incorporated into the bile aggregates (efavirenz). Although FaSSIF-V3 mimics the natural environment better, FaSSIF-V2 behaved overall more robustly across the different analytical tools and the molecular-level picture obtained for the individual media was overall similar.