Browsing by Subject "intestinal absorption"

There is a strong need for new in vitro methods in early drug development that predict in vivo conditions more reliably. One of the prerequisites for successful drug therapy is sufficient permeability. A drug needs to be transported through a cell membrane before it can have a pharmacological effect. Therefore, the drug-cell interactions are studied in the early stage of the drug development process. The literature review of this work covers the traditional in vitro and in silico methods of predicting the permeability of drugs across the intestinal membrane. The widely applied methods are reviewed briefly and the predictability of the methods is evaluated. Moreover, the surface plasmon resonance (SPR) technique is introduced. The principle of SPR and its applications for predicting intestinal permeability using lipid membranes resembling the intestinal membrane and for studying drug-cell interactions are discussed. The advantage of the SPR technique is that it is an optical method which allows real-time monitoring under a constant flow without labeling agents. The aim of the experimental part of this work was to evaluate the suitability of the SPR technique for cell-based studies to monitor drug-cell interactions in native cellular environments. Previously, the SPR technique has been almost merely used in routine biomolecular interaction analysis. Recently, the SPR technique has also been applied to cellbased assays but in those studies the reason for the SPR signal responses is generally poorly discussed. The objective of the experimental study was to evaluate and optimize different cell culturing approaches for living cell sensing for SPR, i.e. cells immobilized on the roof of the PDMS molded flow channel in the SPR instrument and cells immobilized directly on the SPR sensor surface. ARPE-19 cells were immobilized on the PDMS substrates but the challenge of imaging cell monolayers on PDMS molded SPR flow channels suggested that immobilizing the cells directly on the SPR sensor surface would be a more straightforward procedure. Hence, ARPE-19 and MDCKII cell culturing protocols were optimized for successful immobilization of confluent cell monolayers directly on the SPR sensor surface. However, ARPE-19 cells showed poor resistance against shear stress in the flow channel; whereas MDCKII cells showed much better resistance against shear stress in the flow channel. Therefore, only MDCKII cells immobilized on the SPR sensor surfaces were used for drug-cell interaction studies. After three days of culture MDCKII cells were exposed to test compounds in separate SPR measurements. The used test compounds were propranolol, D-mannitol, D-glucose and HSPC:Chol liposomes. During the SPR measurements, the changes in the SPR peak minimum angular position and SPR peak minimum intensity were recorded in real-time, and these were further used for analysis after the measurements. The results showed that clear differences in both SPR signals between propranolol and D-mannitol were observed when the cells were exposed to the test compounds. Propranolol diffuses effectively by the transcellular pathway into cells whereas D-mannitol uses the paracellular pathway. This indicates that the introduced SPR approach may be a potential in vitro method in order to provide real-time information on the permeability of drugs and possibly on cell uptake mechanisms of nanoparticles for a better mechanistic understanding of drug-cell interactions on a cellular level.