The use of the parallel artificial membrane permeability assay method in the field of determining blood-brain barrier permeability assumes that it is possible, with an appropriately designed artificial lipid membrane, to mimic passive transport across the blood-brain barrier. Its applicability is primarily associated with the choice of membrane composition and precise optimization of experimental conditions. Parallel artificial membrane permeability assay cannot replace cell-based methods or in vivo methods, as it does not incorporate transporters, metabolic processes, and other biological factors that determine the passage of compounds into the brain. Nevertheless, it represents a fast, robust and practical tool for early assessment of passive permeability. Therefore, the method represents one of the screening approaches in the development of drugs acting on the central nervous system. The aim of the master's thesis was to establish and evaluate the method for predicting passive transport across the blood-brain barrier and to apply it for testing new, structurally diverse compounds. We evaluated 19 model drugs with known permeability classification and 29 compounds from chemically diverse structural series. In the experiments, we primarily used porcine brain lipid extract with a 4-hour incubation at physiological pH and additionally compared two commercial lipid systems as well as an extended 16-hour incubation. The concentrations of compounds in donor and acceptor solutions were determined spectrophotometrically using calibration curves. The measured values of the permeability coefficient Pe of the model drugs used for system validation spanned three orders of magnitude, confirming sufficient sensitivity of the system. The method was also successfully validated against independent literature data and data from studies at the Faculty of Pharmacy. For model drugs with a determined permeability coefficient, the method classified 75% of drugs in accordance with the prediction of the in silico method. Among the compounds investigated, 87% were classified as well-permeable. The highest absolute permeability was determined for compound KAB-21, and it was comparable to the most permeable model drugs. The main methodological limitations are the limited sensitivity of spectrophotometric detection and the inability to detect active transport. In further research, we therefore recommend the use of a more sensitive chromatographic method and supplementation with a cell-based permeability model.
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