Tuning the predictive capacity of the PAMPA-BBB model.
Due to its robustness and versatility, several variations of the blood-brain barrier specific parallel artificial membrane permeability assay (PAMPA-BBB) have been reported in the central nervous system (CNS) drug discovery practice. In this study, the impact of the main assay parameters on the predictive power of PAMPA-BBB was thoroughly investigated with 27, passively BBB-transported drug molecules with in vivo logBB data. The single and combined effects of the following variables were systematically studied and optimized: incubation time and temperature (4 vs. 18h, RT vs. 37°C), type of the read-out (UV-reader vs. HPLC), solvent composition (n-dodecane/n-hexane), lipid concentration (0-10w/v % PBLE), cholesterol content (0-1.66w/v %), and thickness of the lipid membrane, and the DMSO cosolvent content (5-20v/v %), respectively. Based on our results, solvent-driven and lipid-driven mechanisms of diffusion were identified in different assay conditions. Moreover, the analysis of membrane retention (MR%; the mole fraction of solute "lost" to the membrane) data obtained at various membrane compositions (volume of solvent and concentration of phospholipids) revealed the compound-specific nature of this parameter. The optimized conditions for the PAMPA-BBB were the following: 4h incubation at 37°C, detection by HPLC-DAD, iso-pH conditions (pH=7.4) with 5v/v % DMSO content in buffer solutions, and PBLE (10w/v %; without cholesterol) as membrane dissolved in the mixture of n-hexane:n-dodecane 3:1.
Department of Organic Chemistry and Technology, Budapest University of Technology and Economics, Budafoki út 9-11., Budapest H-1111, Hungary.
Judit
J
Müller
Faculty of Food Science, Corvinus University of Budapest, Villányi út 29-43, Budapest H-1118, Hungary.
Katalin
K
Esső
Department of Organic Chemistry and Technology, Budapest University of Technology and Economics, Budafoki út 9-11., Budapest H-1111, Hungary.
Gergő
G
Dargó
Compound Profiling Laboratory, Gedeon Richter Plc., Gyömrői út 19-21, Budapest H-1103, Hungary.
Árpád
Á
Könczöl
Compound Profiling Laboratory, Gedeon Richter Plc., Gyömrői út 19-21, Budapest H-1103, Hungary. Electronic address: gy.balogh@richter.hu.
György T
GT
Balogh
D001812:Blood-Brain Barrier / Q000378:metabolism*; D018938:Caco-2 Cells / Q000378:metabolism; D006801:Humans; D008567:Membranes, Artificial; D008954:Models, Biological; D010539:Permeability; D010599:Pharmacokinetics
D008567:Membranes, Artificial
Blood–brain barrier; Cholesterol; Cosolvent; Lipid composition; PAMPA; Permeability