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Recently in vivo microdialysis experiments have showed that compound 1 (PMPA) reaches very high brain extracelular fluid concentration probably via active transport through BBB: Nagel, J.; Belozertseva, I.; Greco, S.; Kashkin, V.; Malyshkin, A.; Jirgensons, A.; Shekunova, E.; Eilbacher, B.; Bespalov, A.; Danysz, W. Effects of NAAG peptidase inhibitor 2-PMPA in model chronic pain - relation to brain concentration. Neuropharmacology, 2006, 51(7-8), 1163-71.
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The GCP II inhibitory activity of compounds 5-7 was quantified by assessing the rate of hydrolysis of [3H]-NAAG (NEN Life Science Products) as previously described.5,9 Briefly, the total reaction of 500 μl contained rat forebrain membrane protein 20-40 μg; Tris-HCl 50 mM containing 1 mM ZnCl2, pH 7.4; 4 nM, 3H]-NAAG; inhibitor 10 μM (for IC50 7 point DRC, 37° C; 40-45 min. The reactions were terminated by adding ice cold phosphate buffer (100 mM, pH 7.4, Nonspesific/background activity was determined in the presence of 10 μM 2-PMPA, 3H]-Glutamate was separated from residual [3H]-NAAG on the Dowex AG 1-X8 anion exchange resin. After that the scintillation cocktail OptiPhase Supermix (Perkin Elmer) was added to the respective fractions and the elute radioactivity was determined by scintillation spectrometry [1, 9
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3H]-NAAG on the Dowex AG 1-X8 anion exchange resin. After that the scintillation cocktail OptiPhase "Supermix" (Perkin Elmer) was added to the respective fractions and the elute radioactivity was determined by scintillation spectrometry [1, 9].
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