Ys Mice were housed in groups of five in a controlled environment Le, with free access to food and water and on a 12 H/12 H light / dark cycle, light at 6:00. The procedures for the housing, handling and experimental with the recommendations of the National Institutes of Health Guide for the Care and Use of Laboratory Animals and the Institutional Committee for outlining the care and use of laboratory animals, Universit t meet in Buenos Aires, Argentina. Were made every effort to minimize animal suffering. The number of animals used was the minimum compatible with obtaining meaningful application Ftiger data. The animals were Feeder Llig assigned treatment groups and used only once. The behavioral tests were conducted igf-1r by experimenters who did not approve of the treatment was administered and carried out, evaluated from 10.00 bis 02.00 clock. 2.2. The chemical synthesis of sulfonamide derivatives were prepared as described above and the synthesis procedures were in accordance the sulfonamide substituents selected hlt. 2.3. A biochemical assays radioligand binding assay was used to determine the effect of Mutma Lichen compounds of the benzodiazepine site of the GABA A receptor to assess binding of the complex. The binding of flunitrazepam to the benzodiazepine site binding was raw in lime Performed synaptosomal membranes of rat l Gro Cerebral cortex. The membranes were prepared by. Briefly, the brains were quickly pr on ice parried And the various structures were homogenized in 10 volumes 0.32 M sucrose and centrifuged at 900 g for 10 min. The resulting supernatant was centrifuged at 100,000 g for 30 min and the pellet was min twice in 25 mM Tris HCl, pH 7.4 at 100,000 g for 30, stored at 20 8C until use. The protein determination was performed using the Bradford method.
The compounds were suspended to 0.2 0.3 mg of membrane protein in 1 ml of buffer 25 mM Tris-HCl in the presence of 0.3 nM flunitrazepam. In testing each compound was tested at 300 mM in triplicate. In tests of competition incubations with 10 600 mM of compound 4, 1300 mm of compound 7 and 1 100 mm of compound 10 were carried out. Diazepam was used as positive control at concentrations between 1 and 100 nm. At S Saturation assays increasing concentrations of flunitrazepam in the presence of a vehicle, compound 7 to 50 mM or 10 mM compound 10 are incubated. Specific Binding not measured in the presence of 10 mM and Vascular Disrupting Agent flunitrazepam represented 5-15% of total binding. The incubations were performed at 4 8C for 1 h. After incubation, the tests by vacuum filtration through Whatman GF / B glass fiber filters by washing three times with 3 ml of each terminated incubation medium followed. Individual filters were incubated overnight with scintillation cocktail before measurement of radioactivity t in a Wallac 1214 Rackbeta Flüssigszintillationsz Incubated counter. 2.4. Drugs and administration protocols synthetic starting material and reagents L Solvents were analysenreaktionsf compatibility available and were purchased from Sigma Aldrich and Fluka. Diazepam, flumazenil and compounds 7 and 10 are dissolved St using the sequential addition of dimethyl sulfoxide, an L Solution of 0.25% Tween 80 and saline Solution, final concentrations of up to 5%, 20% and 75%, are . The Mice were intraperitone.
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