acrylamide 0 resolving gel buffer Millipore -water 0 SDS solution 0 l L, 0 APS solution 0 l L, and TEMED l L. A pet-ri dish was used to form the gel with a diameter of Seliciclib cm. Before adding TEMED to the gel preparati sliced tissue pieces were spread out at the glass surface. To ensure a homogenous distribu-ti tissue pieces were mixed within the gel matrix thoroughly. The gel matrix was covered with a layer of Millipore water and al-lowed for polymerization. Forparis a gel without embed-ded tissue pieces was casted for every experiment. . Adsorption of drugs to polyacrylamide-tissue gel The polymerized gel was transferred to a round ground-joint glass dish with ground-glass lid. Only glass laboratory ware was used to avoid any nonspeci binding of the highly lipophilic-pounds to plastic material.
A thin layer of PBS buffer at the bottom of the glass dish maintained the gel humidity. To determine drug adsorpti a deed dose of themercially available nasal formulation was dispersed in ANF. Immediate l L con-taining l L of the drug in this homogenous mixture was applied evenly onto the gel surface . The glass dish was closed and transferred to an orbital platform P450 Inhibitors shak and the rotational speed was set to 5 rpm . The incubation was performed for 0 min at 7 ° C . After the incubation ti the gel was washed thoroughly to en-sureplete removal of unboundpound and not yet dis-solved drug particles. Therefo a self-designed washing device was used which was lathed from a Te?on block so that it tightly onto the ground joint of the glass dish.
The inner part held right atrium elaborate milled-out portions to allow ef ient washing cycles and yet support the gel suf iently withoutpromising its integrity. The gel was washed with approximately mL PBS . The ing buffer was thoroughly mixed with MeOH to dissolve any solid drug particles. For eachpou a gel without embedded tissue was treated accordingly. All samples contained the same proportion of A P and MeOH. Samples were stored at 0 ° C until analysis. . Desorption of drugs from the polyacrylamide-tissue gel The washed gel was transferred into mL human plasma or whole blood of 7 ° C, respective and gently sha-ken with a rocking platform shaker for h at 7 ° C. Samples of mL were drawn after 5, 0, and 0 min . The volume withdrawn was replaced with pre-warmed fresh human plasma or whole blo respectively.
Samples were stored at 0 ° C until analysis Sample preparati analysis and HPLC conditions Samples of adsorption experiments were centrifuged for 0 min at 0 g at 0 ° C . Supernatants were directly injected into the high-perfor-mance liquid chromatography system. Typical 0 l L of sample was injected. Linearity was given from 0 to ng/mL for B and and coef ients of correlation of the calibration curves were at least r = . Plasma samples of mL were mixed with 0 l L internal standard solution and extracted twice with mL diethylether for 0 m using a roller mix followed by centrifugation at g for min at room tempera-ture. Theanic phases werebined and evaporated to dryness under a gentle stream of nitrogen at 5 ° C. The resulting residue was reconstituted in methanol. AZ blood samples of mL were mixed with 0 l L internal standard solution followed by D. Baumann / European Journal of Pharmaceutics and Biopharmaceutics 0 addition of l L NaOH.