CaCO2 cells were maintained by media replacement in both chambers every other

day for 14 days, and subsequently, daily for up to 21 days. The integrity of the monolayer PS-341 manufacturer formed was assessed by trans-epithelial electrical resistance (TEER) readings employing a Millicell (MilliPore, Bedford, MA). Monolayers registering net TEER values ranging between 400 and 500 Ω were used for permeation assay. Before the permeation study, CaCO2 monolayer integrity and permeability were assessed using the Millicell and Lucifer yellow respectively. Permeation was carried out with 10 μg/ml (0.5 ml) of C-DIM-5 or C-DIM-8 (in pH-adjusted HBSS-HEPES buffer) and 1.5 ml of blank HBSS-HEPES buffer (pH 7.4) added to the apical and basolateral compartments respectively. The transwells were perfused with 5% CO2 in a humidified 37 °C atmosphere under constant stirring at 50 rpm. Collection of permeated samples (200 μl) from the basolateral compartments were done at 2 h. The samples were injected into a Symmetry C18 column

of an HPLC under an isocratic AP24534 ic50 flow of 1 ml/min in an acetonitrile:water (70:30) mobile phase and detection done at a wavelength of 240 nm. Apparent permeability (Papp) was computed thus: Papp=(([C]×Vb)/([C]a×Va))/(T×Va/A)Papp=(([C]×Vb)/([C]a×Va))/(T×Va/A) where [C] = drug concentration in acceptor compartment; Vb = volume of fluid in acceptor compartment; [C]a = drug concentration in donor compartment; Va = volume of fluid in donor compartment; T = time; and A = surface area of transwell membrane. Aqueous formulations suitable for nebulization were prepared by dissolving C-DIM-5 (50 mg) in 0.5 ml ethanol and 500 mg of vitamin E TPGS and diluting up to 10 ml with distilled water to obtain a 5 mg/ml solution of Levetiracetam C-DIM-5. This was used for in vitro cytotoxicity

studies and aerodynamic characterization. A 5 mg/ml nebulizing solution was prepared and used for animal studies and comparable formulations of C-DIM-8 were also prepared. An eight-stage Anderson cascade impactor (ACI), Mark II was used for particle size assessment. Impactor plates were coated with 10% pluronic-ethanolic solution to mitigate particle rebound. The formulation was nebulized using a PARI LC STAR jet nebulizer at a dry compressed air flow rate of 4 l/min for 5 min into the cascade impactor at a flow rate of 28.3 l/min. Aerosol particles deposited along the ACI (throat, jet stage, plates on impactor stages 0–7, and filter) were collected by washing with 5 ml of mobile phase comprising acetonitrile:water (70:30) and analyzed by high performance liquid chromatography (HPLC). The analysis was performed on a Waters HPLC system using a Symmetry C18 column (5 μm, 4.6 × 250 mm) with a Nova-Pack C8 guard column at a wavelength of 240 nm and flow rate of 1 ml/min. The mass median aerodynamic diameter (MMAD) and geometric standard deviation (GSD) were computed from the obtained impactor data utilizing a validated protocol ( Patlolla et al., 2010).