| Assay Detail |
PAMPA analysis was conducted following a procedure reported by Lokey and coworkers. Each measurement was conducted at room temperature in an insulated polystyrene box to maintain a constant internal temperature. Measurements were taken in triplicate, and the reported permeability values in Table 1 are the average value over the three runs. Carbamazepine was included in the stock solution of each cyclic peptide to verify the integrity of the membrane as an internal standard. Measurements which did not give consistent permeability values (-Log Papp ±0.1) were repeated in triplicate. In our hands, we consistently achieved permeability values of -Log Papp = ~5.7-5.8 for carbamazepine (Literature value using this method: -Log Papp = 5.1) which indicates that our reported values are at worst case underestimated.
Preparation of stock solution for donor well: Stock solutions were prepared by dissolving the cyclic peptide (0.5 mM) and carbamazepine (0.5 mM) (as an internal standard) in DMSO at a gross concentration of 1 mM. 10 μL of this stock solution was added to 990μL PBS buffer (pH 7.4) to give a 1 mL solution (10 μM, 1% DMSO v/v) for addition to the donor plate wells.Preparation of acceptor plate: A stock solution of 5% DMSO in PBS buffer was prepared, and 300μL of this solution was added to each of the acceptor wells (96-well Teflon acceptor plate (Millipore MSSACCEPTOR)). Preparation of donor plate: 100 mg of lethicin (90%, soybean) was suspended in n-dodecane (10 mL) and sonicated to give a homogenous, clear solution. 5 μL of this solution was carefully applied to the membrane in the 96-well donor plate (with 0.45 μm hydrophobic Immobilon-P membrane supports (Millipore MAIPNTR10)) (with care, without touching the membrane). Shortly after (~5-15 min), 150 μL of the 10 μM stock solution of cyclic peptide and carbamazepine (prepared as above) were added to the donor well. The donor plate was placed on top of the acceptor wells and visually inspected to ensure no air bubbles had formed around the membrane in contact with the acceptor solution. The lid was placed on top of the plates and the system was moved to an insulated polystyrene box with a damp paper towel inside to prevent evaporation. The box was sealed and left overnight (16 hours) with the exact time of acceptor and donor plate separation recorded for analysis. Analysis of donor/acceptor wells: 100μL of the solution in each of the donor and acceptor wells were taken separately and added to a 1-dram vial and sealed immediately to prevent evaporation. The solution was sealed until ready for injection to LC-MS. The solutions were analysed by LC-MS (15 μL injection) using the LC-MS method for PAMPA analysis (see Page 3). Using EIC mode, the peak area was measured for analyte in the donor and acceptor wells, and the Papp value was calculated using the method reported by Lokey and coworkers.
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