| Assay Detail |
PAMPA was performed with a 96-well donor plate with 0.45 μm hydrophobic immobilon-P membrane supports (MultiScreen, MAIPNTR10) at the bottom of plate and a 96-well polystyrene acceptor plate (MultiScreen, MATRNPS50). A 1% (w/v) solution of lecithin in n- dodecane was prepared and sonicated for 5 min before being used for the assay. Lecithin solution (5 μL) was carefully added into the membrane of the donor plate without any touch to the membrane. Each sample was prepared as a 10 μM solution in 5% (v/v) DMSO/PBS buffer. The donor plates were prepared by the addition of a sample solution (150 μL), and the acceptor plates were filled with 5% DMSO/PBS buffer solution (300 μL). The donor plate was placed on top of the acceptor plate without any bubble between the donor and acceptor plates. A lid was placed on the donor plate, and the whole plates were wrapped with a wet paper towel to prevent an evaporation. Then, the plates were incubated at room temperature for 19 h. An aliquot in each donor or acceptor (50 μL) was mixed with 10 μM Fmoc-Tyr(OtBu)–OH in 1:1 (v/ v) water/acetonitrile (50 μL) as an internal standard. The mixture (30 μL) was injected into the LC-MS instrument monitoring the analyte and internal standard with selected ion monitoring (SIM) mode. The analyte to standard peak area ratios were calculated and used to determine the relative concentration. A retention (R) and permeability (Pe) was calculated: R = 1 - (Cd*Vd+Ca*Va)/(C0*Vd), Pe = (Va*Vd)/(V0*A*t) * ln(1-(Ca/Ceq)), Ceq = (Cd*Vd+Ca*Va)/(Vd+Va). (C0, initial concentration (10 μM); CA, concentration of acceptor plate (μM); CD, concentration of donor plate (μM), VA, volume of acceptor plated (300 μL); VD, volume of donor plate (150 μL); A, area of mem- brane (0.24 cm^2), t, time (s), V0: VA + VD).
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