| Assay Detail |
A 96-well donor plate with 0.45 μm hydrophobic Immobilon-P membrane supports (Millipore MAIPNTR10) and a 96-well Teflon acceptor plate (Millipore MSSACCEPTOR) were used in the PAMPA permeability test. The acceptor plate was prepared by adding 300 μL of 5% DMSO in PBS buffer (pH 7.4) to each well. Sample solutions were prepared by diluting DMSO stock solutions to a final 2 µM sample concentration in PBS buffer with a final DMSO concentration of 5%.
A 1% (w/v) solution of lecithin (soybean, 90%) in n-dodecane was prepared and sonicated before use. 5 μL of the n-dodecane / lecithin solution was carefully applied to the underside of membrane supports in the wells of the donor plate, with care taken to not touch the pipet tip to the membrane. After approximately 15 minutes, 150 μL of the 2 µM test compounds were added to the donor wells. The donor plate was then placed on top of the acceptor plate so that the artificial membrane was in contact with the buffer solution below, ensuring that no bubbles form beneath the membrane. A lid was placed on the donor well, and the whole assembly was covered within a sealed chamber and left overnight at room temperature. A wet paper towel was placed inside the chamber to prevent evaporation. After ~15 h (exact time recorded and used for subsequent calculations) the donor and acceptor plates were separated,and 50 µL of each well (donor and acceptor) were mixed with 50 µL MeOH in another 96-well 300 µL conical-bottom plate and sealed. These solutions were analyzed as quadruplicate via UPLC-MS as described above. Permeability (%T) was quantified as the ratio of analyte area in the acceptor well divided by a theoretical equilibrium ratio based on amounts of combined analyte found in the donor and acceptor wells as follows: AnalyteEquil = ((Ia*Va) + (Id*Vd)) / (Va + Vd), %T = (Ia/[AnalyteEquil])*100. Recovery (%R) was quantified as the ratio of total compound identified in the donor and acceptor wells relative to the total compound identified in the original dilution sample. %R = ((Ia*Va) + (Id*Vd)) / (Ir * Vd) * 100. Permeation rates (Papp) were calculated from %T by the following equations: C = (Va * Vd) / ((Va + Vd)*Msa*Ts), Papp(cm/s) = -C*ln(1-(%T/100)). Where: • Active surface area of membrane (mm^2): Msa = 240• Volume of acceptor well (µL): Va = 300• Volume of donor well (µL): Vd = 150• Assay run time (s): Ts• Donor intensity: Id• Acceptor intensity: Ia• Recovery intensity: Ir In addition, propranolol was used as a standard reference to confirm the assay was performed correctly, which had log Papp ~ -5.
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