| Assay Detail |
A 96-well donor plate with 0.45 µm hydrophobic Immobilon-P membrane supports (Millipore) and a 96-well Teflon acceptor plate were used in the PAMPA. The donor wells were prepared by adding 150 uL of each cyclic peptide solution (10 µM in 5% DMSO/PBS at pH 7.4) to the wells in triplicate. A 1% (w/v) solution of lecithin in dodecane was prepared and sonicated for 5 minutes prior to use. The dodecane lecithin solution (5 µL) was applied to the membrane supports in the wells of the acceptor plate. The acceptor plate was prepared by adding 300 uL of 5% DMSO/PBS (pH 7.4) to each well. The donor plate was then placed on top of the acceptor plate so the artificial membrane was in contact with the buffer solution below. A lid was placed on the donor well, and the system was covered with a glass evaporating dish and left for 10 hours at room temperature. A wet paper towel was placed on the inside of the chamber to prevent evaporation.
Once the assay was complete, a sample from each well (90 µL) was diluted in a 10 µM solution of H2N-Tyr(Ot-Bu)-CO2H in MeOH (90 µL), then 20 µL was injected into the LC-MS. Acceptor and donor well concentrations were measured by LC-MS (Thermo LTQ) using selected ion monitoring (SIM) mode to detect the parent masses of both the analyte and the internal standard. Analyte-to-standard peak area ratios from the TIC detector were calculated and used to determine relative concentrations in both donor and acceptor wells. These ratios were then used to calculate an equilibrium value adjusted for retention (ER): ER = (RAVA + RDVD)/VA + VDWhere RA is the analyte-to-standard peak ratio of the acceptor, VA is the volume of the acceptor (cm^3), RD is the analyte-to-standard peak of the donor, and VD is the volume of the donor. Transmittance percentage (%T) was then calculated for each sample:%T = (RA/ER) x 100 The %T values were converted into time-independent Pe values: Pe = [(VA x VD)/(V0 x A x t)] x ln(1-(%T/100)) Where V0 is the total volume (cm3), A is accessible filter area of the membrane (0.24 cm^2), and t is the incubation time (s). Average %T and Pe values were calculated for each compound from at least three data points excluding extreme outlying permeability values. Standard deviations were calculated for the average values. Because percent recovery is accounted for in the ER, we assume no compound loss in the calculation and it holds true that apparent permeability (Pa) and effective permeability (Pe) values are equivalent.
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