| 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 permeability test. The acceptor plate was prepared by adding 300 μL of 5% DMSO/PBS to each well. 100 μM solutions of the cyclic peptides were prepared in 5% DMSO/PBS buffer, and a 1% (w/v) solution of lecithin in dodecane was prepared and sonicated before use. 5 μL of the dodecane lecithin solution were carefully applied to the membrane supports in the wells of the donor plate, with care being taken to not touch the pipet tip to the membrane. Without allowing this solution to evaporate, 150 μL of the 100 μM peptide solutions were added to the donor wells. Each compound was tested in triplicate. 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. A lid was placed on the donor well, and the system was covered with a glass evaporating dish and left overnight at room temperature for 16 hours. A wet paper towel was placed on the inside of the chamber to prevent evaporation.
Acceptor and donor well concentrations were measured by LCMS (Thermo LTQ) using selected ion monitoring (SIM) mode. An internal standard of H2N-Tyr(OtBu)- CO2H was run with each sample so that compound-to-standard peak area ratios from the TIC detector could be used to determine relative concentrations. Next, a solution was prepared containing 150 μL of the 10 μM standard, and 50 μL of either the donor or the acceptor well in a 250 μL LCMS vial insert. 20 μL of this solution were injected into the LCMS. Ratios of analyte-to-standard peak areas were then calculated to represent the concentration of analyte in either well. The logPe values were calculated using the following formula: logPe = log {-C・ln (1 - [drug]_acceptor / [drug]_equilibrium) }. Where C= (V_DonorWell・V_AcceptorWell / (V_DonorWell + V_AcceptorWell) Area_Plate ・time). Prior to running the PAMPA each compound was individually tuned on the instrument using an 80%/20% MeOH/H2O solution containing 20 μM of the compound and 1% Formic Acid.
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