| Assay Detail |
Basic protocols for running PAMPA were based on literature from the Millipore technical library. First a 1% lecithin/dodecane lipid solution (10mg lecithin, 1ml dodecane) was prepared and sonicated for 1 hour to ensure complete dissolution. 5 µL of this solution was then carefully applied to the donor well, avoiding pipette tip contact with the PAMPA membrane. Immediately after artificial membrane application, 150uL of 2.5 µM cyclic peptide containing donor solutions prepared in 0.25% DMSO/PBS were added to the donor plate. The donor plate was then carefully placed on top of the teflon acceptor plate, which was filled with 300 µL of 0.25% DMSO/PBS buffer. The sandwiched donor and acceptor plates were enclosed in a glass container with wet paper towels, in order to avoid evaporation during the 19 hour incubation period. The equation below was used to calculate log Pe values. logPe = log {-C・ln (1 - [drug]_acceptor / [drug]_equilibrium) }. Where C= (V_DonorWell・V_AcceptorWell / (V_DonorWell + V_AcceptorWell) Area_Plate ・time).
1mM stocks of the 14C labeled cyclic peptides were prepared in DMSO. From these, 2.5 µM donor well solutions were prepared in 0.25% DMSO/PBS. To quantitate donor well concentrations, 200 µL of the donor well solutions were added to 10 ml of Scinti Verse II scintillation cocktail (Fischer) in 20 ml scintillation vials. A Beckman LS 6500 scintillation counter, equipped with automatic quench and chemiluminescence correction, was then used to measure disintegrations per minute (DPM). From the starting donor well DPM’s, equilibrium concentrations were calculated. After 19 hours of incubation at room temperature, DPM’s were measured for 200 µL of the acceptor well solutions. Each cyclic peptide was run in three PAMPA wells and averages were taken to obtain the final log Pe values.
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