| Assay Detail |
Basic protocols for running PAMPA were based on literature from the Millipore technical library. These include a 5% DMSO/PBS buffer and a 1% lecithin/dodecane lipid solution. Due to the low solubility of the cyclic peptides in buffer, detection methods were altered. Acceptor and donor well concentrations were measured by an LCMS TIC detector (Finnigan LCQ) using selected ion monitoring (SIM), which only monitors specific masses, giving higher sensitivity than a full scan. In addition, an internal standard of 2-aminofluorene was run with each sample, so that ratios of peak areas of compound to standard from the TIC detector, would be used to measure concentrations. 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). Donor well starting ratios of compound to standard were used to calculate the equilibrium concentration.
1mM stocks of the standard 2-aminofluorene, and compounds 1-9 were prepared in DMSO. From these, 5uM solutions of compounds 1-9 were prepared in 5% DMSO/PBS. Next, a solution was made containing 99% of the 5uM stocks and 1% of the 1mM standard. After vortexing the solution thoroughly, 80uL was injected onto the LCMS, utilizing an SIM scan, which only monitors the mass of compounds 1-9 (m/z 713±5) and the standard (m/z 182±5). Ratios of peak areas were then calculated as starting donor well concentrations. From this, the equilibrium concentration was determined. 76 hour time points of the acceptor wells were taken in this same manner (99uL of acceptor well, 1uL of 1mM standard, 80uL injection onto LCMS). Each cyclic peptide was run in three PAMPA wells and averages were taken to obtain the final log Pe values. New solutions of 1mM 2-aminofluorene in DMSO were made at every time point. Compound 1 and 9 were also ran in PAMPA with a 1%DMSO PBS buffer, in which Log Pe values were calculated after 48 hours of incubation.
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