| Assay Detail |
PAMPA was performed and analyzed as in Naylor et al. from 2017 with a few differences introduced by sink conditions and in the concentration of analytes. All other details were consistent. The donor well solution was prepared with the addition of 0.2% polysorbate-80 and the acceptor solution had 0.2% TGPS added. Sample preparation was complicated by the need to equalize the polymer presence in the final donor and acceptor solutions. After plating the 100 µL each from the donor and acceptor wells of the experiment, 100 µL of ACN and 100 µL of PBS buffer with 5% DMSO and either 0.2% polysorbate-80 or 0.2% TPGS was added to each well such that both polymers were present in each sample before quantitation on the Velos Pro Orbitrap LC-MS system. PAMPA on mixtures was performed at 500µM total concentration, resulting in a maximum theoretical 3.3 µM concentration for each putative peptide. 1NMe3 was added as an internal standard at 1 µM (exact mass 754.50, 1.98 ± 0.14 * 10^-6 cm/s under these conditions). Where there was insufficient signal (heptamer sub-libraries DDAL, DLMAL), injection volumes were increased from 5 µL to 20 µL. Our initial PAMPA data set showed a strong correlation between the pure resynthesized compound permeabilities and the library permeabilities. Combined with the low deviation observed in section 5.1 for PAMPA on the same mixture at varying concentrations, we found it unnecessary to perform further replicates of the library data. Pure compounds were assayed at a concentration of 10 µM in the donor well with four replicates to establish a standard deviation after any bad wells were removed. Carbamazepine was used as an internal standard at 10 µM.
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