| Assay Detail |
Artificial phospholipid membranes were prepared by adding 5 μL of phospholipid organic solvent solution consisting of L-alpha-phosphatidylcholine (Avanti, Cat. 840051P, 1.67%) and 1,2 -dioleoyl-sn-glycero-3-phospho-L-serine and n-dodecane/1-octanol (=10/1) on filter plate (Merck Millipore, Cat. MAIPN4550). Next, 25 μL of DMSO solution containing the peptide compound at a concentration of 20 μM was diluted to a final concentration of 1 μM by adding 475 μL of 50 mMKPB (potassium phosphate buffer, pH 7.4 or pH 6.5). 300 µL of the compound solution was added to the lower side (donor side) across the artificial phospholipid membrane of a PAMPA 96-well plate (Filter Plate (Merck Millipore, Cat. MAIPN4550)). 200 μL of 5% DMSO KPB (pH 7.4) was added to the upper side (acceptor side) across the artificial phospholipid membrane. Permeation test through the artificial phospholipid membrane was performed at 25°C for 4 hours. The concentration of the compound in solution in the donor and acceptor plates was measured by LC/MS/MS, and the membrane permeation rate (Pe) of the compound was calculated from the following equation, where t is the test time, A is the membrane filter area = 0.3 cm^2, VD is the donor solution volume = 300 μL, VA is the acceptor solution volume = 200 μL, CD(t) is the compound concentration in the donor solution at time t, CA(t) is the compound concentration in the acceptor solution at time t. Cequilibrium = [CD(t)*VD+CA(t)*VA]/(VD+VA) Pe = -ln[1-CA(t)/ Cequilibrium]/[A*(1/VD+1/VA)*t] Pe is synonymous with Papp. Permeability (in unit of cm/s)
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