| Assay Detail |
A PAMPA (Parallel Artificial Membrane Permeability Assay) test was performed for the purpose of comparing and examining the membrane permeability of the synthesized cyclized peptides. An artificial phospholipid membrane was prepared by adding 4 μL of a phospholipid organic solvent solution consisting of millipore 96-well membrane filter (hydrophobic PVDF (polyvinylidene difluoride), pore size 0.45 μm) (purchased from Nihon Millipore) was added with 10% (w/v) egg lecithin, 0.5% (w/v) cholesterol and dodecane (both purchased from Fluka).
To a pH 6.5 50 mM MOPSO buffer containing 5.0% (w/v) of glycocholic acid, a DMSO solution containing a compound at a concentration of 10 mM was added at a ratio of 0.5% (v/v). 330 μL was added to a 96-well Teflon plate (donor plate). The above artificial phospholipid membrane plate was mounted on the donor plate, and DMSO was added to pH 6.5 50 mM MOPSO buffer containing 5.0% (w/w) glycocholic acid at a ratio of 0.5% (v/v). 280 μL of the solution added in 1 was added onto the artificial phospholipid membrane (acceptor plate). After allowing these to stand at 37°C for 18 hours, the concentration of the compound in solution in the donor plate and acceptor plate was measured by LC/MS or LC/UV. The membrane permeation rate (Pe) of the compound was calculated from the following formulas (1) and (2). (1): Pe = (2.303 * V_D / A * t ) * (1 / (1 + V_D / V_A)) * log10 [1 - ((1 + V_A / V_D) / (1 - R)) * (C_A,t / C_D,t=0)]. (2): R = 1 - ((C_D,t + (C_A,t * V_A / V_D)) / C_D,t=0). Here, t is the test time, A is the membrane filter area, V_D is the amount of the donor solution, V_A is the amount of the acceptor solution, C_D,t is the concentration of the compound in the donor solution at time t, C_A,t is the concentration of the compound in the acceptor solution at time t.
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