Source Name: 2021_Lee
Literature Information
| Literature Title | Interplay among Conformation, Intramolecular Hydrogen Bonds, and Chameleonicity in the Membrane Permeability and Cyclophilin A Binding of Macrocyclic Peptide Cyclosporin O Derivatives |
| Doi | 10.1021/acs.jmedchem.1c00211 |
| Research Group |
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| Data Number | 5 |
| Minimum Molecular Weight | 1160.6 |
| Maximum Molecular Weight | 1231.7 |
Assay Information 1
| Assay Type | PAMPA |
| Permeability Type | logPe |
| Membrane Measurement Direction (Exclude PAMPA) | - |
| Assay Detail |
PAMPA was conducted with a 96-well donor plate with 0.45 μm hydrophobic immobilon-P membrane supports and a 96-well polystyrene acceptor plate in triplicate. A 1% (w/v) solution of lecithin in n-dodecane was prepared and sonicated for 5 min before being used for PAMPA. Lecithin solution (5 μL) was carefully added into each membrane support in the bottom of the donor plate without the pipet tip touching the membrane. Each sample was prepared as a 10 μM solution in 5% (v/v) DMSO/phosphate-buffered saline (PBS) buffer. The donor plates were prepared by the addition of a sample solution (150 μL), and the acceptor plates were filled with 5% DMSO/PBS buffer (300 μL). The donor plate was placed on top of the acceptor plate without any bubbles between the donor and acceptor plates. A lid was placed on the donor plate, and the whole plates were covered with a wet paper towel to prevent evaporation. Then, the plates were incubated for 18 h at room temperature. Next, an aliquot (50 μL) from each donor or acceptor was mixed with 50 μM Fmoc-Tyr(OtBu)-OH dissolved in 1:1 (v/v) water/acetonitrile (50 μL) as an internal standard. The mixture (30 μL) was injected into the LC-MS instrument, and the analyte and internal standard were monitored in selected ion monitoring (SIM) mode. The analyte- to-standard peak area ratios were calculated and used to determine the relative concentrations. These concentrations were used to calculate the mass retention (R) and permeability (Pe): R = 1 - (Cd*Vd+Ca*Va)/(C0*Vd), Pe = (Va*Vd)/(V0*A*t) * ln(1-(Ca/Ceq)), Ceq = (Cd*Vd+Ca*Va)/(Vd+Va).
wherer C0 is the initial concentration of donor plate (10 μM), Ca is the concentration of acceptor plate (μM), Cd is the concentration of donor plate (μM), Va is the volume of acceptor plate (300 μL), Vd is the volume of donor plate (150 μL), A is the area of membrane (0.24 cm^2), t is the time (s), and V0 = Va + Vd. |
Assay Information 2
| Assay Type | Caco2 |
| Permeability Type | logPapp |
| Membrane Measurement Direction (Exclude PAMPA) | Average of Apical to Basolateral (AB) and Basolateral to Apical (BA) |
| Assay Detail |
Caco-2 cells (passage 33-34) were incubated in DMEM (pH 7.3) containing 10% fetal bovine serum, 100 U/mL penicillin, and 100 μg/mL streptomycin. The cells were grown in 100-mm tissue culture Petri dishes (Eppendorf AG, Hamburg, Germany) and maintained in an atmosphere of 5% CO2 and 90% relative humidity at 37 °C. For Caco-2 permeability assay, 2.5 × 105 Caco-2 cells/well were seeded into each insert in an HTS Transwell 96-well plate. The cells were incubated on the inserts in DMEM for 3 weeks, and the culture medium was replaced with fresh medium every 3 days until the whole area of the membrane was covered. After 21 days, the inserts and the plate were washed twice with HBSS buffer (pH 7.4) and incubated in fresh HBSS buffer at 37°C for 1 h (120 μL was dispensed into the inserts and 250 μL into the receiver plate). For apical to basolateral (AtoB) permeability with verapamil, the cells were preincubated in HBSS buffer containing verapamil (200 μM, 60 μL) for 30 min before permeability assay. After the HBSS buffer was drained, the sample solution (10 μMin1% DMSO/HBSS) was added to the inserts (120 μL) or the receiver plate (250 μL). For AtoB permeability with verapamil, the sample solution (20 μM in 2% DMSO/HBSS) was added to the inserts (60 μL). The opposite side was filled with HBSS buffer. AtoB permeability, BtoA permeability, and AtoB permeability with verapamil were measured to evaluate the apparent permeability, permeation pathway, and efflux ratio, respectively. The well plate was incubated at 37 °C without shaking for 2 h. Aliquots (20 μL) taken at each time point (0.5, 1, 1.5, 2 h) were mixed with Fmoc-Tyr(OtBu)- OH in acetonitrile solution (60 μL) as internal standards. The mixture was centrifuged at 1000 rpm for 15 min and then analyzed by LC-MS in selected ion monitoring (SIM) mode. Each experiment was performed in triplicate. In addition, aliquots of the donor were taken at 2 h to calculate the recovery (%). The analyte-to-standard peak area ratio was calculated and used to determine permeability (Papp) and recovery (%): Papp = dQ/dt * (1/(A*C0)), recovery(%) = (Cd+C2h)/C0 * 100, where dQ/dt is the permeability rate (cm/s), A is the area of insert (0.143 cm^2), C0 is the initial concentration (10 μM), Cd is the concentration of donor at 2 h (μM), C2h is the concentration of acceptor at 2 h (μM).
Caco-2 monolayers and tight junctions were confirmed by a test using Lucifer yellow, which could not pass through Caco-2 cells. After permeability assay, Caco-2 cells were incubated in HBSS buffer at 37°C for 1 h for the Lucifer yellow assay. The inserts were filled with Lucifer yellow solution in HBSS buffer (60 μM, 75 μL), and a receiver plate was prepared by HBSS buffer (235 μL). The plate was incubated at 37 °C for 1 Hyposmocoma lucifer yellow passage (%) was determined by monitoring fluorescence at 530 nm (excitation: 480 nm). Inserts that showed less than 3% Lucifer yellow passage were valid for permeability assay. |