Source Name: 2015_Bockus_1
Literature Information
| Literature Title | Probing the Physicochemical Boundaries of Cell Permeability and Oral Bioavailability in Lipophilic Macrocycles Inspired by Natural Products |
| Doi | 10.1021/acs.jmedchem.5b00128 |
| Research Group |
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| Data Number | 16 |
| Minimum Molecular Weight | 755.0 |
| Maximum Molecular Weight | 1202.6 |
Assay Information 1
| Assay Type | PAMPA |
| Permeability Type | logPe |
| Membrane Measurement Direction (Exclude PAMPA) | - |
| Assay Detail |
A 96-well donor plate with 0.45 um hydrophobic Immobilon-P membrane supports (Millipore) and a 96-well Teflon acceptor plate were used in the PAMPA. A 1% (w/v) solution of lecithin in dodecane was prepared and sonicated for 5 minutes prior to use. The dodecane lecithin solution (5 uL) was carefully applied to the membrane supports in the wells of the donor plate, with care being taken not to touch the pipet tip to the membrane. The acceptor plate was prepared by adding 300 uL of 5% DMSO/PBS (pH 7.4) to each well. The donor wells were prepared by adding 150uL of each cyclic peptide solution (10 uM in 5% DMSO/PBS at pH 7.4) to the wells in quadruplicate. The donor plate was then placed on top of the acceptorplate so the artificial membrane was in contact with the buffer solution below. A lid was placed on the donor well, and the system was covered with a glass evaporating dish and left for 5 hours at room temperature. A wet paper towel was placed on the inside of the chamber to prevent evaporation.
Once the assay was complete, a sample from each well (50 uL) was diluted in a 10 uM solution of H2N-Tyr(Ot-Bu)-CO2H in 1:1 ACN:H2O (50 uL), then 20 uL was injected into the LC-MS. Acceptor and donor well concentrations were measured by LC-MS (Thermo LTQ) using selected ion monitoring (SIM) mode to detect the parent masses of both the analyte and the internal standard. Analyte-to-standard peak area ratios from the TIC detector were calculated and used to determine relative concentrations in both donor and acceptor wells. These ratios were then used to calculate an equilibrium value adjusted for retention (E_R): E_R = (R_A*V_A + R_D*V_D)/V_A + V_DWhere R_A is the analyte-to-standard peak ratio of the acceptor, V_A is the volume of the acceptor (cm^3), R_D is the analyte-to-standard peak of the donor, and V_D isthe volume of the donor. Transmittance percentage (%T) was then calculated for each sample:%T = (R_A/E_R) x 100 The %T values were converted into time-independent Pe values:Pe = [(V_A x V_D)/(V_0 x A x t)] x ln(1-(%T/100))Where V_0 is the total volume (cm^3), A is accessible filter area of the membrane (0.24 cm^2), and t is the incubation time (s). Average %T and Pe values were calculated for each compound from at least three data points excluding extreme outlying permeability values. Standard deviations were calculated for the average values. Because percent recovery is accounted for in the ER, we can assume no compound loss in the calculation and it holds true that apparent permeability (Pa) and effective permeability (Pe) values are equivalent.To calculate membrane/plate retention of each compound: %R = ([(R_A*V_A)+(R_D*V_D)]/(V_0*R_D0))*100)Where %R is the percent recovery and R_D0 is the initial analyte-to-standard ratio in the donor well. |
Assay Information 2
| Assay Type | RRCK |
| Permeability Type | logPapp |
| Membrane Measurement Direction (Exclude PAMPA) | Apical to Basolateral (AB) |
| Assay Detail |
Cell permeability was determined using MDCK-LE cells (Pfizer, Inc., Groton, CT). MDCK-LE cells were generated in house as a subclone of Madin-Darby Canine Kidney wild-type (MDCK-WT) cells that displayed low expression of endogenous P-glycoprotein (approximately 1-2% of MDCK-WT cells, based on mRNA level). Cells were cultured in minimal essential medium with supplements and passaged when 70-80% confluent. Cell monolayer flux studies were conducted five days after seeding in 24-well transwell inserts (MDCK-LE in 1.0 um pore size (Becton Dickinson, Cowley, UK)) at 4.2 x 10^4 cells/cm^2. In the MDCK-LE assay, donor and acceptor solutionswere prepared from HBSS containing HEPES at 20 mM, pH 7.4. Stock solutions of test compounds were prepared at 10 mM in DMSO and used to prepare donor solutions of 2 mM compound in 0.5% (v/v) DMSO. Apparent permeability (Papp) was determined in apical to basolateral (AB) direction in triplicate by incubating with compound for 2 h at 37 °C. Samples of the medium were analyzed using the LC-MS/MS method above. Papp values were calculated according to the equation Papp = (Q/t) x 1/Co x 1/A), where Q is the sampled concentration in the acceptor compartment, t is the incubation time, Co is the initial concentration in the donor compartment, and A is the area of the filter of the trans-well plate.
For compound 6, the initial MDCK-LE flux measured was 2.0 x 10^-6 cm/s, which was significantly lower than the expected permeability based on its high PAMPA permeability. We re-assayed 1, 4, 5, 6, 12, 13, 14 in MDCK-LE cells using plates designed to minimize nonspecific binding, with 0.4% BSA and protease inhibitor, and low-binding tips, and found the permeability to be similar except for 6, which showed a greater than 2-fold improvement in permeability. Compound 5, which had low permeability in the MDCK-LE system (1.4 x 10^-6 cm/s), had virtually identical permeability when the assay was re-run using low-binding plates (1.3 x 10^-6 cm/s). |