Source Name: 2018_Naylor

Literature Information

Literature Title Lipophilic Permeability Efficiency Reconciles the Opposing Roles of Lipophilicity in Membrane Permeability and Aqueous Solubility
Doi 10.1021/acs.jmedchem.8b01259
Research Group
  1. Department of Chemistry and Biochemistry, University of California Santa Cruz
  2. Daiichi Sankyo Co., Ltd.
  3. Sage Therapeutics Inc.
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Data Number 81
Minimum Molecular Weight 578.8
Maximum Molecular Weight 1218.6

Assay Information 1

Assay Type RRCK
Permeability Type logPapp
Membrane Measurement Direction (Exclude PAMPA) Apical to Basolateral (AB)
Assay Detail Assay materials used were as described by Di et. Al.(2) with minor deviations. Fetal Bovine Serum (FBS) and L-Glutamine 200 mM were purchased from Corning Inc. (Corning, NY). Penicillin-Streptomycin (Pen/Strep) was purchased from the Lonza Group (Basel, Switzerland). Hanks Balanced Salt Solution (HBSS), HEPES, and Calcium Chloride (CaCl2) were purchased from Fisher Scientific (Hampton, NH). 1M Anhydrous Dextrose was used in place of 1M D-Glucose and was purchased from EMD Millipore (Ontario, Canada). Magnesium Chloride Hexahydrate was used in place of Magnesium Chloride and was purchased from Sigma-Aldritch (St. Louis, MO). The MEM-α media and Buffer B solution were delivered with a 12-Channel pipette. Cell culturing was performed as described by Di et. Al.(2) with minor modifications. MDCKII-LE cells were cultured at 37°C, 5% CO2, 95% relative humidity in T25 flasks prior to splitting which took place between 3-5 days or when cells reach approximately 90% confluency. Under sterile conditions in a biological safety cabinet, media were removed from the T25 flask containing the cells, followed by two 5mL rinses with 0.5mL of 0.25% Trypsin (prewarmed to 37°C) evenly across the bottom of the flask. The flasks were incubated for 2-5 minutes, or until cells no longer adhered to the flask. Four mL of MEM-alpha were added to the flask to dilute the trypsin and quench proteolysis. This was followed by appropriate dilution of cells into new flasks (T25/T50/T75) to continue the cell line.The cells were typically cultured in one or two T75 flasks when prepared for the assay. Media was aspirated, followed by spreading 1.5mL of 0.25% Trypsin and incubation for 2-5 minutes or until cells dislodged from the surface of the flask. 8.5mL of MEM-alpha media was added to the trypsinized cells to get a total of 10mL MEM-alpha. Cells were counted using a hemocytometer and diluted to a volume of 5 x 10^5 cells/mL in complete MEM-alpha media. Cells were then plated using the same methods as Di et al(2). The seeded plates were left in the incubator for 5 days. On the day of the permeability assay, media of assay plates is removed, and plates are rinsed with Buffer B. Lucifer Yellow (LY) (excitation wavelength: 425-430 nm1; emission wavelength: 515-520nm(3, 4)) was incorporated into the assay to provide a rapid measure of monolayer quality. A 15mM stock of LY was prepared in Buffer B a day prior to the assay. From this stock, a 15uM stock was prepared, followed by a serial 1:3 dilution with Buffer B into a 96-well plate—giving a range from 15uM to 20.5nM with the final well containing only Buffer B. The series was repeated in triplicate and read in a PerkinElmer Envision Plate Reader, alongside 4-5 wells containing the 5uM LY in Buffer B stock used in the assay.
Sample compound mixtures were prepared as described above in a 2 mM stock in DMSO, delivering 10 uL into a 96-well conical plate. 90 uL of Buffer B with 5 uM LY was added to the 10 uL DMSO stock to yield a 200 uM compound concentration in 10% DMSO, with thorough mixing (precipitation is evident at this concentration for some library mixtures). 10 uL of this mixture was diluted with 990 uL of Buffer B with 5 uM LY, with thorough mixing, for a final concentration of 2 uM compound and 0.1% DMSO, as the “t=0” dilution.An initial fluorescence reading of the “t=0” dilution was collected as LYt=0. 75uL of the final dilution was added to the apical layer of the assay plates. 250uL of Buffer B (without LY) was added to the basal/collector plate. The assay plate was incubated for 90 minutes then separated. 100uL of the basal layer was separated for a fluorescence reading as LYt=90. 50uL of the basal layer was transferred to a 96-well conical plate, mixed with 50uL of acetonitrile, and sealed with a pierceable seal for UPLC-MS quantification (Ibasal) . The same process was performed for the “t=0” dilution before application to the cell monolayer (It=0). Protein contamination in the basal layer collected on the column was backflushed from the column after analyzing plates (30:30:30:10 MeCN:MeOH:iPrOH:H2O). Permeability rates across the cell monolayer were calculated via the following equation: Papp = dMr/dt * 1/(Msa*CD(0)) [6]. Where:dMr/dt = flux of the compound across the cell monolayer. Msa = surface area of filter membrane = 0.11 cm^2. CD(0) = concentration of compound in the donor well at “t=0”.

Assay Information 2

Assay Type PAMPA
Permeability Type logPapp
Membrane Measurement Direction (Exclude PAMPA) -
Assay Detail A 96-well donor plate with 0.45 μ hydrophobic Immobilon-P membrane supports (Millipore MAIPNTR10) and a 96-well Teflon acceptor plate (Millipore MSSACCEPTOR) were used in the PAMPA permeability test. The acceptor plate was prepared by adding 300 μL of 5% DMSO in pH = 7.4 phosphate-buffered saline (PBS) to each well. Donor well solutions of were prepared by diluting DMSO stock solutions prepared above to a final volume of 1000 μL with PBS (pH 7.4), and mixed thoroughly.A 1% (w/v) solution of lecithin (soybean, 90%) in n-dodecane was prepared and sonicated before use. 5 μL of the dodecane / lecithin solution was carefully applied to the underside of membrane supports in the wells of the donor plate, with care taken to not touch the pipet tip to the membrane. After approximately 15 minutes, 150 μL of the peptide solutions were added to the donor wells. The donor plate was then placed on top of the acceptor plate so that the artificial membrane was in contact with the buffer solution below, ensuring that no bubbles form beneath the membrane. A lid was placed on the donor well, the system was covered within a sealed chamber and left overnight at room temperature. A wet paper towel was placed inside the chamber to prevent evaporation.
After ~16 h (exact time recorded and used for subsequent calculations) the donor and acceptor plates were separated and 100μL of each well (donor and acceptor) were transferred to a 96 well plate for quantification. The plate was immediately sealed with a pierceable plate cover (without adhesive to prevent sample evaporation. These solutions were analyzed and quantified via UPLC (see above). Permeability (%T) was quantified as the ratio of analyte area in the acceptor well divided by a theoretical equilibrium ratio based on amounts of combined analyte found in the donor and acceptor wells as follows: AnalyteEquil = {(Ia*Va)+(Id*Vd)} / (Va+Vd) [1], %T = (Ia/[AnalyteEquil]) [2]. Recovery (%R) was quantified as the ratio of total compound identified in the donor and acceptor wells relative to the total compound identified in the original dilution sample. %R = {(Ia*Va)+(Id*Vd)} / (Ir*Vd) [3]. Permeation rates (Papp) were calculated from %T by thefollowing equations: Papp(cm/s) = -C * ln(1-%T) [5] often displayed in log scale. Where: Active surface area of membrane (mm^2): Msa = 240, Volume of acceptor well (uL): Va = 300, Volume of donor well (uL): Vd = 150, Assay run time (s): Ts = plate-specific, approx. 16 hr. In addition to visual inspection, permeation standards were included in each well at 1 uM to discard wells with poor membranes: carbamazepine (Papp = 7.5 x10^-6 cm/s) and propranolol (Papp = 3.9 x10^-6 cm/s). Additionally, any individual measurement with %T > 98 was discarded; the rate cannot be assigned at equilibrium.