Source Name: 2017_Pye

Literature Information

Literature Title Nonclassical Size Dependence of Permeation Defines Bounds for Passive Adsorption of Large Drug Molecules
Doi 10.1021/acs.jmedchem.6b01483
Research Group
  1. Department of Chemistry and Biochemistry, University of California Santa Cruz
  2. Worldwide Medicinal Chemistry, Groton Laboratories, Pfizer Inc.
  3. Worldwide Medicinal Chemistry, Cambridge Laboratories, Pfizer Inc.
  4. Daiichi Sankyo Co., Ltd.
  5. -
  6. -
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Data Number 21
Minimum Molecular Weight 785.0
Maximum Molecular Weight 1151.5

Assay Information 1

Assay Type PAMPA
Permeability Type logPapp
Membrane Measurement Direction (Exclude PAMPA) -
Assay Detail A 96-well donor plate with 0.45 μ hydrophobic Immobilon-Pmembrane supports (Millipore) and a 96-well Teflon acceptor plate 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 the cyclic peptide libraries were prepared by diluting 10 μL of the DMSO stock solutions prepared above to a final volume of 200 μL with PBS (pH 7.4). The suspensions were centrifuged to remove any insoluble material. A 1% (w/v) solution of lecithin in dodecane was prepared and sonicated before use. 5 μL of the dodecane / lecithin solution was carefully applied to the membrane supports in the wells of the donor plate, with care being taken to not touch the pipet tip to the membrane. Without allowing this solution to evaporate, 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. A lid was placed on the donor well, and the system was covered with a glass evaporating dish and left overnight (~18 h) at room temperature. A wet paper towel was placed on the inside of the chamber to prevent evaporation.
After ~18hr (exact time recorded and used for subsequent calculateions) the donor and acceptor plate 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 piercable plate cover to prevent any sample evaporation. These solutions were analyzed by UPLC on a C18 Thermo HyperSil column (2.1x30mm, 3µm) with an accurate mass MS detector (Thermo Scientific Orbitrap VelosPro) using +/- 0.02 AMU windows for integration. Permeability (%T) was quantified as the ratio of analyte areas 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: %T = RA / ((RA*VA+RD*VD)/(VA+VD)). Where RA and RD are the integrations analyte in the acceptor and donor wells, respectively, and VA and VD are the volumes of the acceptor (300 µL) and donor (150 µL) wells, respectively. Permeation rates (Papp) were calculated from %T by the following equations: Papp = - (VA*VD)/(VA+VD) * (ln(1-%T))/At Where A is the active surface area of the filter support (0.24 cm^2), and t is time of the incubation period in seconds.

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 RRCK cells (Pfizer, Inc. Groton, CT). RRCK 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 (~ 1-2% of MDCK-WT cells, based on mRNA level). Cells were cultured in minimal essential medium alpha with supplements and passaged when 70-80% confluent. Cell monolayer flux studies were conducted five days after seeding in 24-well transwell inserts, 1.0 μm pore size, (Becton Dickinson, Cowley, UK) at 4.2 x 10^4 cells/cm^2. Donor and acceptor solutions were prepared from HBSS containing HEPES at 20 mM, pH 7.4. Stock solutions of test compounds, prepared at 10 mM in DMSO, were used to prepare donor solutions of 2 μM compound in 0.05% (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 by liquid-chromatography tandem mass spectrometry (LC-MS/MS). Papp values were calculated according to the equation Papp = (Q/t) x 1/C0 x 1/A, where Q is the sampled concentration in the acceptor compartment, t is the incubation time, C0 is the initial concentration in the donor compartment and A is the area of the filter of the transwell plate.