Source Name: 2015_Marelli

Literature Information

Literature Title Enantiomeric Cyclic Peptides with Different Caco-2 Permeability Suggest Carrier-Mediated Transport
Doi 10.1002/chem.201501270
Research Group
  1. Department of Chemistry, Technical University of Munich
  2. Institute of Molecular Pharmacy, University of Basel
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Data Number 10
Minimum Molecular Weight 454.5
Maximum Molecular Weight 849.0

Assay Information 1

Assay Type PAMPA
Permeability Type logPe
Membrane Measurement Direction (Exclude PAMPA) -
Assay Detail Values of log Pe were determined in a 96-well format with the PAMPA permeability assay. For each compound, measurements were performed in quadruplicate at pH value 7.4 (for compound 4, 4*, 5, and 5* pH 3.0 and 5.0 were measured additionally). For this purpose, wells of a deep-well plate were filled with 650 μL System Solution. Samples (150 μL) were withdrawn from each well to determine the blank spectra by UV spectroscopy (SpectraMax 190). Then, analyte dissolved in DMSO was added to the remaining System Solution to yield 50 μM solutions. To exclude precipitation, the optical density was measured at 650 nm, with 0.01 being the threshold value. Solutions exceeding this threshold were filtered. Afterwards, samples (150 μL) were withdrawn to determine the reference spectra. Further 200 μL were transferred to each well of the donor plate of the PAMPA sandwich P/N 110 163 (pIon, Woburn MA, USA). The filter membranes at the bottom of the acceptor plate were impregnated with 5 μL of GIT-0 Lipid Solution each, and 200 μL of Acceptor Sink Buffer were filled into each acceptor well. The sandwich was assembled, placed in the Gut-Box, and left undisturbed for 16 h. It was then disassembled, and samples (150 μL) were transferred from each donor and acceptor well to 96 well plates. Quantification was performed by LC-MS; log Pe values were calculated with the aid of the PAMPA Explorer Software (pIon, version 3.5 and 3.7).

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 were cultivated in tissue culture flasks (BD Biosciences, Franklin Lakes, NJ, USA) with DMEM high-glucose medium containing L-glutamine (2 mM), nonessential amino acids (0.1 mM), penicillin (100 U/mL), streptomycin (100 μg/mL), and FBS (10%). The cells were kept at 37 °C in humidified air containing 5% CO2, and the medium was changed every second day. When ca. 90% confluence was reached, the cells were split in a 1:10 ratio and distributed to new tissue culture flasks. At passage numbers between 60 and 65, they were seeded at a density of 5.3x10^5 cells per well to Transwell 6-well plates (Corning, NY, USA) with 2.5 mL culture medium in the basolateral and 1.5 mL in the apical compartment. The medium was renewed on alternate days (2 mL apical, 2.5 mL basolateral). Permeation experiments were performed between days 19 and 21 post-seeding. Prior to the experiment, the integrity of the Caco-2 monolayers was evaluated by measuring the transepithelial electrical resistance (TEER) with an Endohm tissue resistance instrument (WORLD Precision Instruments Inc., Sarasota, FL, USA). Only wells with TEER values > 250 Ωcm^2 were used.
Experiments were performed in the apical-to-basolateral (absorptive) and basolateral-to-apical (secretory) directions in triplicate at a compound concentration of 62.5 µM. Transport medium (DMEM without sodium pyruvate and phenol red) was withdrawn from the donor compartments and the compound solution was added. The Transwell plate was then shaken (600 rpm) inthe incubator. Samples (40 μL) were withdrawn after 15, 30, and 60 min from the donor and acceptor compartments. The concentrations were determined by LC-MS. Apparent permeability coefficients (Papp) were calculated according to the equation: Papp = dQ/dt * 1/(A*c0) where dQ/dt is the permeability rate, A the surface area of the monolayer, and c0 the initial concentration in the donor compartment. After the experiment, TEER values were assessed again for each well and results from wells with values < 250 Ωcm^2were discarded.