Source Name: 2015_Wang
Literature Information
| Literature Title | Exploring experimental and computational markers of cyclic peptides: Charting islands of permeability |
| Doi | 10.1016/j.ejmech.2015.04.049 |
| Research Group |
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| Data Number | 62 |
| Minimum Molecular Weight | 454.5 |
| Maximum Molecular Weight | 882.1 |
Assay Information 1
| Assay Type | PAMPA |
| Permeability Type | logPapp |
| Membrane Measurement Direction (Exclude PAMPA) | - |
| Assay Detail |
A 96-well donor and accepter plate system pre-loaded with artificial lipid membrane (BD Gentest™ Pre-coated PAMPA Plate) was used. In these plates, each well consists of a polyvinylidene fluoride (PVDF) filter coated with a lipid/oil/lipid trilayer, where the lipid layer is composed of 1,2-dioleoyl-sn-glycero-3- phosphocholine and the oil layer is composed of hexadecane. 300 uL of 50 uM peptide (5% (v/v) DMSO in Hank's Balanced Salt Solution [HBSS])was added to the donor wells and 200 uL of buffer (5% (v/v) DMSO in HBSS) was added in the acceptor wells. The acceptor plate was lowered onto the donor plate so that the artificial membrane was in contact with the peptide solution below. A cover plate was placed on the acceptor plate and the assay plate covered with foil. Incubation of peptides was carried out at room temperature for 5 h. Acceptor and donor well concentrations were measured using an UHPLC/MS system equipped with a C18 Kinetex (2.10 x 50 mm, 7 um). The eluant was acetonitrile/water 10/90 with 0.045% v/v formic acid and was pumped through the column at 0.4 mL min^-1. The visible spectra were recorded at 214 and 280 nm. Mass spectrometer parameters were set as follows for positive ion mode: gas temperature 250 °C; drying gas (N2), 15 L min^-1; capillary voltage 4.5 kV. The mass range used for selective ion monitoring was changed depending on the peptide.
As the concentration differences between acceptor and donor wells are significant, they were analyzed separately. Dilution series of each peptide sample were prepared and injected into the LC-MS to generate a standard curve for quantification. Five mL from donor or 40 mL from acceptor wells were injected into the LC-MS and the analyte peak areas were recorded. The limit of detection of the peptides varies; however, most gave a value of ~0.05 uM. Percentage permeability was calculated relative to the equilibrium concentration. The permeability coefficient (Papp-pampa) values were calculatedaccording to the equation: Papp-pampa = (-ln[1-CA/Cequilibrium])/(A*(1/VD+1/VA)*t). Where CA is the sampled concentration in the acceptor compartment, VA is the volume in the acceptor compartment, VD is the volume ofthe donor compartment, t is the incubation time, A is the area of the filter and: Cequilibrium = [CD*VD + CA*VA]/(VD + VA). Each PAMPA measurement was typically an average of triplicates and repeated to n = 3. For some peptides with low permeability (e.g. Papp-pampa < 0.1 x 10^-6 cm/s), measurements were not repeated more than n = 2. Permeability values could not be determined for peptides 29, 30, 31, and 46 due to poor sample solubility. |
Assay Information 2
| Assay Type | Caco2 |
| Permeability Type | logPapp |
| Membrane Measurement Direction (Exclude PAMPA) | Apical to Basolateral (AB) |
| Assay Detail |
Caco-2 cells were cultured in an atmosphere of 5% CO2 and 90% relative humidity in T75 flasks (Nunc) with 10 mL of media (10% fetal bovine serum (FBS), 1% non-essential amino acids (NEAA), 1% penicillin/streptomyocin). Cells were passaged 1:10 every five days, or when 80-90% confluent, using 0.25% trypsin-EDTA (Invitrogen, Gibco Laboratories). Caco-2 cells were seeded onto each membrane of the transwell permeable support 12-well plate insert to give 50,000 cells/well. The individual feeding tray wells received 1.5 mL ofcell culture medium. The cell culture mediumwas changed every second day. Caco-2 cell monolayers were used for experimentation 21 days post-seeding. To prepare the cells for transport studies (apical to basolateral, A to B) cell culture media was removed from both compartments and the cells were washed once with HBSS in both the A and B compartments. The washing buffer was removed and 0.75 mL of fresh HBSS placed in compartment A and 1.5 mL of HBSS in compartment B for pre-incubation, carried out at 37 °C for 30 min under agitation. Transepithelial electrical resistance (TEER) was measured across the cell membranes at the beginning of the experiment. HBSS was subsequently removed from compartment A, replaced with 0.75 mL of the peptide solution prepared in HBSS. Monolayers were incubated and continuously shaken. 100 mL samples were taken from the A and B compartments at 45 min and 90 min intervals, and the buffer volume replenished. After 90 min, 200 mL of compartment A and B was also collected for Lucifer Yellow (LY) measurements. Amount ofLYpermeatedwas measure on a Tecan plate reader (excitation 485 nm, emission 530 nm). At the conclusion of the experiment TEER was measured again to confirm the integrity of the Caco-2 monolayers (TEER > 300 Ω cm^2). Acceptor and donor well concentrations were measured by LC-MS as per the PAMPA Assay.
The permeability coefficient (Papp-caco) values were calculated according to the equation: Papp-caco = VA/t * CA/C0 * 1/A Where CA is the sampled concentration in the acceptor compartment, VA is the volume 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. Each Caco-2 measurement was typically an average of triplicates and repeated to n = 3. For permeability values less than 1 * 10^-6 cm/s, some samples were not repeated more than n = 2. Permeability values could not be determined for peptides 29, 30 and 31 due to poor sample solubility. |