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Parallel Artificial Membrane Permeability (PAMPA) Assay

 

 

Readout: log Pe, % recovery

Controls: Propranolol, Warfarin, and Furosemide

 

Assay Description:

Working solutions of each compound are prepared from 10 mM stock solution in DMSO diluted to a final concentration of 10 μM in PBS buffer (pH 7.4, 1% DMSO).

1% (w/v) lecithin/dodecane is added to the donor side of the Multi Screen Filter Plate, then 10 μM control or test compound working solution is added. The receiver side of the Multi Screen Filter Plates is filled with PBS buffer containing 1% DMSO. The plates are kept at room temperature for 24 h. Samples are collected from the donor and receiver sides. The donor sides samples are diluted 20-fold with PBS (1% DMSO).  All receiver and diluted donor side samples are mixed with ACN/MeOH (1:1, v/v) containing 25 ng/mL terfenadine and 50 ng/mL tolbutamide as internal standards. Samples are vortexed and then centrifuged at 4 °C. An aliquot of the supernatant is transferred to a 0.65 ml tube for LC-MS/MS analysis.

The MS detection is performed using a Sciex API 4000 instrument. Each compound is analyzed by reversed phase HPLC using a Kinetex 2.6μ C18 100Å column (3.0 mm X 30 mm, Phenomenex). Mobile phase – Solvent A: water with 0.1% formic acid,  solvent B: ACN with 0.1% formic acid.

 

Data Analysis:

The amount of compound is determined on the basis of the peak area ratio (compound area to IS area) for the two sides.

LogPe is determined using the following equations:

LogPe = Log {C*-Ln(1-[drug]acceptor/[drug]equilibrium)}

C = VD*VA /{(VD+VA)*Area*time}

Recovery is calculated as follows:

%Recovery = (Total compound mass in donor and receiver compartments at the end of the incubation / Initial compound mass in the donor compartment) x 100

 

Abbreviations:

ACN                         Acetonitrile

DMSO                     Dimethylsulfoxide

HPLC                       High-performance liquid chromatography

LC                             Liquid Chromatography

MS                           Mass spectrometry

PBS                          Phosphate-buffered saline

VA                             Volume of the acceptor well

VD                            Volume of the donor well

 

Literature:

  1. Kansy, M.; Senner, F.; Gubernator, K.; “Physicochemical high throughput screening: parallel artificial membrane permeation assay in the description of passive absorption processes”; Med. Chem. 41, 1007, (1998).
  2. Ottaviani, G.; Martel, S.; Carrupt, P.-A.; “Parallel Artificial Membrane Permeability Assay: A New Membrane for the Fast Prediction of Passive Human Skin Permeability”; Med. Chem. 49, 3948, (2006).
  3. Faller, B. “Artificial Membrane Assays to Assess Permeability”; Curr. Drug Metab., 9, 886, (2008).
  4. Reis, J. M.; Sinko, B.; Serra, C. H.R.; “Parallel Artificial Membrane Permeability Assay (PAMPA) – Is it Better than Caco-2 for Human Passive Permeability Prediction?”; Mini-Rev. Med.Chem. 10, 1071, (2010).