Comparison of PAMPA and Caco2 Cells 541 Parallel Artificial Membrane Permeability Assay

PAMPA model was first introduced in 1998 (Kansy et al., 1998), and since then numerous reports have been published illustrating the general applicability of this model as a high-throughput permeability screening tool (Avdeef, 2001; Di et al., 2003; Ruell et al., 2003; Kerns et al., 2004; Balimane et al., 2006). The model consists of a hydrophobic filter material coated with a mixture of lecithin/phospholipids dissolved in an inert organic solvent such as dodecane creating an artificial lipid membrane barrier that mimics the intestinal epithelium. The rate of permeation across the membrane barrier was shown to correlate well with the extent of drug absorption in humans. The use of 96-well microtiter plates coupled with the rapid analysis using a spectrophotometric plate reader makes this system a very attractive model for screening a large number of compounds and libraries. PAMPA is much less labor intensive than cell culture methods but it appears to show similar predictability. One of the main limitations of this model is that PAMPA underestimates the absorption of compounds that are actively absorbed via drug transporters. Despite the limitation, PAMPA may serve as an invaluable primary permeability screen during early drug discovery process because of its high-throughput capability.

5.4.1.1 PAMPA Study Protocol

A 96-well microtiter plate and a 96-well filter plate (Millipore, Bedford, MA, USA) were assembled into a "sandwich" such that each composite well was separated by a 125 |im microfilter disc (0.45 |im pores). The hydrophobic filter material of the 96-well filter plate was coated with 5 |L of the plON lipid solution and gently shaken to ensure uniform coating. Subsequently, the filter plate was placed on the microtiter plate containing 200 |L of 100 |M test compounds (dissolved in 1% DMSO/buffer), which constituted the donor well. The donor solution containing the test compound was prepared by dilution (100-fold) from a 10 mM stock solution in DMSO using the pIONĀ© buffer solution at pH 7.4 followed by filtration through a 0.20 |im polyvinylidene fluoride (PVDF) 96-well filter plate (Corning Costar, Corning, NY, USA). The receiver wells (i.e., the top of the wells) of the sandwich were hydrated with 200 |L of the specialized ionic buffer solution. The system was then incubated at room temperature for 4 h. At the end of the incubation time, samples were removed from the receiver and donor wells and analyzed by a UV-plate reader. The permeability studies were performed in triplicates (i.e., three wells per compound). The apparent permeability coefficient was estimated using the software provided by pION.

Was this article helpful?

0 0

Post a comment