Caco-2 permeation assay

The evaluation of the apparent ionization constants (i) can indicate in partition experiments the extent to which a charged form of the drug partitions into the octanol or liposome bilayer domains, (ii) can indicate in solubility measurements, the presence of aggregates in saturated solutions and whether the aggregates are ionized or neutral and the extent to which salts of dmgs form, and (iii) can indicate in permeability measurements, whether the aqueous boundary layer adjacent to the membrane barrier, Umits the transport of drugs across artificial phospholipid membranes [parallel artificial membrane permeation assay (PAMPA)] or across monolayers of cultured cells [Caco-2, Madin-Darby canine kidney (MDCK), etc.]. 

There are several approaches to estimating absorption using in vitro methods, notably Caco-2 and MDCK cell-based methods or using methods that assess passive permeability, for example the parallel artificial membrane permeation assay (PAMPA) method. These are reviewed elsewhere in this book. The assays are very useful, and usually have an important role in the screening cascades for drug discovery projects. However, as discussed below, the cell-based assays are not without their drawbacks, and it is often appropriate to use ex vivo and/or in vivo absorption assays. 

Another in vitro method for permeability screening was parallel artificial membrane permeation assay (PAMPA) initially reported by Kansy. In a PAMPA permeability screen, the Caco-2 cell mono-layer membrane is replaced by an artificially generated membrane. Versions of different artificial membranes that lack active transporter systems and pores have been developed to mimic the in vivo transcellular intestinal epithelial cell barrier. Therefore, the PAMPA screen only measures the intrinsic... 

Kansy et al. (1998) proposed the use a parallel artificial membrane permeation assay (PAMPA) as a high-throughput alternative to Caco-2 monolayers for the prediction of passive drug permeation. In the PAMPA approach, aqueous... 

The prediction of the important structural features that affect intestinal permeability is useful information to obtain early in the drug discovery process. The two most common models used to obtain fast, high-throughput measurements are the parallel artificial membrane permeation assay (PAMPA) and the cell line assays that feature cultured human colon adenocarcinoma cells (Caco-2). Each method uses a surrogate model to mimic intestinal absorption followed by LC-MS analysis. 

Artificial Systems (PAMPA) Some artificial systems are widely used as an alternative to Caco-2 cell culture to evaluate drug permeability. An example of these is the parallel artificial membrane permeation assay (PAMPA). PAMPA is usually performed in a 96-well microtiter plate format that consists of test wells fixed with an artificial hydrophobic membrane and reference wells (Kansy et al., 1998). Test compounds are applied at one side of the membrane, and the permeability rates are monitored at the receiving side. The properties of the membrane (lipophilicity and thickness) control the rate of permeability. Different membranes could be designed to mimic the passive... 

The use of a faster-growing cell line, MDCK (Madin-Darby canine kidney) cells, appears to be a good replacement for Caco-2 cells (Irvine et al. 1999). The parallel artificial membrane permeation assay (PAMPA) is a rapid in vitro assay, in which transcellular permeation is evaluated (Kansy et al. 1998). PAMPA may also be used to predict oral absorption, blood-brain barrier penetration, and human skin permeability (Fujikawa et al. 2007) by using QSAR models. To our knowledge, neither PAMPA, Caco-2 cell monolayers nor MDCK cells have been used to examine the absorption/permeability of the pyrethroids. The advantages and limitations of the Caco-2 model were reviewed by Artursson et al. (1996) and Delie and Rubas (1997). 

One of the main in vitro permeability assays used in the pharmaceutical industry has been for many years the Caco-2 monolayer. Therefore, most of the in silica models developed to predict permeability were based on Caco-2 data. Hou and Johnson produced a couple of reviews that comprehensibly summarizes the recent efforts using Caco-2 permeability data [92, 94]. All those models are designed to predict the influx or apparent permeability of drugs in the same direction as intestinal absorption occurs, that is, from the apical to the basal side of the cell line, regardless of the extent of active transport involved in the permeation process. 

The parallel artificial membrane permeability assay (PAMPA) is a recent development in the area of artificial membranes that appears to offer considerable potential. Measuring the flux values (membrane permeation levels) of a range of test compounds by PAMPA and relating these values to the flux curves obtained in Caco-2 studies have shown good correlations, indicating that the PAMPA assay could be a good alternative to Caco-2 cells for the measurement of passively diffusing compounds. 

PAMPA Parallel Artificial Membrane Permeability Assay is a method which determines the permeability of substances from a donor compartment, through a Upid-infused artificial membrane into an acceptor compartment. PAMPA models regularly exhibit a high degree of correlation with permeation across a variety of barriers including Caco-2 cultmes, the G1 tract, blood-brain barriers and skin... 

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