PAMPA permeation assay

PAMPA parallel artificial membrane 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. 

The use of artificial membranes to investigate passive permeation processes has a long history, going back more than 40 years [68], The parallel artificial membrane permeation assay (PAMPA) is an application of the filter-supported lipid membrane system [149] and was first introduced by Kansy and... 

Batzl-Hartmann C, Hurst L, Maas R (2002) Method for the improvement of parallel artificial membrane permeation assay (PAMPA) by using an additional hydrophilic membrane. German Patent Application 4. 

Solubility and permeability were measured by a high throughput solubility assay and parallel artificial membrane permeation assay (PAMPA), respectively [56], The assays categorized 14 out of 18 drugs based on the BCS consistent with their known solubility and permeability characteristics [56],... 

HTS plates permit to determine drug permeability across a cell monolayer with a throughput similar to that of the parallel artificial membrane permeation assay (PAMPA), which measures rate of diffusion across a lipid layer.46 As is the case with PAMPA, the tiny surface area of the filters of the 96-well HTS presents an analytical challenge for compounds with low-to-moderate permeability. 

The successful application of in vitro models of intestinal drug absorption depends on the ability of the in vitro model to mimic the relevant characteristics of the in vivo biological barrier. Most compounds are absorbed by passive transcellular diffusion. To undergo tran-scellular transport a molecule must cross the lipid bilayer of the apical and basolateral cell membranes. In recent years, there has been a widespread acceptance of a technique, artificial membrane permeation assay (PAMPA), to estimate intestinal permeability.117118 The principle of the PAMPA is that, diffusion across a lipid layer, mimics transepithelial permeation. Experiments are conducted by applying a drug solution on top of a lipid layer covering a filter that separates top (donor) and bottom (receiver) chambers. The rate of drug appearance in the bottom wells should reflect the diffusion across the lipid layer, and by extrapolation, across the epithelial cell layer. 

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... 

One decade has passed since the parallel artificial membrane permeation assay (PAM PA) was first introduced in 1998 [47]. Since then, PAM PA rapidly gained wide popularity in drug discovery [3, 48-51]. Today, PAMPA is the most widely used physicochemical membrane permeation model. The term PAMPA is nowusedas the general name for a plate-based (HTS enabled), biter-supported (filter immobilized) artificial membrane. Typically, phospholipids dissolved in an organic solvent are impregnated into the filter to construct a PAMPA membrane. 

Parallel Artificial Membrane Permeation Assay (PAMPA)... 

Several in vitro assays using filter immobilized artificial membranes exist for the estimation of permeability (Parallel artificial membrane permeation assay, PAMPA). In these assays the permeation of a compound is followed directly by estimating the amount of compound on either side of the membrane barrier. The results of these experiments are expressed as permeability values rather than lipophilicity values. 

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). 

The in vitro measurements of permeability by the cultured-cell or PAMPA model underestimate true membrane permeability, because of the UWL, which ranges in thickness from 1500 to 2500 pm. The corresponding in vivo value is 30-100 pm in the GIT and nil in the BBB (Table 7.22). The consequence of this is that highly permeable molecules are (aqueous) diffusion limited in the in vitro assays, whereas the membrane-limited permeation is operative in the in vivo case. Correcting the in vitro data for the UWL effect is important for both GIT and BBB absorption modeling. 

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