PAMPA membrane permeation

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.]. 

Passive diffusion through the lipid bilayer of the epithelium can be described using the partition coefficient between octanol/water (log P) and A log P (the difference between the partition into octanol/water and heptane/ethylene glycol or heptane/ octanol) [157, 158], The lipophilicity of the drug (log P) (or rather log D at a certain pH) can easily be either measured or calculated, and is therefore generally used as a predictor of drug permeability. Recently, a method using artificial membrane permeation (PAMPA) has also been found to describe the passive diffusion in a similar manner to the Caco-2 cell monolayers [159]. 

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. 

Recently, there is some negativity towards PAMPA [52], seemingly due to an overexpectation and misunderstanding of PAM PA and the science of passive membrane permeation [53]. PAMPA is a refined descendant of log Poet and is an improved surrogate measurement for passive transcellular permeation. PAMPA permeability usually correlates well with passive transcellular permeation. It is important to correctly understand the pros and cons of this tool and to use it appropriately in drug discovery. 

PAMPA membranes typically consist of phospholipids dissolved in an organic solvent. Both of them affect chemical selectivity. Phospholipids facilitate the permeability of moderately hydrophilic molecules by ionic or hydrogen-bonding interactions (phopholipids are hydrogen bond acceptors). This allows permeation of moderately lipophilic compounds. Recently, it was shown that anionic phospholip-id(s) increases the permeation of basic compounds by ion pair mechanism [54—56]. Many PAMPA variants (and other artificial membrane tools) add anionic phospho-lipid(s) to increase the in vivo predictability. 

Falten et al. recently reported that phospholipid membrane vesicles can be con-stmcted on a filter scaffold without any organic solvent [57-60]. In this system, the phospholipid vesicle occupies the filter pores to form a permeation barrier. This is more relevant to the cellular membrane than PAMPA membranes with organic solvent. The membrane can be stored up to two weeks without significant change and is stable at pH 2-8. The Fa% predictability was compared with BM-PAMPA, DS-PAMPA, Caco-2 and immobilized liposome chromatography, resulting in promising predictability. 

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. 

On the other hand, PAMPA is a purely artificial method and PAMPA membranes do not reassemble real lipid bilayer structures as barriers for permeation but much thicker barriers. The thickness and material of the supporting PVDF filters also influences artificially the permeation of compounds depending on the lipophilicity of the compounds more than the thin polycarbonate filter does in CACo2 experiments. Also the best choice of membrane constituents for PAMPA experiments is still under investigation and it seems that it will depend a lot on the goal of the PAMPA experiment which membrane is used (e.g. blood brain barrier permeation or intestinal absorption). One has to take into account that PAMPA today is a summary term on a lot of different methods applied in different laboratories using different membrane constituents, sink conditions, permeation times etc., which makes inter laboratory comparison difficult. 

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. 

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. 

---