Parallel Artificial Membrane Permeability Assay PAMPA

Parallel Artificial Membrane Permeability Assay (PAMPA) 

The use of partition coefficients between water and lipophilic media is of wide use in pharmaceutical research. As discussed in the last chapters, different lipophilicity scales are used to describe the lipophilicity of a compound and relate it to its absorption behaviour in vivo. Differences between the logPow and partitioning between phospholipids and water (mainly determined using liposomes) for diverse compounds have been described leading to the development of the immobilized artificial membrane chromatography system. However, also the predictivity of the IAM system is limited and only a small number of membrane systems are available. 

PAMPA is generally carried out in the 96 well format using a PAMPA sandwich construction. The sandwich consists of a standard 96 well plate and a filter plate put on top (Microtiter plate filter plates that are commercially available). The bottom standard 96 well plate is filled with buffer so that the liquid surface will be in contact with the filter material of the filter plate put later on top. The filter material was impregnated 

PAMPA is often used at various pH values in order to measure permeability pH profiles as the permeability of ionisable compounds depends heavily on the pH of the buffer. As the pH range of the intestinal tract varies between pH 6 and pH 8 this is the range of pH values that mostly is used. Kerns (2004) recommended to measure from pH 4 to pH 7,4 in order to predict both bases and acids correctly. Ruell (2003) used permeation pH profiles from pH 4 to pH 9 together with the pKa values of the compounds under investigation to establish the optimum pH value for a single pH PAMPA measurement. 

The permeation in PAMPA experiments can be followed directly as the percentage of compound permeated into the acceptor compartment after a given time (%T) as described in the original work by Kansy et al. (1998) which reflects an equilibrium analysis at a given time point. 

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PARALLEL ARTIFICIAL-MEMBRANE PERMEABILITY ASSAY (PAMPA)... 

This book is written for the practicing pharmaceutical scientist involved in absorption-distribution-metabolism-excretion (ADME) measurements who needs to communicate with medicinal chemists persuasively, so that newly synthesized molecules will be more drug-like. ADME is all about a day in the life of a drug molecule (absorption, distribution, metabolism, and excretion). Specifically, this book attempts to describe the state of the art in measurement of ionization constants (p Ka), oil-water partition coefficients (log PI log D), solubility, and permeability (artificial phospholipid membrane barriers). Permeability is covered in considerable detail, based on a newly developed methodology known as parallel artificial membrane permeability assay (PAMPA). 

Table 18.3 Parallel artificial membrane permeability assay (PAMPA) permeability and human absorption for 19 drug compounds.

Avdeef, A., Bendels, S., Di, L., Fallen B., Kansy, M., Sugano, K. and Yamauchi, Y. (2007) Parallel artificial membrane permeability assay (PAMPA)-critical factors for better predictions of absorption. Journal of Pharmaceutical Sciences, 96, 2893-2909. 

To predict in vivo permeabilities, the parallel artificial membrane permeability assay (PAMPA) was introduced in 1998. Since then, it has gathered considerable interest in the pharmaceutical industry [41-47]. This method uses a phospholipid-coated filter separating two aqueous compartments to mimic the passive transport of small molecules. It readily provides information about passive-transport perme-... 

II.E.3 Parallel Artificial Membrane Permeability Assay (PAMPA) 471... 

In vitro experiments can sometimes provide valuable insight into what is happening in vivo that is limiting oral bioavailability. The typical experiments, often employed in tandem, to understand bioavailablilty are determinations of compound solubility, membrane permeability, and stability in subcellular fractions. The membrane permeability assays that are most often employed are either a measurement of permeability through an artificial membrane (Parallel artificial membrane permeability assay, PAMPA, is the most common technique) or a cell monolayer (Caco-2, a human colon carcinoma-derived cell line, is the most common cell monolayer). The subcellular fractions most often employed are plasma (for ester-containing compounds) and liver microsomes with the addition of either reduced nicotinamide adenine dinucleotide phosphate (NADPH) or uridine diphosphoglucuronic acid (UDPGA) as cofactor. 

Van Dijck A, Masungi C, MenschJ, Borremans C, Willems B, Mackie C, Brewster M, and Noppe, M. Parallel Artificial Membrane Permeability Assay (PAMPA) Combined with a 10-Day Multiscreen CACO-2 Cell Culture for Prediction of Passive and Active Absorption of Drugs. 2003 American Association of Pharmaceutical Scientists Annual Meeting and Exposition, Salt Lake City, Utah, USA, October 26-30, 2003. 

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. 

In 1998, Dr Manfred Kansy and co-workers at Hoffinann-La Roche published a high-throughput method for determining how well compounds can passively diffuse through a model of a membrane bilayer. They called it parallel artificial membrane permeability assay (PAMPA). ... 

Another assay that has gained popularity and acceptance for the evaluation of permeability is the parallel artificial membrane permeability assay (PAMPA). Earlier versions of this system coated polyvinylidene fluoride (PVDF) filter plates with artificial membrane using dioleoyl-sn-glycerol-3-phosphocholine (Chen et al., 2008). Several companies attempted to develop this technology in the late 1990s with limited success (Kansy et al., 1998). These earlier versions suffered poor correlation to cell models, poor correlation to human absorption, and poor reproducibility. More modem systems have been developed with different lipid formulations and solvation techniques that seem to correlate better with Caco-2 and human data and are more reproducible (Chan et al., 2005). Some companies use the PAMPA as a tier 1 prescreen discovery... 

Another approach commonly used to measure permeability is the parallel artificial membrane permeability assay (PAMPA) (Avdeef, 2005 Avdeef and Tsinman, 2006 Avdeef et al., 2007 Chen et al., 2008). The main advantage of PAMPA is that it is an artificial membrane, so it is easier to do the test. Once... 

Masungi C, Mensch J, Van Dijck A, Borremans C, Willems B, Mackie C, Noppe M, Brewster ME. Parallel artificial membrane permeability assay (PAMPA) combined with a 10-day multiscreen Caco-2 cell culture as a tool for assessing new drug candidates. Pharmazie 2008 63(3) 194-199. 

Permeability and Associated Assays Orally administered drugs need to cross the intestinal epithelium cell layer to reach the systemic circulation. Therefore, membrane permeability is a major determining factor of intestinal absorption and oral bioavailability for drug molecules. Several in vitro systems mimicking the epithelium cell layer are routinely used in ADME profiling to assess membrane permeability of NCEs. The parallel artificial membrane permeability assay (PAMPA) uses a dual chamber sandwich plate separated by an artificial lipid membrane to simulate the epithelium layer (Figure 6.6A) [75]. Compounds are... 

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