Understanding PAMPA

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. 

Though phospholipids may add some similarity to the biological membrane, the organic solvent remaining in the membrane largely affects its permeability. The BDSD theory would support the use of an alkane or alkyldiene [43]. 

Composition of the PAM PA membrane varies from a purely organic solvent membrane to a purely phospholipid membrane. At the first international conference of PAM PA in 2002 (www.pampa2002.com/), it was agreed that these variations would be notated as initials or a short adjective at the head of PAMPA (e.g., BM-PAMPA for biomimetic PAMPA). The original PAMPA (Egg-PAMPA) [47], hexadecane membrane PAMPA (HDM-PAMPA) [48], BM-PAMPA [49], double sink PAMPA (DS-PAMPA) and blood-brain barrier PAMPA (BBB-PAMPA) [51] are reviewed elsewhere [3]. 

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PAMPA is typically used to make a prediction of the passive, transcellular absorption of a compound. Compounds which may be absorbed by a paracellular mechanism or may be substrates for active transport (uptake or efflux) are usually better assessed in a cell based system. A combination of assays can be applied to gain a greater understanding of the permeability and transport properties of a compound. 

This agrees to internal VolSurf models derived for PAMPA membrane transport [163] to understand passive transcellular transport across membranes. One of our internal models based on 29 compounds characterized by immobilized artificial membrane chromatography by Salminen etal. ]164] shows an of 0.81 and = 0.70 for two PLS components derived using VolSurf descriptors. This is one of the rare examples where ionized starting molecules led to slightly better PLS statistics, while the general chemical interpretation is not affected. 

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 data were also used to understand permeation into the pharmacological and toxicological target cells. A discrepancy between the enzyme level assay and cell-based assay is often observed in drug discovery, resulting in a misreading of the structure-activity relationship [69]. 

Accordingly, we will briefly outline the use of MIEs in three research areas. In absorption, the use of MIEs to understand and model permeability through PAMPA, Caco-2, MDCK cells as well as BBMEC cells has been demonstrated. However, this approach is limited to a passive mechanism. 

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. 

Many organizations use colon adenocarcinoma (Caco-2) for detailed study of permeability however, this method can be resource intensive. Parallel artificial-membrane permeability (PAMPA) [19] has proven to be a reliable predictor of passive transcellular permeability for intestinal absorption prediction. It is also useful to interpret results of cell-based discovery assays, in which cell-membrane permeability is limiting. Finally, pTf provides insight into the pH dependence of solubility and permeability. It can be measured [20] or calculated to get an understanding of the regions of the intestine in which the compound will be best absorbed, as well as to anticipate the effect of pH on solubility and pemieability. Permeability at the blood-brain barrier (BBB) also can be rapidly profiled [21]. 

Drug absorption generally occurs either through passive transcellular or paracellu-lar diffusion, active carrier transport, or active efflux mechanisms. Several methods have been developed to aid in the understanding of the absorption of new lead compotmds. The most common ones use an immortalized cell line (e.g., Caco-2, Madin-Darby canine kidney, and the like) to mimic the intestinal epithelium. These in vitro models provide more predictive permeability information than the artificial membrane systems (i.e., PAMPA and permeability assays, described previously) based on the cells ability to promote (active transport) or resist (efflux) transport. Various in vitro methods are listed in the U.S. FDA guidelines. These are acceptable to evaluate the permeability of a drug substance, and includes a monolayer of suitable epithelial cells, and one such epithelial cell line that has been widely used as a model system of intestinal permeability is the Caco-2 cell line. 

So Caco-2 assays constitute more of a composite system than the relatively pure passive transcellular diffusion that PAMPA and lAM reflect. In light of this additional complexity, the trend shown in Figure 9.10 becomes quite understandable. The two methods tend to correlate with each other fairly well, except when active uptake (diamonds) or active efflux (triangles) mechanisms play a role. As most of the drugs are passively absorbed, however, this kind of analysis explains why initial screening of discovery compounds is often done by the somewhat higher throughput and cheaper PAMPA method, with Caco-2 reserved as a secondary assay for compounds of interest." ... 

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