Membrane-Based Models PAMPA

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 

No animals required Prediction of intestinal absorption before synthesis of compound 

Difficult to model complex interplay of a multitude of factors 

High throughput Relatively inexpensive Different lipid compositions available 

Animal or human origin Relatively easy to perform 

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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. 

The development of a new coculture-based model of human BBB that enables the prediction of passive and active transport of molecules into the CNS has recently been reported (Josserand et al., 2006). This new model consists of primary cultures of human brain capillary endothelial cells cocultured with primary human glial cells (Megard et al., 2002 Josserand et al., 2006). The advantage of this systan includes the use of human primary cells, avoiding species, age, and interindi-vidual differences since the two cell types are removed from the same human donor and because of the danonstrated expression of functional efflux transporters such as P-gp, MRP-1, MRP-4, MRP-5, and BCRP. Such models have potential for the assessment of permeability of drug and specific transport mechanisms, which is not possible in artificial membrane assays (e.g., PAMPA) or other cell models due to incomplete expression of active transporters. 

The above iso-pH measurements are based on the 2% DOPC/dodecane system (model 1.0 over pH 3-10 range). Another membrane model was also explored by us. Table 7.16 lists iso-pH effective permeability measurements using the soy lecithin (20% wt/vol in dodecane) membrane PAMPA (models 17.1, 24.1, and 25.1) The negative membrane charge, the multicomponent phospholipid mixture, and the acceptor sink condition (Table 7.1) result in different intrinsic permeabilities for the probe molecules. Figure 7.40 shows the relationship between the 2% DOPC and the 20% soy iso-pH PAMPA systems for ketoprofen. Since the intrinsic permeability of ketoprofen in the soy lecithin membrane is about 20 times greater than in DOPC membrane, the flat diffusion-limited transport region of the log Pe... 

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. 

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. 

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