Transport, active permeability ratios

The applicability range of any model should be limited to molecules having a similar mechanism of transport. Therefore, we have selected from the literature only those compounds with well-characterized Caco-2 cell permeability and excluded compounds with a high efflux ratio. Known P-gp substrates and actively transported compounds were also excluded from the list. 

In the transport assays, the permeability of a compound in both absorption and secretion directions is measured using polarized epithelial cells that constitutively express high levels of P-gp (e.g. Caco-2) or have been transfected with the gene for a specific P-gp (e.g. MDR1-transfected MDCK or LLC-PK1 cells). Since P-gp is expressed on the apical membrane, ratios ofbasolateral-to-apical (B —> A) permeability versus apical-to-basolateral (A —> B) permeability greater than 1 may indicate an active efflux transport process. Bidirectional permeability measurements can also be performed in the presence of a specific P-gp inhibitor. Thus, apical-to-basolateral permeability increases and basolateral-to-apical permeability decreases such that... 

In real cells, multiple transmembrane pumps and channels maintain and regulate the transmembrane potential. Furthermore, those processes are at best only in a quasi-steady state, not truly at equilibrium. Thus, electrophoresis of an ionic solute across a membrane may be a passive equilibrative diffusion process in itself, but is effectively an active and concentra-tive process when the cell is considered as a whole. Other factors that influence transport across membranes include pH gradients, differences in binding, and coupled reactions that convert the transported substrate into another chemical form. In each case, transport is governed by the concentration of free and permeable substrate available in each compartment. The effect of pH on transport will depend on whether the permeant species is the protonated form (e.g., acids) or the unprotonated form (e.g., bases), on the pfQ of the compound, and on the pH in each compartment. The effects can be predicted with reference to the Henderson-Hasselbach equation (Equation 14.2), which states that the ratio of acid and base forms changes by a factor of 10 for each unit change in either pH or pfCt ... 

The Calu-3 human submucosal gland cell line forms polarized cell monolayers with tight junctions, produces mucus, and develops apical cilia when grown at an air interface. Transport studies can be performed after 10-14 days in culture, and it has been shown that Calu-3 cells express P-gp and actively transport amino acids, nucleosides, and dipeptide analogs organic anions, organic cations, polyamines, and efflux pump substrates are not actively transported.43,51,53-56 Because Calu-3 cells are not subject to the influence of multiple in vivo cell types, the expression of carrier proteins and enzymes may not reflect in vivo levels. Nevertheless, values obtained in Calu-3 permeability studies correlate well with those obtained from primary cultured rabbit tracheal epithelial cells and in vivo rat lung absorption studies.54 Mannitol permeation in Calu-3 cells is about 10 times less than that in vivo, but this is the same ratio difference between Caco-2 cells and in vivo intestinal epithelium.51... 

Larger coupling ratios can also erroneously be obtained when calcium uptake ceases before the splitting of ATP is interrupted. In this case, the calcium-dependent ATPase activity is underestimated. Similar deviations can be expected if the preparation contains an enzyme which transports calcium without being activated by calcium ions, as it is the case for sarcoplasmic reticulum preparations isolated from red skeletal muscles [34]. On the other hand, smaller ratios were found when the preparation contained a sizeable fraction of open vesicles this fraction only contributes to the calcium-dependent ATPase activity and not to calcium transport. A reduced coupling ratio can also result from an increase in calcium permeability occurring for instance, at elevated temperatures or at alkaline pH [72]. 

In addition to the unidirectional mode described above, Caco-2 assays can also be performed in the bidirectional mode. A bidirectional assay essentially consists of two independent unidirectional assays, with compounds added to the apical side in one experiment, and to the basolateral side in the other. Unidirectional Caco-2 assays yield permeability values only in A > B direction, whereas bidirectional assays yield values in both A > B and B > A directions. Since many active transporter proteins are expressed in the Caco-2 cell line, the ratios of Pa>b to Pb>a in bidirectional assays can be used to assess transporter involvement in drug permeability. If Pa>b Pb>a, then the compound is likely to be actively transported for uptake if Pb>a/Pa>b > 3, then the compound is likely a substrate for efflux transporters. Additional assays using the same bidirectional format can be used to further identify whether the test compounds are substrates or inhibitors of the transporters, therefore providing liabihty assessments of transporter-related DDI potential as either victims (substrates) or perpetrators (inhibitors) [77]. 

In a review on membrane proteins, Guidotti (1972) has classified membranes into three types on the basis of their protein content. The first class is the simple, inert membrane represented by myelin. It consists primarily of lipid with little protein, acts as a permeability barrier and insulator, and has only three known enzymatic activities (Beck et al., 1968 Olafson et al., 1969 Kurihara and Tsukada, 1967 Gammer et al., 1976 Yandrasitz et al., 1976). The large second class of membranes which have a protein-to-lipid ratio of about 1 1 (w w) are typified by most mammalian plasma membranes. They have many enzymatic activities and sophisticated transport systems associated with them, in addition to the permeability factor. The third class of membranes has bacterial and inner mitochondrial membranes as its models. These membranes have proportionately larger amounts of protein than lipid and have added functions such as oxidative phosphorylation and nucleic acid synthesis. In general, the specialization and enzyme function of the membrane increases in proportion to its protein content. Table 4 gives the amino acid composition of some isolated membrane proteins. Total membrane protein (intrinsic + extrinsic) often has an amino acid composition which falls into the range of other nonmembrane, "soluble" proteins (Vanderkooi and Capaldi, 1972). 

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