In transport assays

To illustrate the role of passive influx in transport assays, we plotted the flux ratio Jp, a/Ja b as a function of the passive flux ((b). Passive flux varies enormously... 

Phloretin is the aglycon of phlorizin and inhibits the facilitated diffusion of glucose catalyzed by GLUT1 or GLUT4. It has been used to terminate the uptake of glucose in timed assays with isolated membranes or reconstituted transporters. 

SNAPs is an acronym for soluble NSF attachment proteins. They were originally discovered as cofactors for NSF that mediate the membrane binding of NSF in in vitro transport assays. Several isoforms of SNAPs exist in mammalian cells. SNAPs are also highly conserved proteins. Crystallographic studies indicated that the proteins form a very stiff and twisted sheet that is formed by a series of antiparallel and tightly packed helices connected by short loops. 

The study of active transport mechanisms has grown substantially in recent years, with transport proteins such as P-gp, BCRP, and MRP-2 among the most studied [59]. Several types of in vitro assays to assess substrates of transporters have been established these include assays directed toward intestinal and biliary efflux [60]. Assays that measure passive and active transport are also used to assess penetration of the blood-brain barrier. In addition to the assays described above, transfected cell lines that overexpress transporters present in the blood-brain barrier are also employed [61]. 

The equations used to calculate permeability coefficients depend on the design of the in vitro assay to measure the transport of molecules across membrane barriers. It is important to take into account factors such as pH conditions (e.g., pH gradients), buffer capacity, acceptor sink conditions (physical or chemical), any precipitate of the solute in the donor well, presence of cosolvent in the donor compartment, geometry of the compartments, stirring speeds, filter thickness, porosity, pore size, and tortuosity. 

Highly insoluble molecules are in part transported in the GIT by partitioning into the mixed micelles injected into the lumen from the biliary duct in the duodenum (Fig. 2.3). Mixed micelles consist of a 4 1 mixture of bile salts and phospholipids (Fig. 7.13). In contrast, at the point of absorption in the BBB, highly insoluble molecules are transported by serum proteins. This distinction is expected to be important in in vitro assay modeling. The use of simulated intestinal fluids is appealing. 

In conclusion, there are several drawbacks to the use of Caco-2 cells in studies of active drug transport. Despite these drawbacks, we note that a recent comprehensive study comparing various P-glycoprotein drug efflux assays in drug discovery came to the conclusion that the Caco-2 transport assay is the method of choice, since it displays a biased responsiveness towards compounds with low or moderate permeability - in other words, towards compounds whose intestinal permeability is most likely to be significantly affected by drug efflux mechanisms [101]. 

Here, we briefly describe the automated Caco-2 assay used at the research site in AstraZeneca R D Molndal. The solubility of the test compounds is measured (or theoretically predicted) before they are run in the Caco-2 assay. In order to be able to make correct determinations of the permeability coefficient, the substance must be dissolved when added to cell monolayer in the transport experiment. Compounds with insufficient solubility are therefore not tested. We generally apply a test concentration of 10 pM, but in specific projects or under certain circumstances a concentration of only 1 pM is applied. The test compounds are first prepared in DM SO solution (1 mM) on a parent plate and are then diluted in transport buffer to give a final drug concentration of 10 pM (solution containing 1% DMSO) when added to the cell monolayers. 

Fig. 14.1. Schematic representation of drug transport assay in cell monolayers cultured on a culture insert containing permeable membrane.

A variety of in vitro assays are available to identify compounds as substrates and inhibitors of P-gp. These assays, which have been reviewed elsewhere in great detail [20-24], can be classified into three general categories (1) transport, (2) accumulation/ efflux and (3) ATPase activity [20-28]. It is important to note that these in vitro model systems can be adapted for measuring the interaction of dmgs with other important drug transporter systems [22]. 

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... 

Accumulation/efflux studies can be performed on different cell systems or membrane vesicle preparations. In the accumulation assays, uptake of a probe over time, typically either fluorescent (e.g. calcein-AM (CAM) [25-27]) or radiolabeled, into the cell or membrane vesicles is measured in the presence or absence of a known P-gp inhibitor. As P-gp transports substrates out of the cells, the inhibition of the protein would result in an increase in the amount of the probe in the cell. Accumulation studies in cells that overexpress P-gp can be compared to those obtained in the parental cell line that does not have as high a level of P-gp expression. The probe in the absence of inhibitors shows lower accumulation in P-gp expressing cells than in P-gp deficient cells. Similarly, probe accumulation is increased under conditions where P-gp is inhibited such that the difference in accumulation in P-gp deficient and overexpressing cells, respectively, becomes smaller. Accumulation assays poorly distinguish substrates and inhibitors of P-gp and, as far as transport assays are concerned, are also influenced by a passive diffusion property of molecules [20]. In contrast to transport assays, both accumulation (i.e. calcein-AM assay) and ATPase assays tend to fail in the identification ofrelatively low permeable compounds as P-gp active compounds [20]. 

The applicability of an in vitro assay to classify drug substances is verified by demonstrating a rank-order relationship between the extent of human absorption and experimental permeability values with 20 model drugs. The model drugs should represent ranges of 0-50%, 50-89%, and 90-100% absorption (/a) and the results should clearly differentiate between HP and LP drugs. The model can also be characterized for the presence of functional active transporters (e.g., amino acids, di/tripeptides, monocarboxylic acids, nucleosides) and efflux mechanisms (e.g., P-gp, MRP). 

Interestingly, these assays have been employed to address the functional consequences of polymorphisms in the ABCC family of transporters but no notable alterations in transport capacity have been found (43). It seems that although ABCC transporters contain several potentially important polymorphisms and are important in drug transport overall, functional variability is actually quite low. This is perhaps the reason for the multiple negative studies that have assessed ABCC polymorphisms as they relate to drug bioavailability (20). 

Discrimination of efflux, active or passive transport is already feasible by suitable in vitro experiments. For instance, the PAMPA assay detects passive transport only, while Caco-2 cells include transporters. A comparison between transport in PAMPA and Caco-2 cells by a calibration plot reveals compounds with greater or less transport in Caco-2 cells than in PAMPA. These compounds should be tested in uptake and efflux transport assays in order to gain deeper insight into absorption fate. 

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