In vivo and in vitro models for the investigation of substrate specificity of ABC-T from different mammal species, and their reciprocal correlations have been a subject of intense research, and have been revised by several authors. We base the following brief and basic introduction to some frequently used models on some notable reviews:
Polli et al., 2001; Schinkel & Jonker, 2003; Fricker & Miller, 2004; Garberg et al., 2005;
Löscher & Potschka, 2005b; Dallas et al., 2006; Balaz, 2009.
Frequently used in vitro (direct) transport experiments are performed whether on lipidic vesicles, on cells or on cell monolayers. Transport in lipidic vesicles utilize inside-out, membranous vesicles that are purified from cells which were previously transfected for the expression of a particular transporter. The transporters are directed to the inner part of the vesicle, so that the substrates are translocated to the interior of this lipidic complex, i.e., they are accumulated inside. Incubation with a standard inhibitor of the transporter allows for discriminating between transport and absence of transport.
Amongst common in vitro assays that utilize whole cells, but that do not require the formation of a tight monolayer are the (direct) uptake assay of the drug in question, and the calcein uptake assay in combination with the investigated drug (indirect method). In both cases, cells are either chemically selected or transfected for the expression of ABC-T, and compared for drug transport with non-selected (non-resistant) or parental cells, and with cells incubated with a standard inhibitor. In the first case, the direct accumulation of the drug in the cells is measured after an appropriate incubation period.
In principle, drugs that are substrates of the transporter are actively effluxed, and thus, less accumulated within the cells, and this condition can be reversed with the inhibitor.
A disadvantage of this assay is that a highly lipophilic drug can diffuse quickly through cell membrane, limiting the concentration inside the cell. As a consequence, negative results are not to be discarded when highly permeable compounds are investigated. In the second case, the calcein uptake assay, the transport of a drug is indirectly measured. Calcein acetoxymethyl ester (calcein-AM) is a non-fluorescent, lipophilic substance that rapidly diffuses into the cells, where it is cleaved by intracellular esterases resulting in the fluorescent, impermeable calcein, which is trapped within the cell unless actively extruded by Pgp, or MRP1 (Szakács et al., 1998; Dogan et al., 2004). This assay relies on the theory that if the tested drug is a substrate for Pgp, for instance, it will compete with calcein for affinity sites in the transporter. Hence, a Pgp 21
substrate can decrease calcein efflux, and as a consequence, it can increase calcein accumulation. This assay has the advantage that no high-cost analytic equipment other than a fluorometer is needed. The biggest disadvantage of the calcein uptake assay is that if a drug does not inhibit calcein efflux, it does not necessarily mean that the drug is not transported. No necessary correlation exists between substrates and inhibitors (Feng et al., 2008).
In the last years the use of cell monolayers has been largely extended, especially because they may better simulate the in vivo situation (Liu et al., 2008). In this case, polarized cell monolayers are grown on the microporous membrane of inserts, which are located within a larger compartment (i.e., a well), so that a tridimensional, two-compartment (i.e., apical and basolateral) model is created (Fig. 1.4). For the widely used bidirectional transport assay, the drug is diluted in the medium and applied in one of both compartments, and then samples are taken from the opposite compartment at given time intervals. This way, the permeability of a drug can be assessed from basolateral to apical and vice versa. An obvious requisite for this model is that the transporter must have a polarized expression. For instance, Pgp is expressed on the apical membrane in the kidney epithelial cell lines MDCK II and LLC-PK1. In this example, it is expected that a substrate will penetrate more quickly from basolateral to apical direction, than from apical to basolateral. A ratio of the permeabilities in both directions is easily calculated, and the ratio obtained in the transfectants (e.g., MDR1 transfected cells) is corrected with the ratio obtained in the parental cells, in order to find the difference that is attributable to the overexpressed transporter. This model has been used not only for drug screening of ABC transporter substrates (typically using Caco-2, MDCK II and/or LLC-PK1 cells), but also for the investigation of drug permeability across the BBB (including the former cell lines, and/or primary cultures of rat, porcine or bovine brain capillary endothelial cells). One of the biggest disadvantages of this Transwell® model is that the only available cells so far display relatively high paracellular permeability, because of suboptimal expression of TJ (Liu et al., 2008). For monitoring the tightness of the monolayer, the permeability of polar substances, such as fluorescein, mannitol or sucrose, can be measured. This parameter indicates the rate of drug diffusion via the paracellular route (paracellular markers). Another important parameter to be tested is the transepithelial or transendothelial electrical resistance (TEER) which has a direct relation with the formation of TJ; i.e., tighter monolayers show higher TEER values, but the values vary for each cell line. The safest method is a combination of TEER measurement and the use of paracellular markers.
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“Blood-side”
Figure 1.4 Transwell® model for bidirectional transport assay. Polarized cell monolayers that express apical transporters (orange dots) are grown on the microporous membrane of an insert (apical [A]), which is located within a basolateral compartment (B). Given the polarization of the cells and their transporters, the basolateral chamber represents the “brain side” and the apical chamber, the “blood side” in this model. The permeability of a drug from basolateral to apical (purple arrow and ploted line), and from apical to basolateral (green arrow and ploted line) are measured, and a transport ratio between them (TR) is obtained. The corrected transport ratio (cTR) of a drug is the ratio of the TR from Pgp over-expressing cells divided by the TR from parental cell lines, and indicates the magnitude of transport mediated by the over-expressed transporter. The graph shows typical results for the standard Pgp substrate vinblastine sulphate.
A drug is considered to be a good substrate for Pgp, if the cTR is higher than 2.5 in this model.
A cTR ≥ 1.5 is considered as the cut-line to classify the drug as substrate.
One of the most destacable in vitro systems for BBB permeability is the dynamic model described by Santaguida et al. (2006) and Cucullo et al. (2007) which consists of sets of capillaries. Endothelial cells are cultured onto the lumen of the capillaries, while astrocytes are grown on the abluminal part of them. But most importantly, the medium can flow through the system, recapitulating the physiological shear stress, and
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improving the formation of tight junctions. Endothelial cells from human beings have been used for drug permeability assays across this in vitro BBB (Cucullo et al., 2007).
In vivo studies of brain permeability and impact of MDTs at the BBB are obviously limited, and the approach of this topic is beyond the scope of this work, but some illustrative examples are mentioned next. A feasible technique for in vivo studies are inhibitory assays, where MDTs are systemically inhibited and the increment in the brain permeation of the drug is measured indirectly by using a specific end-point; i.e., a drug-specific pharmacological effect. One of the few available clinical examples is an inhibitory assay with the opioid drug loperamide, which was performed in healthy volunteers. Loperamide can not permeate into the brain tissue because it is a substrate for Pgp. In order to investigate this, human beings were administered with the opioid, while the Pgp was inhibited with quinidine. This resulted in increased brain availability, with the concomitant opioid effect. The end-point measured in this test was the respiratory rate, with respiratory depression observed after Pgp inhibition, because of the activity of loperamide on the respiratory center (Sadeque et al., 2000). On the other hand, a promising tool for the evaluantion of CNS drug permeation are imaging techniques, such as positron emission tomography, where 11C-radiolabeled drugs can be measured non-invasively in human beings. With this approach, inhibitory studies for drug permeation across the BBB in vivo can be carried out. This technique is still under development.
The most remarkable animal model for studies of drug transport mediated by ABC-T is the knockout-mice model, in which brain concentrations of the investigated drug are compared between wildtype mice and knockout-mice. An increase of drug brain-uptake in the latter indicates involvement of the silenced transporter in the efflux of such a drug (Rizzi et al., 2002; Doran et al., 2005).