Cellular Models and In Vitro Assays for the Screening of modulators of P-gp, MRP1 and BCRP – PMC

 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

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For this reason, a phosphate-mimicking transport inhibitor e. The rate of the formation of this transition state can be assessed stopping the catalytic reaction by excess ATP and UV cross-linking. This formation is proportional to the rate of transport. When the substrates are efficiently transported, there is an increase in the formation of the trapped nucleotide [ 32 ]. Since both direct photoaffinity labeling and nucleotide trapping experiments are complicated techniques associated with complex protocols and are not routinely applied in the pharmaceutical industry, these techniques are important tools for studying details of the molecular mechanism.

Direct photolabeling is generally not adequate for distinguishing between substrates and inhibitors [ 1 , 32 ]. On the other hand, ABC transporters form low-affinity interactions with a wide variety of hydrophobic compounds. The interaction sites and intensities may directly depend on the test drug and actual conformation of the transporter [ 32 ].

Cell-based assays may provide more clear information about the interaction between compounds and ABC transporters, applied in the evaluation of the following kinetic parameters: K m and V max for substrates, and K i and IC 50 for inhibitors Table 2.

The cytotoxicity assay is, by far, the most widely applied cell-based approach for investigating ABC transporters function. This test compound can be an inhibitor, activator or inducer of the ABC carrier under study. These assays allow a high-throughput screening of compounds due to reduced time consumption and cost, when compared, for example, with the in vivo assays, which have a high cost, are time-consuming, and have ethical restrictions.

However, cell-based assays are more labor and time consuming than the membrane-based assays. It is important to consider the following features: a particular cell line can express multiple transporters, although there are modified cell lines expressing one specific transporter; the culture conditions and number of cell passages may change the transporters expression levels; and the cells need to be maintained under culture conditions prior to use Table 2 [ 1 ].

Tissue localization and changes in gene expression after cells stimulation can be monitored by Northern blot analysis, dot-blot analysis, competitive PCR, RNase protection assays or in situ hybridization. Although these methods require large RNA amounts and starting material, not allowing a rapid analysis of multiple genes and large sample numbers, they are widely accepted and reliable and can be applied to the evaluation of ABC transporters gene expression [ ].

Real-time RT-PCR is commonly used in molecular biology for mRNA analysis, including detection and quantitation, by the use of fluorescent probes [ ]. This technique is sensitive enough to enable precise and reproducible mRNA quantitation both rare and abundant from a single cell [ ]. The evaluation of the gene expression is based on cycle threshold Ct values rather than end-point detection [ ]. There are two main classes of chemistry compounds, i. The PCR product accumulation corresponds to an increase in the fluorescence intensity.

Although requiring extensive optimization, this is the most economical and the easiest method. The need of optimization is related to the SYBR Green ability for binding to any double-stranded DNA during reaction, including primer-dimers and other non-specific reaction products, resulting in an overestimation of the target gene concentration. On the other hand, there are hydrolysis and hybridization FRET-based probes [ ].

The proximity of the dyes, during unhybridized state, does not completely quench the fluorescence, being possible to observe a background fluorescence.

During the PCR reaction, the probe anneals specifically between the primers forward and reverse to the desired target region of the gene. Then, the polymerase carries out the extension of the primer and replicates the template. This process is repeated in every cycle and fluorescence increases in proportion to the amount of probe cleavage.

TaqMan probe does not need extensive optimization. The second FRET-based technique is based on two probes, one labeled with a fluorescent donor dye and other labeled with an acceptor dye. Once in close vicinity 3 to 5 base pairs , the donor dye emits energy that excites the acceptor dye.

Consequently, there is emission of fluorescence at a different wavelength, which is monitored with a specific equipment. After each cycle, additional hybridization probes anneal, increasing the fluorescence intensity, which is measured during the exponential phase of the PCR reaction. The fluorescence intensity is proportional to the amount of input target DNA [ ].

Real-time PCR allows sample processing in a multi-well plate, automatically and with high-throughput. Glyceraldehyde 3-phosphate dehydrogenase GAPDH is used as a reference gene for expression analysis in human tissues, but alternative reference genes can be used for other cell systems [ ]. Langmann and colleagues developed a rapid, accurate and highly sensitive real-time PCR method for detection and quantification of all ABC transporters using a TaqMan probe.

The method allows a rapid and complete analysis of all ABC transporters in obtained RNA samples, from twenty different human tissues. As a result, authors identified tissues involved in secretory adrenal gland , metabolic liver and kidney , barrier lung, trachea and small intestine and reproductive and tropic placenta, uterus, prostate and testis functions with high transcriptional activity for ABC transporters [ ]. Flow cytometry is a rapid and specific technique that provides complete cellular analysis, being used as a tool for understanding the regulation and interaction of cell systems, mainly based in the use of fluorescent antibodies.

Light emitted from these antibodies allow the identification of a wide array of cell surface and even cytoplasmic antigens [ ]. Flow cytometry provides quantitative measurements of cells and other particles at a high speed, being suitable for the study of single mammalian cells in suspension by measuring their optical and fluorescence characteristics [ ].

Some physical properties, such as cell size and internal complexity, can be measured by flow cytometry [ ]. Additionally, antibodies conjugated with fluorescent dyes can bind to specific proteins on cell membranes intact cells or inside cells permeabilized cells. Also, the use of fluorescent substrates, such as rhodamine , may be useful for the evaluation of membrane transporters activity.

The labeled cells are passed by a light source and the fluorescent molecules are excited to a state of higher energy. When returning to their resting states, the fluorochromes emit light energy at higher wavelengths. The emitted fluorescence is collected using a flow cytometer, spectrally filtered and detected using photomultiplier tubes.

It is possible to simultaneously measure several cell properties, using multiple fluorochromes, each one emitting light at different wavelengths, although being excited with similar wavelengths. Propidium iodide, phycoerythrin and fluorescein are commonly used dyes [ ]. Flow cytometry assays can be applied to the study of ABC transporters, allowing the characterization of the interactions between drugs and ABC carriers, and usually involve the use of fluorescent transporter substrates, such as rhodamine and calcein acetoxymethyl ester calcein-AM for P-gp [ ].

Vilas-Boas and colleagues evaluated the influence of aging in P-gp expression and activity, in human lymphocytes isolated from whole blood samples of 65 healthy caucasian male donors, comparing two different methodologies. P-gp expression was analyzed using an anti-P-gp monoclonal antibody UIC2 , in the presence and absence of vinblastine.

P-gp activity was studied by measuring the efflux rate of the P-gp fluorescent substrate, rhodamine , and by using the UIC2 shift assay. The results obtained in both studies were compared and showed a significant age-dependent increase in mean P-gp expression and no differences were found in P-gp activity.

Moreover, the UIC2 shift assay proved to be more selective than the rhodamine efflux assay, in the analysis of P-gp activity [ ]. The researchers also used flow cytometry to study, in RBE4 cells, the putative modulatory effect of rifampicin and three rifampicin derivatives over P-gp function, using rhodamine as a fluorescent substrate [ 20 ]. Recently, Silva and co-authors have been using a flow cytometry-based approach to study the ability of different compounds, such as doxorubicin, colchicine, X and TX, to modulate P-gp expression and activity, using the Caco-2 cell model.

In these studies, the UIC2 monoclonal antibody conjugated with fluorescein isothiocyanate was used to study P-gp expression, and rhodamine was used to evaluate P-gp activity [ 16 , 21 , 22 , ]. Despite flow cytometry usefulness in expression and functional studies of ABC transporters in live cells, most dyes used as indicators have limited applicability as they do not simultaneously detect all types of ABC carriers [ ].

Beyond flow cytometry, other accumulation and efflux assays are suitable for the screening of compounds that interfere with efflux transporters. These assays can be performed using cell suspensions, cell monolayers or membrane vesicle preparations [ ]. Upon loading of the cells with lipophilic dye s , with diffusion capacity across cell membranes, the resulting fluorescence intensity of the cell s will depend upon the activity of the ABC transporters [ ].

The accumulation of the fluorescent substrates can be measured in the presence and absence of specific inhibitors or activators, in order to understand the effect of the transporters activity [ ]. The intracellular accumulation of the dye is inversely proportional to the ABC carrier activity and can be measured by fluorescence spectrophotometry [ ]. Therefore, an increased intracellular accumulation of a given substrate higher intracellular fluorescence can be observed in the presence of an inhibitor, while the opposite decreased intracellular accumulation is characteristic of an ABC transporter inducer and activator.

However, the discrimination between an inducer and an activator is only related with the time of contact of such compounds with the cells. On the other hand, the effect of an inducer in the pump activity requires an increased incubation period, since the de novo synthesis of the protein is needed.

Moreover, to note that although an increased expression could be observed after incubation with an inducer, it will not necessarily be translated in an increased activity of a given transporter [ 3 , 16 ]. The efflux studies comprise the pre-load of the cells with the dye of interest.

The amount of dye in the extracellular environment is measured under various conditions known to influence the transporter activity. In the presence of an inhibitor of the efflux transporter, the amount of dye expelled from the cells will be smaller than that observed for control cells. The change in the intracellular accumulation of the fluorescent compounds when co-administered with inhibitors, inducers or activators, is considered to be mainly due to their effect on the efflux pumps located in the cellular membrane, such as P-gp.

It is important to notice that the analysis of the inhibition of P-gp may depend on the nature of the used substrate, since at least two binding sites, H and R, are considered to exist and inhibitors may differently interact with them.

Consequently, inhibition assays may be performed with various P-gp substrates [ 38 , , ]. The analysis of the efflux transporters activity may be based on the evaluation of the dye accumulation, efflux or both. For example, one protocol routinely used for the evaluation of the effect of inducers or activators consists in two phases: i the accumulation phase, in the presence of the dye, and in which the ABC transporter activity is blocked with an inhibitor of energy production e.

The first phase results in maximum substrate accumulation inside the cells. The second phase consists in restoring the normal function of the transporter, which is now able to transport the fluorescent substrate out of the cells. By analyzing the cells both after the inhibited accumulation phase and after the efflux phase, is possible to infer the amount of substrate transported by the pump.

For transfected cells or drug-induced cells that over-express a particular drug efflux transporter, accumulation or efflux studies can be compared to the wild-type or parental cell line that does not have as high a level of drug efflux transporter expression [ ]. It is important the selection of specific inhibitors and specific fluorescent substrates. In P-gp activity studies, rhodamine is frequently used as a fluorescent substrate, and cyclosporine A or PSC as P-gp inhibitors [ 16 , 19 , 20 , , , , , ].

Western blotting or protein blotting or immunoblotting is an important technique used for the immunodetection of proteins post-electrophoresis, particularly those at low abundance [ ]. Western blotting analysis is commonly performed in ABC proteins expression studies [ 22 , , ]. Western blotting is characterized by the following specific advantages: a wet membranes are flexible and of easy handling; b the proteins immobilized on the membrane are easily accessible to different ligands; c only a small amount of reagents is required for transfer analysis; d it is possible to obtain multiple replicas of a gel; e it is possible to storage transferred patterns, prior to use; f the same protein transfer can be used in multiple successive analysis [ ].

Transport assays are the most direct tool for the evaluation of transporter function and permeability of the test compound [ 1 ]. When cells reach confluency, they differentiate and become ready to be used in permeability studies.

The two compartments are designated as apical and basolateral, denoting the membrane orientation of polarized cell layers. These two chambers are connected only through the cells monolayer and their semipermeable support.

The transport differences between the basolateral-to-apical and the apical-to-basolateral compartments are easily measured. The calculated ratio is referred to as efflux ratio and for results greater than 2 the test compound is considered substrate of the active efflux transporters [ 1 , 32 , , , , , ]. The experimental protocol is initiated by the addition of a solution containing the test compound to either the apical upper chamber or basolateral lower chamber compartment, for the study of the apical-to-basolateral A-to-B or basolateral-to-apical B-to-A transport, respectively [ 1 , , , , ].

On the other side is added a buffer. At desired time points, aliquots of added solution are removed from the lower chamber for studies of A-to-B transport or from the upper chamber for studies of B-to-A transport.

In the presence of efflux transporters expression on the apical membrane, P app, A-to-B is smaller than P app, B-to-A. These results will be contradicted if the transporter is localized on the basolateral cell membrane [ 1 , ]. Passively diffused compounds present P app values that are independent on its concentration. The flux rate is linearly correlated with the concentration of the compound. The flux rate of actively transported compounds is saturable with increasing of its concentration.

The determination of kinetic parameters, such as K m and V max , is possible [ 1 ]. Primary cultured cells, such as primary cultured brain endothelial cells, conjunctiva and alveola epithelial cells are cell types used in these studies [ 1 , ].

The cell type suitable for these assays must be polarized [ 1 ]. During transport assays several points should be taken into consideration, such as the selected cell line, pore size, pore density and filter material [ 32 ]. Many ABC carriers are constitutively expressed at the apical membrane of epithelial cells of different organs, including those that function as body barriers, such as the liver, brain, kidney and intestinal tract [ , ].

In the small intestine and colon, P-gp is one of the most important efflux proteins and may play a major contribution for several orally administered drugs bioavailability [ ].

Ex vivo methodologies are an experimental approach where an organ or tissue is removed from the animal and placed in chambers where physiological conditions found in the living body are mimicked, namely the access to nutrients and oxygen, allowing the viability of the organ or tissue during the experimentation time. ABC function can be accurately evaluated by using ex vivo approaches Table 2. Serosal to mucosal transport of the fluorescent substrate, in the presence or absence of the putative ABC carrier modulator, is evaluated in each intestinal sac by determining the substrate concentration, by spectrofluorometry, in samples of mucosal medium, over time.

Rhodamine is a dye usually used as P-gp substrate [ , , ]. Given the relevance of the ABC transporters in the toxicokinetics and pharmacokinetics, namely in the absorption, distribution BBB permeation and excretion processes, as well as their involvement in diverse pathophysiological conditions, the search for new modulators of these carrier proteins is of particular importance in both pharmacological and toxicological fields. Thereby, computational models are very valuable tools, allowing the identification of new putative ligands and, at the same time, being a relevant alternative to excessive animal testing and a preliminary approach to the in vitro and ex vivo experiments, very often expensive, laborious and time-consuming.

In silico models provide rapid and inexpensive screening platforms, and can include the development of quantitative structure-activity relationship QSAR models, as well as docking studies for ligand-carrier interactions prediction, and also the development of pharmacophores for ABC transporters inducers and activators [ 3 ]. Docking studies have long been used to predict the interaction of compounds with their potential targets proteins, nucleic acids, carbohydrates and lipids.

Several docking models were developed to map potential modulators of P-gp, BCRP and MRP1, thus allowing to evaluate the potential binding modes of such compounds in a given transporter [ 20 , 21 , 22 , , , , , , ]. Indeed, newly synthetized thio xanthonic derivatives demonstrated the ability to immediately increase P-gp activity after a short incubation period, an effect compatible with P-gp activation, resulting in a significant decrease in the toxicity of a P-gp substrate, PQ.

The possibility of a co-transport mechanism between TXs and PQ was further supported by docking studies using a validated P-gp model [ 22 ]. However, although numerous computational models, based on QSAR analysis, pharmacophore modelling and molecular docking techniques, have been developed to predict ABC transporters substrates and inhibitors, particularly in what concerns to P-gp, the search for new inducers and activators has been mainly performed by random screening [ 21 ].

Noteworthy, and in an attempt to address this gap, pharmacophores for P-gp inducers and activators were recently developed, which can be of utmost importance, in the future, in predicting new ligands [ 22 , ]. In fact, based on the in vitro P-gp activation ability of newly synthetized thioxanthonic derivatives [ 22 ] and on a set of known P-gp activators described in the literature, the authors developed and validated common feature pharmacophore models for P-gp activation.

The best ranked pharmacophore reported was composed of three features one hydrophobic feature, one aromatic ring, and one hydrogen bond acceptor group and can be a very useful tool to efficiently and rapidly predict new ligands with the ability to activate P-gp.

Additionally, pharmacophore construction was also performed for P-gp inducers. Briefly, the pharmacophores were validated using known P-gp inducers and can be used to map new compounds, as it was the case of newly synthetized TXs, for which there was previous indication from data of in vitro assays about their potential to activate and induce P-gp. However, since many signalling transduction pathways can be considered in regulating the expression of a given transporter, fact that is particular evident for ABC transporters, and given the structural diversity of the compounds, finding a pharmacophore for P-gp inducers can be a challenging task.

Noteworthy, by using such pharmacophores for P-gp inducers and activators, a perfect match between in silico and in vitro studies was observed [ 21 , 22 ], thus further reinforcing the idea that the use of such in silico strategies can help to predict the P-gp modulatory effects of new drugs that can be initially screened through these newly developed pharmacophores. Also, in vitro data on the ability of newly synthetized dihydroxylated xanthones to activate P-gp and protect Caco-2 cells against the cytotoxicity induced by a P-gp substrate, PQ, triggered the development of a 2D QSAR model, which demonstrated that the maximal partial charge for oxygen atoms is related with the P-gp activation ability of such compounds [ 21 ].

Furthermore, a perfect match was again observed, with both the docking studies and the QSAR model being in accordance with the reported in vitro data [ 21 ].

Taken together, the in silico models disclose new possibilities in drug discovery and can be a valuable and complementary tool in the prediction of new ligands, allowing a more rational use of in vitro, ex vivo and in vivo assays. In vitro and in vivo studies with inducers and activators of the ABC transporters have shown that the use of these compounds may be an effective antidotal pathway against xenobiotic-induced toxicity.

The action mechanisms of both are not clear. Therefore, it is important to conduct more research involving putative inducers and activators of the ABC transporters, in order to understand: 1 their mechanism of action; 2 their specificity and 3 their toxicity in tissues with toxicological relevance. During the assessment of new modulators of the ABC transporters it is important to use adequate in vitro assays, high throughput and low-cost alternatives to excessive animal testing, evaluating their main effects on the expression and activity of the ABC transporters.

Using only one technique or one concentration of the test compound could lead to false results. To all financing sources the authors are greatly indebted. Published online Apr 8. Author information Article notes Copyright and License information Disclaimer. Received Jan 30; Accepted Mar Abstract Adenosine triphosphate ATP -binding cassette ABC transporters are highly expressed in tumor cells, as well as in organs involved in absorption and secretion processes, mediating the ATP-dependent efflux of compounds, both endogenous substances and xenobiotics, including drugs.

Keywords: inducers, activators, ATP-binding cassette transporters, cellular models, membrane assays, cell-based assays, in vitro assays, P-glycoprotein, multidrug resistance-associated protein 1, breast cancer resistance protein.

Introduction The bioavailability of a wide variety of compounds that cannot permeate the membrane by passive diffusion e. Open in a separate window. Figure 1. Figure 2. Figure 3. Overview of Modulators of the ABC Transporters: Activators and Inducers Compounds that interact with ABC transporters can act as substrates being moved across membranes via the transporter , inhibitors impairing the transporter-mediated efflux of other compounds , inducers enhancing the transporter expression levels or activators enhancing the transporter activity , but one compound can also have overlapping modes of action [ 9 ].

Study Models for ABC Transporters According to in vivo and in vitro results obtained, inducers and activators of the ABC transporters can represent an important protection tool against xenobiotic-induced toxicity and an antidotal pathway to be explored [ 3 , 15 , 16 , 19 , 20 , 21 , 22 ].

Cellular Models 3. Figure 4. Cardiovascular System The cardiac endothelial cells are characterized by expression of uptake and efflux transporters, which control the transport of a wide range of compounds, including drugs and toxins, into and out of the heart, respectively [ ]. Liver The liver is an important tissue involved in the synthesis and secretion of bile acids, metabolism and transport of cholesterol, as well as in the metabolism and efflux of endogenous and exogenous substances [ , ].

Figure 5. Kidney The kidney is responsible for maintaining fluid and electrolyte homeostasis, maintaining the essential nutrients and eliminating both potentially toxic compounds and metabolic waste products from the body. Figure 6. Intestine The intestine, in addition to the liver, is an important tissue that regulates the extent of absorption of orally administered drugs [ , ].

Figure 7. In Vitro Assays Appropriate in vitro assays for transport studies can be divided in two major groups: membrane-based assays and cell-based assays. Membrane-Based Assays The study of the function of the ABC efflux transporters and the identification of their substrates and inhibitors has been performed by using membranes, prepared from cells expressing ABC transporters. Table 2 Main advantages versus disadvantages of the described in vitro and ex vivo assays adapted from [ 1 ].

Advantages Disadvantages In vitro assays Cell-based assays Allows to screen for P-gp inducers, activators, inhibitors and substrates. Cell-based transport assays are a classic assay to determine substrates or inhibitors and, more recently, activators.

However, to note that an increased expression of a given transporter may not necessarily result in an increase in its transport activity. May provide more information on the interaction between xenobiotics and transporters, due to the intact cell structure.

Can be employed to assess kinetic parameters, such as the half maximal inhibitory concentration IC 50 for inhibitors. Can be easily adapted to a high throughput mode with automation and cell culture in multi-well plates. Additional information may be obtained, such as information on the xenobiotic permeability and transporter localization in cells. It is more difficult to characterize the xenobiotic effects on one specific efflux transporter, given the expression of multiple transporters in a particular cell line including cell lines that have been engineered to express a given transporter.

The transporters expression levels can change according to the cell culture conditions and number of passages in culture. Cell culture media can be expensive, according to the specific supplementation requirements of a given cell line.

These assays are more laborious and time consuming than the ATPase assay and membrane vesicular transport studies. In the transport assays, polarized epithelium cells with well-defined tight junctions are needed. In the particular case of Caco-2 cells, the development of a proper polarized cell monolayer requires a long-time culture and the cells have multiple efflux transporters expressed.

False negative results can be obtained in the transport assays for xenobiotic with high passive diffusion. ATPase assays can be used as a high throughput screening tool to identify ligands for ABC transporters—a positive result either stimulation or inhibition indicates that the test xenobiotic is a ligand for a specific efflux pump. The membrane vesicular transport assays, contrarily to the ATPase assays, are functional assays and, thus, can be used to distinguish a transporter inhibitor from a substrate.

Do not allow to screen for P-gp inducers, since de novo synthesis of these proteins cannot be detected. ATPase assays are not functional assays and cannot be used to distinguish between substrates and inhibitors. In the ATPase assays, the xenobiotics effects should be evaluated at several concentrations to avoid false negative results, since the stimulation or inhibition can occur at either low or high concentrations. False negative results may also be observed for low affinity ligands, since the concentration tested can be limited by the xenobiotic solubility.

Membrane-based assays aiming the evaluation of membrane vesicular transport mediated by a given transporter may also give false negative results for lipophilic xenobiotics, which have high nonspecific binding and high passive diffusion. A more accurate determination of the transporter functions in absorption, biliary elimination, renal excretion and brain penetration can be obtained by using isolated perfused intestine, liver, kidney or brain.

The use of a perfused organ assay allows a much simpler understanding of the role of a transporter in a given organ, when compared with the use of the whole animal, since the concentration of the drug in the target organ can be controlled and the effect from other organs can be avoided. It is more difficult to characterize the xenobiotic effects on one specific efflux transporter.

The organ integrity and enzyme activity may become fragile and compromised during long-term perfusions. Important to evaluate the potential interspecies differences in transporters when extrapolating data from animal to humans. ATPase Assays The determination of the ABC transporters ATPase activity can be performed either in isolated membranes containing the desired transporter insect or mammalian cell membranes , or in reconstituted ABC protein preparations [ 32 ]. Cell-Based Assays Cell-based assays may provide more clear information about the interaction between compounds and ABC transporters, applied in the evaluation of the following kinetic parameters: K m and V max for substrates, and K i and IC 50 for inhibitors Table 2.

ABC Transporter Gene Expression Tissue localization and changes in gene expression after cells stimulation can be monitored by Northern blot analysis, dot-blot analysis, competitive PCR, RNase protection assays or in situ hybridization.

Flow Cytometry Assays Flow cytometry is a rapid and specific technique that provides complete cellular analysis, being used as a tool for understanding the regulation and interaction of cell systems, mainly based in the use of fluorescent antibodies. Accumulation and Efflux Assays Beyond flow cytometry, other accumulation and efflux assays are suitable for the screening of compounds that interfere with efflux transporters.

Western Blotting Western blotting or protein blotting or immunoblotting is an important technique used for the immunodetection of proteins post-electrophoresis, particularly those at low abundance [ ].

Transport Assays Across Polarized Cell Monolayers Transport assays are the most direct tool for the evaluation of transporter function and permeability of the test compound [ 1 ]. Ex Vivo Assays Many ABC carriers are constitutively expressed at the apical membrane of epithelial cells of different organs, including those that function as body barriers, such as the liver, brain, kidney and intestinal tract [ , ].

In Silico Studies for ABC Transporters Inducers and Activators Given the relevance of the ABC transporters in the toxicokinetics and pharmacokinetics, namely in the absorption, distribution BBB permeation and excretion processes, as well as their involvement in diverse pathophysiological conditions, the search for new modulators of these carrier proteins is of particular importance in both pharmacological and toxicological fields.

Conclusions In vitro and in vivo studies with inducers and activators of the ABC transporters have shown that the use of these compounds may be an effective antidotal pathway against xenobiotic-induced toxicity. Conflicts of Interest The authors declare no conflicts of interest.

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