The objectives of this study were to determine whether there are differences in the in vitro permeability, pgp substrate profiles, and physicochemical properties of drugs for central nervous system cns and noncns indications, and whether these differences are useful criteria in. The efflux ratio of permeability b to aa to b is calculated. The pgpcoupled atp hydrolysis curves were fit in igor pro 6. Frontiers lysosomal pgpmdr1 confers drug resistance of. Passive permeability and pglycoproteinmediated efflux.
Natural flavonoids silymarin and quercetin improve the. The presence of p gp efflux transporter at the bbb may restrict the entry of several p gp substrates into the brain. More formally, it is an atpdependent efflux pump with broad substrate. Pglycoprotein pgp plays an important role in the in vivo disposition of a wide range of.
Mdckmdr1 permeability pglycoprotein assay cyprotex. The in vivo braintoplasma concentration ratio of vincristine in ddy mice was decreased by coadministration of 0. An assessment of the brain plasma area under the curve auc ratio in knockout mice as compared to wildtype mice has become a standard experimental methodology to determine whether p gp mediated efflux poses a possible obstacle to the activity of cns targeted drugs in vivo 1, 59, 60. Structural modifications that alter the pglycoprotein efflux. A similar model, previously used to describe apical efflux in caco.
To understand the relationship between drug binding and efflux, ett and stt were used to probe the structural basis of triptan transport. Pgp expression has been linked to the efflux of chemotherapeutic drugs in human cancers leading to multidrug resistance. P gp is overexpressed in many intrinsically resistant tumors leukemias, lymphomas, adult and. Osthol and curcumin as inhibitors of human pgp and multidrug efflux pumps of staphylococcus aureus. Efflux ratios obtained in vitro should be used for risk assessment in the early phases and once validated in vivo, can be used for progression of compounds through the screening cascade. Caco2 permeability, pglycoprotein transport ratios and. For efficient drug discovery and screening, it is necessary to simplify pglycoprotein p gp substrate assays and to provide in silico models that predict the transport potential of p gp. Wh en added to the caco2 cells alone, all compounds tested were. For cns delivery, a drug should ideally have an in vitro passive permeability 150 nms and not be a good b 3aa 3b ratio 2. Mdr1 or bcrp substrates exhibit a greater permeability in the b to a direction than the a to b direction and therefore have an efflux ratio greater than 2. Pluronic block copolymer, p85, inhibits the pglycoprotein pgp drug efflux system and increases the permeability of a broad spectrum of drugs in the bloodbrain barrier bbb. Overexpression of pgp was linked to multidrug resistance mdr in mammalian cell lines and human cancers, evoking intense interest first from molecular and cell biologists, and later, when purified pgp became available, from biochemists and biophysicists. Strategies for in vitro transporter testing and alignment. Pgp substrate efflux ratio, is more clinically relevant than qualitative classification.
Mechanism of pluronic effect on pglycoprotein efflux. Mdr1mdck permeability assay, in vitro drug metabolism. P gp is the efflux pump discovered in 1976 2 due to its expression in various types of mdr tumors 3. Predicting efflux ratios and bloodbrain barrier penetration from. There are two models, an artificial neural network. The efflux ratio er, defined as the ratio of papp in the ba direction to the. If the efflux ratio is greater than two then this indicates drug efflux. Perhaps the drug has a carrier for absorption if the efflux ratio equal to 1. More formally, it is an atpdependent efflux pump with broad substrate specificity. Melchior, the fluorosome company, worcester, ma 01605, usa frances j. Combining passive permeability with active efflux by pglycoprotein. Today this fascinating protein, which is proposed to operate as an atppowered drug efflux. Using nonlinear regression software scientist ver sion 2.
Abcb1, a member of the atpbinding cassette abc superfamily, exports structurally diverse hydrophobic compounds from the cell driven by atp hydrolysis. Multiple timepoint sampling is required in transcellular transport studies to accurately calculate the appropriate efflux ratio er. As shown in figure 5b, the efflux ratio of digoxin in untreated mdckiiwt cells er8. In each case, the efflux ratio of the compound decreases to unity in the appropriate ko cell line versus the wt cell line figures 1, 2, and 3. Simplified method to determine the efflux ratio on p. At a low pgp efflux ratio a high brain penetration and at a high pgp efflux ratio a low brain penetration is expected. The goal for cns drug discovery programs is to design compounds with. Comparing the efflux ratios generated in the presence and absence of a pgp inhibitor identifies whether the test article is a pgp substrate. Hepg2 human liver hepatocellular carcinoma, hep3b2 human hepatocellular carcinoma, h226 human lung squamous cell carcinoma, n87 human gastric.
The ratio may be optimized using in silico models friden et al. Pglycoprotein mediated efflux limits the transport of the novel anti. Strategies to assess bloodbrain barrier penetration. Efflux pumps generally consist of an outer membrane protein, middle periplasmic protein, inner membrane protein, and transmembrane duct. Efflux and uptake transporters involved in the disposition. In vitro pglycoprotein efflux ratio can predict the in. Functional interactions between pglycoprotein and cyp3a. Sharom guelphwaterloo centre for graduate work in chemistry and biochemistry, university of guelph, guelph, ontario canada n1g 2w1. In vitroin vivo extrapolation of key transporter activity at the blood.
If the efflux ratio is greater than or equal to 2 then this indicates drug efflux is. Mdr1mdck permeability assay, in vitro drug metabolism bioduro. Caco2 permeability assay, in vitro drug metabolism bioduro. The result is typically reported as an efflux ratio i. Mdckmdr1 permeability this assay model allows for an in vitro approach for evaluating possible interactions with the efflux protein pgp, by monitoring the permeability of a known substrate, using the mdckmdr1 cell line in the presence of the study compound. Predicting efflux ratios and bloodbrain barrier penetration from chemical structure. Pglycoprotein pgp inhibition measurement bmg labtech. In addition, cimetidine was identified as a dual substrate for both mdr1 and bcrp transporters using the single and double ko cell lines compared to wt figure 4. Assessing transport in both directions apical to basolateral ab and basolateral to apical ba across the cell monolayer enables an efflux ratio to be.
Additionally, the permeability for these compounds was measured in two in vitro. Membrane permeability and pglycoprotein pgp can be limiting factors for bloodbrain barrier penetration. In this study, we developed a simplified in vitro screening method to evaluate p gp substrates by unidirectional membrane transport in p gp overexpressing cells. Osthol and curcumin as inhibitors of human pgp and. Once the pshpgppsa ratio is selected as the criterion for pgp. Midazolam and felodipine had efflux ratios of 1 and, thus, were confirmed not to be pglycoprotein substrates. The efflux ratio of cpt in mdckiipgp in the presence of 0. Pgp surface expression, as determined by the immunofluorescence staining intensity ratio using the antibody 4e3. The ratio of the p app values bl to apap to bl provides a means to assess the permeability process.
Transcellular transport studies were conducted with 10 non. Efflux transport of pgp mechanism was similar to that of enzymatic reactions, except that. In transport studies only pgp blocker effectively inhibited the efflux of flz, which was. Pglycoprotein 1 permeability glycoprotein, abbreviated as p gp or pgp also known as multidrug resistance protein 1 mdr1 or atpbinding cassette subfamily b member 1 abcb1 or cluster of differentiation 243 cd243 is an important protein of the cell membrane that pumps many foreign substances out of cells.
Briefly, a panel of svr models was built by the libsvm package software. Pdf osthol and curcumin as inhibitors of human pgp and. Perhaps the drug has simple diffusion for transformation. Ratios 1 suggest an efflux transport process, approximately 1 passive diffusion, and pgp efflux ratio, and log octanolwater partition coefficients clogp in determining the extent of central nervous system cns penetration observed in vivo. Our study sought to develop a simplified method to determine the er in caco2 cells. Pglycoprotein pgp plays an important role in the in vivo disposition of a wide range of clinically relevant drugs. In vitro substrate assessment for p gp is frequently used to predict the in vivo relevance of p gp mediated efflux at the bbb. Dell, bmg labtech, 00 weston parkway, cary nc 275, usa introduction. The equation for the er was derived from a threecompartment model of apical to basal and basal to apical transport. Multidrugresistance phenotype and clinical responses to.
A compound is considered to be a pgp substrate when the efflux ratio in the absence of inhibitor is 1. Structural modifications that alter the pglycoprotein. The molecular docking studies of curcumin and osthol with the human pgp and s. Molecules free fulltext theoretical prediction of the complex p. Curcumin and osthol showed significant inhibition of the s. The caco2 transport assay showed large and significant changes in the measured efflux ratios of bazedoxifene when selective transporter inhibitors were applied. Sharom, department of molecular and cellular biology, university of guelph, guelph on canada n1g 2w1 e. Amplification curves were analyzed using lightcycler 480 basic software version. The transmembrane duct is located in the outer membrane of the cell. Thirtyone structurally diverse marketed central nervous system cnsactive drugs, one active metabolite, and seven noncnsactive compounds were tested in three pglycoprotein pgp in vitro assays. Models to study p gp interactions assay type tissues parameters comments bidirectional transport caco2 cells mdckmdr1 cells llcpk1mdr1cells net drug flux ratio of b to a and a to b directly measure efflux across cell barrier evaluation of p gp transport and inhibition. P app bap app ab is calculated by performing a bidirectional mdckmdr1 permeability assay where the transport of the compound is measured in the apical to basolateral direction as well as the basolateral to apical direction. A fast and simple method for measuring pglycoprotein pgp inhibition donald l.
Understand the potential of your compound to be a substrate of the pgp efflux transporter using our pgp substrate identification assay. The in vivo pgp efflux ratio is calculated by dividing the bp ratio of. The present results demonstrate that compounds often considered to be pgp. In vitro pglycoprotein efflux ratio can predict the in vivo brain penetration regardless of biopharmaceutics drug disposition classification system class. Phorbol 12myristate acetate inhibits pglycoprotein. Encoded by the multidrug resistance gene 1, mdr1 in humans, mdr1, pgp is found in the intestine, liver, kidney, and brain, and has been shown to transport compounds exhibiting a wide range of chemical diversity.
A bidirectional caco2 permeability assay is performed where the transport of the compound is measured in the apical to basolateral direction as well as the basolateral to apical direction. Studyofmetabolicenzymeeffluxtransporter relationswith in. The efflux ratio er was calculated from p app, bap app, ab tumor cell lines. A average efflux ratio and percentage of compounds defined as pgp. Reversing the resistance against frontline antibacterial drugs.
The role of intestinal efflux transporters in drug. In vitro pglycoprotein assays to predict the in vivo. From the presented data it becomes clear that brain penetration data are not always consistant with the measured efflux ratios yamazaki et al. Based on these equations, the efflux ratio er for any compound can be. It is encoded by mdr1 gene in humans and the mdr1a and mdr1b genes in rodents.
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