Characterization of functional assays of multidrug resistance P-glycoprotein transport activity

Bosch, Irene; Crankshaw, Carolyn L.; Piwnica‐Worms, David; Croop, James M.

doi:10.1038/sj.leu.2400695

Cited by 29 publications

(17 citation statements)

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“…The K i for reserpine and cyclosporin A blocking Pgp uptake of vinblastine in L-MDR1 cells was very similar to the K i for enhancing vinblastine or daunomycin accumulation in P388 cells expressing MDR1 (Lan et al, 1996). For most drugs, lower concentrations of reversal agent were required to inhibit the uptake of the radiolabeled probe [ 3 H]VBL, compared with the fluorescent probe calcein-AM, similar to another report comparing Pgp functional assays (Bosch et al, 1997).…”

Section: More Traditional Assay Of Measuring Pgp Inhibition Is To Anasupporting

confidence: 63%

Interaction of Cytochrome P450 3A Inhibitors with P-Glycoprotein

Yasuda

Lan²,

Sanglard³

et al. 2002

J Pharmacol Exp Ther

128

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Many clinically important drug interactions occur due to inhibition of human liver cytochrome P450 3A (CYP3A) metabolism. The drug efflux pump P-glycoprotein (Pgp) can be an additional locus contributing to these drug interactions because there is overlap in drugs that are substrates for both proteins. We screened a number of CYP3A inhibitors (macrolide antibiotics, azole antifungals, and ergotpeptides) for their ability to interact with Pgp, compared with prototypical Pgp inhibitors. We used cell lines expressing human, mouse, and rat mdr1 genes. Pgp antagonism was defined by interactions of the drugs with four cell lines (LLC-PK1, L-MDR1, L-mdr1a, and L-mdr1b) using a microfluorometric calcein-AM assay and characterized for their inhibitor constant (K i ) toward calcein-AM. The compounds were further defined for their ability to inhibit MDR1 by their effect on vinblastine accumulation into L-MDR1 cells. Representative compounds from each class of drugs were further tested as Pgp substrates, defined by the ability of human Pgp or mouse mdr1a/Pgp to transport them across a polarized kidney epithelial cell in vitro. These same compounds were administered radiolabeled in vivo to mdr1a (ϩ/ϩ) and (Ϫ/Ϫ) mice and the distribution of radioactivity compared. The results are summarized as follows: 1) Some drug interactions with Pgp were substrate-and/or assay-dependent. 2) Ergot alkaloids were identified as a class of MDR1/Pgp chemosensitizers. 3) The Ergot alkaloids revealed species differences in the structureactivity relationships for inhibition of Pgp. Simultaneous inhibition of Pgp by many CYP3A inhibitors contributes to human variation in the extent of drug-drug interactions.

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Section: More Traditional Assay Of Measuring Pgp Inhibition Is To Anasupporting

confidence: 63%

Interaction of Cytochrome P450 3A Inhibitors with P-Glycoprotein

Yasuda

Lan²,

Sanglard³

et al. 2002

J Pharmacol Exp Ther

128

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“…This was achieved thanks to a high sensitivity, dynamic range, and real-time detection without the necessity to separate cells from the reaction mixture, in contrast to methods employing radiolabeled or other fluorescent substrates, including anthracyclines and rhodamine derivatives (3,23,24,27).…”

Section: Discussionmentioning

confidence: 99%

“…9; Table 2) was highest in the most active aminophenothiazines. These contained a Cl atom, whereas substitution with CF 3 and H strongly decreased the inhibition potency in both series containing butylene or propylene acyl chains. The substituents at position 2 also had a strong influence on the sensitivity of particular transporters to inhibition.…”

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confidence: 99%

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New High-Throughput Screening Assay To Reveal Similarities and Differences in Inhibitory Sensitivities of Multidrug ATP-Binding Cassette Transporters

Kołaczkowski

Kołaczkowska

Motohashi

et al. 2009

Antimicrob Agents Chemother

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Cdr1p is the major ATP-binding cassette multidrug transporter conferring resistance to azoles and other antifungals in Candida albicans. In this study, the identification of new Cdr1p inhibitors by use of a newly developed high-throughput fluorescence-based assay is reported. The assay also allowed monitoring of the activity and inhibition of the related transporters Pdr5p and Snq2p of Saccharomyces cerevisiae, which made it possible to compare its performance with those of previously established procedures. A high sensitivity, resulting from a wide dynamic range, was achieved upon high-level expression of the Cdr1p, Pdr5p, and Snq2p transporters in an S. cerevisiae strain in which the endogenous interfering activities were further reduced by genetic manipulation. An analysis of a set of therapeutically used and newly synthesized phenothiazine derivatives revealed different pharmacological profiles for Cdr1p, Pdr5p, and Snq2p. All transporters showed similar sensitivities to M961 inhibition. In contrast, Cdr1p was less sensitive to inhibition by fluphenazine, whereas phenothiazine selectively inhibited Snq2p. The inhibition potencies measured by the new assay reflected the ability of the compounds to potentiate the antifungal effect of ketoconazole (KTC), which was detoxified by the overproduced transporters. They also correlated with the 50% inhibitory concentration for inhibition of Pdr5p-mediated transport of rhodamine 6G in isolated plasma membranes. The most active derivative, M961, potentiated the activity of KTC against an azole-resistant CDR1-overexpressing C. albicans isolate.Candida yeasts are the fourth most common pathogens responsible for systemic bloodstream infections. Candida albicans is the most frequently isolated species, contributing to Ͼ50% of cases (41). It generally shows little permeability to a large variety of toxic compounds, which is believed to result to a large degree from the existence of an active permeability barrier (42). Cdr1p and Cdr2p are two homologous ATPbinding cassette (ABC) multidrug resistance (MDR) transporters of broad specificity that confer resistance to the most widely used azole antifungals as well as to terbinafine, amorolfine, and many other metabolic inhibitors (43,48). CDR1 is constitutively expressed in azole-sensitive isolates, where it modifies the intrinsic level of susceptibility to antifungals, as its inactivation by deletion increases sensitivity (47). The effectiveness of the small number of antifungals available for the treatment of life-threatening systemic mycoses is further reduced by overexpression of both CDR1 and CDR2 in many azole-resistant clinical isolates (46, 59).Cdr1p and Cdr2p are structural and functional homologues of the Pdr5p and Snq2p MDR transporters of the model yeast Saccharomyces cerevisiae (25,29,50). A large-scale screening of Pdr5p substrate specificity identified it as the most important MDR ABC transporter, conferring resistance to most classes of currently available antifungals and other xenobiotics, with some overlap in sp...

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“…Cellular Accumulation of Tc-Sestamibi-Transport function and modulation of class I Pgp were assayed with 99 m Tc-Sestamibi as described previously (26,27). Data are reported as fmol of Tc-Sestamibi (mg of protein)…”

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confidence: 99%