Minor Compensatory Changes in SAGE Mdr1a (P-gp), Bcrp, and Mrp2 Knockout Rats Do Not Detract from Their Utility in the Study of Transporter-Mediated Pharmacokinetics

Zamek‐Gliszczynski, Maciej J.; Goldstein, Keith M.; Paulman, April; Baker, Thomas K.; Ryan, Timothy P.

doi:10.1124/dmd.113.051409

Cited by 17 publications

(21 citation statements)

References 20 publications

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“…Up to now, only limited literature on the brain penetration of P-gp substrates in Mdr1a knockout rats exists. Zamek-Gliszczynski et al (2013) showed a 4-fold increase of paclitaxel brain partitioning in Mdr1a knockout rats. Similarly to the results in this study for WEB 2086, no increase of the brain distribution of paclitaxel was observed in Bcrp knockout rats.…”

Section: Resultsmentioning

confidence: 93%

“…Thus, lack of P-gp at the BBB on a functional level was demonstrated in these knockout rats. Only minor and most probably functionally irrelevant compensatory changes of the respective knockout strains were reported (Zamek-Gliszczynski et al, 2013). Up to now, only limited literature on the brain penetration of P-gp substrates in Mdr1a knockout rats exists.…”

Section: Resultsmentioning

confidence: 99%

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Brain Penetration of WEB 2086 (Apafant) and Dantrolene in Mdr1a (P-Glycoprotein) and Bcrp Knockout Rats

Fuchs

Kishimoto

Ganßer

et al. 2014

Drug Metabolism and Disposition

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Transporter gene knockout rat models are attracting increasing interest for mechanistic studies of new drugs as transporter substrates or inhibitors in vivo. However, limited data are available on the functional validity of such models at the blood-brain barrier. Therefore, the present study evaluated Mdr1a [P-glycoprotein (P-gp)], Bcrp, and combined Mdr1a/Bcrp knockout rat strains for the influence of P-gp and breast cancer resistance protein (BCRP) transport proteins on brain penetration of the selective test substrates [ .5-fold in double Mdr1a/Bcrp knockouts, and 7.3-fold in zosuquidar-treated wild-type rats, but was unchanged in Bcrp knockout rats. Mean BPR of dantrolene increased 3.3-fold in Bcrp knockouts and 3.9-fold in double Mdr1a/Bcrp knockouts compared with wild type, but was unchanged in the Mdr1a knockouts. The human intestinal CaCo-2 cell bidirectional transport system in vitro confirmed the in vivo finding that [ 14 C]WEB 2086 is a substrate of P-gp but not of BCRP. Therefore, Mdr1a, Bcrp, and combined Mdr1a/Bcrp knockout rats provide functional absence of these efflux transporters at the blood-brain barrier and are a suitable model for mechanistic studies on the brain penetration of drug candidates.

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Section: Resultsmentioning

confidence: 93%

Section: Resultsmentioning

confidence: 99%

Brain Penetration of WEB 2086 (Apafant) and Dantrolene in Mdr1a (P-Glycoprotein) and Bcrp Knockout Rats

Fuchs

Kishimoto

Ganßer

et al. 2014

Drug Metabolism and Disposition

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“…In contrast, the accumulation of Hg 2+ in the OSOM was similar in SD and bcrp −/− rats exposed to 0.5 or 1.5 μmol • kg −1 HgCl 2 . Since some proteins have been shown to be upregulated in bcrp −/− rats (Zamek-Gliszczynski et al , 2013), we initially postulated that the expression of Mrp2 may also be increased in order to compensate for the loss of Bcrp activity. Surprisingly, our Western blot data suggest that Mrp2 protein is not greater in bcrp −/− rats.…”

Section: 0 Discussionmentioning

confidence: 99%

Toxicological significance of renal Bcrp: Another potential transporter in the elimination of mercuric ions from proximal tubular cells

Bridges

Zalups

Joshee

2015

Toxicology and Applied Pharmacology

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Secretion of inorganic mercury (Hg2+) from proximal tubular cells into the tubular lumen has been shown to involve the multidrug resistance-associated protein 2 (Mrp2). Considering similarities in localization and substrate specificity between Mrp2 and the breast cancer resistance protein (Bcrp), we hypothesize that Bcrp may also play a role in the proximal tubular secretion of mercuric species. In order to test this hypothesis, the uptake of Hg2+ was examined initially using inside-out membrane vesicles containing Bcrp. The results of these studies suggest that Bcrp may be capable of transporting certain conjugates of Hg2+. To further characterize the role of Bcrp in the handling of mercuric ions and in the induction of Hg2+-induced nephropathy, Sprague-Dawley and Bcrp knockout (bcrp−/−) rats were exposed intravenously to a non-nephrotoxic (0.5 μmol • kg−1), a moderately nephrotoxic (1.5 μmol • kg−1) or a significantly nephrotoxic (2.0 μmol • kg−1) dose of HgCl2. In general, the accumulation of Hg2+ was greater in organs of bcrp−/− rats than in Sprague-Dawley rats, suggesting that Bcrp may play a role in the export of Hg2+ from target cells. Within the kidney, cellular injury and necrosis was more severe in bcrp−/− rats than in controls. The pattern of necrosis, which was localized in the inner cortex and the outer stripe of the outer medulla was significantly different from that observed in Mrp2-deficient animals. These findings suggest that Bcrp may be involved in the cellular export of select mercuric species and that its role in this export may differ from that of Mrp2.

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“…Our data suggest little if any impact on the expression level of these transporters; however, only a few genes were examined in the present in vitro study, and the possibility of compensation at the protein expression level cannot be definitively ruled out. Comparative analyses have been carried out in rat models in which P-gp, BCRP, or MRP2 have been knocked out using ZFN technology (Chu et al, 2012;Huang et al, 2012;Zamek-Gliszczynski et al, 2013). ZamekGliszczynski et al (2013) reported that expression analyses of a set of 112 genes relevant to absorption, distribution, metabolism, and elimination in liver, kidney, intestine, and brain tissues of the three KO rat lines demonstrated only modest compensatory changes and did not preclude their general application to study transporter-mediated pharmacokinetics.…”

Section: Discussionmentioning

confidence: 99%

Zinc Finger Nuclease–Mediated Gene Knockout Results in Loss of Transport Activity for P-Glycoprotein, BCRP, and MRP2 in Caco-2 Cells

Sampson¹,

Brinker²,

Pratt³

et al. 2015

Drug Metabolism and Disposition

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Membrane transporters P-glycoprotein [P-gp; multidrug resistance 1 (MDR1)], multidrug resistance-associated protein (MRP) 2, and breast cancer resistance protein (BCRP) affect drug absorption and disposition and can also mediate drug-drug interactions leading to safety/toxicity concerns in the clinic. Challenges arise with interpreting cell-based transporter assays when substrates or inhibitors affect more than one actively expressed transporter and when endogenous or residual transporter activity remains following overexpression or knockdown of a given transporter. The objective of this study was to selectively knock out three drug efflux transporter genes (MDR1, MRP2, and BCRP), both individually as well as in combination, in a subclone of Caco-2 cells (C2BBe1) using zinc finger nuclease technology. The wild-type parent and knockout cell lines were tested for transporter function in Transwell bidirectional assays using probe substrates at 5 or 10 mM for 2 hours at 37°C. P-gp substrates digoxin and erythromycin, BCRP substrates estrone 3-sulfate and nitrofurantoin, and MRP2 substrate 5-(and-6)-carboxy-29,79-dichlorofluorescein each showed a loss of asymmetric transport in the MDR1, BCRP, and MRP2 knockout cell lines, respectively. Furthermore, transporter interactions were deduced for cimetidine, ranitidine, fexofenadine, and colchicine. Compared with the knockout cell lines, standard transporter inhibitors showed substrate-specific variation in reducing the efflux ratios of the test compounds. These data confirm the generation of a panel of stable Caco-2 cell lines with single or double knockout of human efflux transporter genes and a complete loss of specific transport activity. These cell lines may prove useful in clarifying complex drug-transporter interactions without some of the limitations of current chemical or genetic knockdown approaches.

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Minor Compensatory Changes in SAGE Mdr1a (P-gp), Bcrp, and Mrp2 Knockout Rats Do Not Detract from Their Utility in the Study of Transporter-Mediated Pharmacokinetics

Cited by 17 publications

References 20 publications

Brain Penetration of WEB 2086 (Apafant) and Dantrolene in Mdr1a (P-Glycoprotein) and Bcrp Knockout Rats

Brain Penetration of WEB 2086 (Apafant) and Dantrolene in Mdr1a (P-Glycoprotein) and Bcrp Knockout Rats

Toxicological significance of renal Bcrp: Another potential transporter in the elimination of mercuric ions from proximal tubular cells

Zinc Finger Nuclease–Mediated Gene Knockout Results in Loss of Transport Activity for P-Glycoprotein, BCRP, and MRP2 in Caco-2 Cells

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