Kinetic validation of the use of carboxydichlorofluorescein as a drug surrogate for MRP5-mediated transport
Introduction
Transporters play an important role in the entry and exit of drugs into and out of cells that ultimately determines the pharmacokinetics and pharmacodynamics of drugs. The ATP binding cassette (ABC) transporter superfamily contains 49 mammalian plasma membrane located, energy-driven efflux transporters. Several are drug transporters whose primary role is to efflux structurally diverse compounds, usually xenobiotic molecules, out of the cell and thereby to provide tissues a protective barrier against potentially harmful chemical agents (Kruh et al., 2001, Gottesman et al., 2002, Bodo et al., 2003). A number of these transporters efflux anti-cancer agents or their drug conjugates and play a role in drug sensitivity of tumors. P-glycoprotein (ABCB1) and multidrug resistance proteins (ABCC) are transporters that are expressed in normal and tumor tissues and are capable of influencing the efficacy of cancer chemotherapeutic treatments. P-glycoprotein was the first multidrug resistance protein to be characterized and preferentially transports cationic or neutral molecules out of the cell. Expression of P-glycoprotein in tumor tissue correlates with decreased responsiveness to treatment with chemotherapeutic agents, and expression in drug sensitive cells results in reduced sensitivity to a broad range of drugs. P-glycoprotein is also highly expressed in the intestine, liver, and kidney, as well as the blood–brain barrier, blood–testis barrier and placenta where the transporter can limit absorption and enhance elimination of xenobiotics (Fromm, 2004).
Multidrug resistance-associated proteins (MRP1-8, ABCC1-6, ABCC10, 11 respectively) are a related family of plasma membrane ABC efflux transporters. These family members preferentially transport negatively charged molecules that may result from conjugation or phosphorylation, or co-transport neutral compounds with anions such as GSH (Haimeur et al., 2004). Although MRP proteins transport organic anions, each has its own substrate specificity. When there is substrate overlap between transport proteins, the transporters are often distinguished by their affinities for the substrates. Most MRP proteins are able to confer resistance to otherwise drug sensitive cells, and tissue-specific expression of MRP proteins contributes to the absorption, elimination and/or barrier properties of the tissues.
MRP5 is expressed in most normal tissues, and is overexpressed in colon, lung, breast and pancreatic cancers (Kool et al., 1997, McAleer et al., 1999, Konig et al., 2005, Sandusky, 2002). When transfected into drug-sensitive cells, MRP5 confers resistance to antifolate drugs such as methotrexate (MTX) and pemetrexed (Alimta™), (Pratt et al., 2005) and to nucleoside-based drugs such as 6-mercaptopurine, 6-thioguanine, 9-(2-phosphonyl-methoxyethyl)adenine (PMEA), azidothymidine (AZT), cytosine arabinoside (AraC), 5-fluorouracil (5-FU), and gemcitabine (Wijnholds et al., 2000, Davidson et al., 2002, Wielinga et al., 2002, Pratt et al., 2005). MRP5-expressing inside-out membrane vesicles demonstrate direct transport of monophosphate metabolites of nucleoside-based drugs such as 5-FU and 6-thioguanine (Wielinga et al., 2002, Pratt et al., 2005).
5-Chloromethylfluorescein diacetate (CMFDA) is a non-fluorescent, membrane permeable compound that is hydrolyzed by esterases intracellularly to a thiol-reactive, fluorescent, negatively charged intermediate that is membrane impermeable. McAleer et al. (1999) reported reduced fluorescent labeling of MRP5-transfected cells after incubation with CMFDA and proposed that MRP5 mediated efflux of the fluorescent CMFDA hydrolysis product even though coincubation of the cells with probenecid, a non-specific inhibitor of MRPs, had no effect on cellular fluorescence. Carboxydichlorofluorescein (CDCF) is an impermeable fluorescent compound that resembles the hydrolysis product of CMFDA except the sulfhydryl reactive chloromethyl group is substituted by a carboxylic acid (Fig. 1). The present study demonstrates that MRP5 transports the fluorescent compound CDCF into inside-out MRP5-expressing membrane vesicles. Consistent with an earlier report that MRP5 confers resistance to methotrexate (MTX) and pemetrexed (Pratt et al., 2005), MRP5 also is shown to transport these two cytotoxic drugs into membrane vesicles (Wielinga et al., 2005). The transport of these two drugs and that of CDCF is characterized kinetically and compared. These studies validate the use of CDCF as a drug surrogate for the study of MRP5 mediated transport.
Section snippets
Materials
Cell culture medium and components were obtained from Gibco-BRL (Invitrogen, Carlsbad, CA, USA). Biocoat poly-d-lysine coated plates were from BD Biosciences, (Bedford, MA, USA) and Packard GF/B glass fiber plates were purchased from Perkin Elmer (Boston, MA, USA). 5-Chloromethylfluorescein diacetate (CMFDA, CellTracker Green) and 5-(and-6)-carboxy-2′,7′-dichlorofluorescein (CDCF, mixed isomers) were purchased from Molecular Probes (Invitrogen, Carlsbad, CA, USA). [3H]Pemetrexed was custom
Cellular accumulation of CMFDA
The cellular accumulation of CMFDA and retention of its fluorescent product was examined after incubation of stably transfected MRP5- and vector-HEK 293 cells with 2.5 μM CMFDA. As shown in Fig. 2, vector control cells accumulated approximately three-fold higher levels of the fluorescent hydrolysis product than did the MRP5-transfected cells. Treatment of both cell lines with increasing concentrations of probenecid, a non-specific inhibitor of MRP transporters (Haimeur et al., 2004), increased
Discussion
In these studies, we demonstrate for the first time the direct transport of pemetrexed and the fluorescent substrate CDCF by membrane vesicles expressing MRP5 and we confirm the recently reported transport of MTX by MRP5 (Wielinga et al., 2005). Kinetic characterization of these substrates reveals that CDCF is a higher affinity substrate in comparison to MTX, pemetrexed and other reported substrates of MRP5. In addition, CDCF has other properties which make it useful as a drug surrogate for
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2018, Experimental Eye ResearchCitation Excerpt :Although we are unsure as to why this may be, this release of GSH was able to be significantly blocked using Mrp inhibitors, demonstrating that GSH release is transporter-mediated, and not simply a result of GSH leakage from the lens (Qin et al., 1996). Due to the uncertainty concerning Mrp isoform specificity of MK571 (Jedlitschky et al., 2000; Luna-Tortos et al., 2010; Pratt et al., 2006; Reid et al., 2003), we were unable to determine the relative contributions of the two Mrp isoforms (Mrp1 and 5 (Umapathy et al., 2015);), which we previously localised in the rat lens, in mediating GSH release. Interestingly, Mrp1 is also able to mediate GSSG efflux, with GSSG transported more efficiently than GSH (Leier et al., 1996).
Quantification of biliary excretion and sinusoidal excretion of 5(6)-carboxy-2',7'-dichlorofluorescein (CDF) in cultured hepatocytes isolated from Sprague Dawley, Wistar and Mrp2-deficient Wistar (TR<sup>-</sup>) rats
2014, Toxicology in VitroCitation Excerpt :On the other hand, biliary excretion of CDF over the canalicular membrane could also be due to other transporters on the canalicular membrane e.g. P-gp, bcrp, mate1 and mdr3, as previous reports have shown that CDF is not a specific Mrp2 substrate (Nezasa et al., 2006; Pratt et al., 2006; Zamek-Gliszczynski et al., 2003). It seems likely that Mrp3 is responsible for the CDF efflux at the sinusoidal membrane of these TR− rat hepatocytes, as CDF is a substrate for Mrp3 and it is also up-regulated in TR− deficient rats (Pratt et al., 2006; Zamek-Gliszczynski et al., 2003). Future in vitro and in vivo studies are required to confirm this.