Although glucuronide and sulfate conjugates of many drugs and endogenous compounds undergo appreciable hepatic basolateral excretion into sinusoidal blood, the mechanisms that govern basolateral ...translocation of these hydrophilic metabolites have not been completely elucidated. In the present study, the involvement in this process of Mrp3 and Mrp4, two basolateral efflux transporters, was evaluated by analyzing the hepatic basolateral excretion of the glucuronide and sulfate metabolites of acetaminophen, 4-methylumbelliferone, and harmol in Abcc3(-/-) and Abcc4(-/-) mice using a cassette dosing approach. In the livers of Abcc3(-/-) and Abcc4(-/-) mice, the basolateral excretory clearance of acetaminophen sulfate was reduced approximately 20 and approximately 20%, 4-methylumbelliferyl sulfate was reduced approximately 50 and approximately 65%, and harmol sulfate was decreased approximately 30 and approximately 45%, respectively. The basolateral excretory clearance of acetaminophen glucuronide, 4-methylumbelliferyl glucuronide, and harmol glucuronide was reduced by approximately 96, approximately 85, and approximately 40%, respectively, in the livers of Abcc3(-/-) mice. In contrast, basolateral excretory clearance of these glucuronide conjugates was unaffected by the absence of Mrp4. These results provide the first direct evidence that Mrp3 and Mrp4 participate in the hepatic basolateral excretion of sulfate conjugates, although additional mechanism(s) are likely involved. In addition, they reveal that Mrp3 mediates the hepatic basolateral excretion of diverse glucuronide conjugates.
Methotrexate (MTX) is an anticancer agent that is widely used in a variety of human cancers including primary central nervous system lymphoma (PCNSL). Important pharmacological properties that ...directly bear on the use of MTX in PCNSL, such as mechanisms that govern its uptake into brain tumors, are poorly defined, but are amenable to investigation in mouse models. In order to pursue such preclinical pharmacological studies, a rapid and sensitive liquid chromatography-tandem mass spectrometry (LC/MS/MS) method for the determination of MTX and its metabolite, 7-hydroxymethotrexate (7-OH MTX) in plasma and microdialysate samples from brain tumors and cerebrospinal fluid (CSF) is needed. The plasma assay was based on 10
μl samples and following a protein precipitation procedure enabled direct injection onto a LC/MS/MS system using positive electrospray ionization. A column switching technique was employed for desalting and the clean-up of microdialysate samples from brain tissues.
The methods were validated for MTX and 7-OH MTX in both plasma and microdialysate samples from brain tumor and CSF, and produced lower limits of quantification (LLOQ) in plasma of 3.7
ng/ml for MTX and 7.4
ng/ml for 7-OH MTX, and in microdialysate samples of 0.7
ng/ml for both MTX and 7-OH MTX. The utility of the method was demonstrated by estimation of pharmacokinetic (PK) and brain distribution properties of MTX and 7-OH MTX in conscious mice. The method has the advantages of low sample volume, rapid clean-up, and the simultaneous measurement of MTX and 7-OH MTX in plasma and brain tissues allowing detailed PK studies to be completed in individual mice.
The disposition of fexofenadine, a commonly used antihistamine drug, is governed primarily by active transport. Biliary excretion of the parent compound is the major route of systemic clearance. ...Previous studies demonstrated that fexofenadine hepatic uptake is mediated by organic anion transporting polypeptides. Recently, we showed that in mice fexofenadine is excreted into bile primarily by multidrug resistance-associated protein (Mrp) 2 (Abcc2). In the present study, the roles of Mrp3 (Abcc3) and Mrp4 (Abcc4) in the hepatobiliary disposition of fexofenadine were examined in knockout mice using in situ liver perfusion. Compared with that in wild-type mice, basolateral excretion of fexofenadine was impaired, resulting in a approximately 50% decrease in perfusate recovery in Abcc3(-/-) mice; in contrast, fexofenadine hepatobiliary disposition was unaltered in Abcc4(-/-) mice. As expected, in Abcc2(-/-) mice, fexofenadine was redirected from the canalicular to the basolateral membrane for excretion. In Abcc2(-/-)/Abcc3(-/-) double-knockout mice, fexofenadine biliary excretion was impaired, but perfusate recovery was similar to that in wild-type mice and more than 2-fold higher than that in Abcc3(-/-) mice, presumably due to compensatory basolateral transport mechanism(s). These results demonstrate that multiple transport proteins are involved in the hepatobiliary disposition of fexofenadine. In addition to Mrp2 and Mrp3, other transport proteins play an important role in the biliary and hepatic basolateral excretion of this zwitterionic drug.
The MRP subfamily of ABC transporters currently consists of at least six members, several of which have been demonstrated to transport amphipathic anions and to confer in vitro resistance to ...chemotherapeutic agents. In searching the data bases we identified the product of a cDNA sequencing project that bears significant similarity to MRP subfamily transporters. In this report the predicted coding sequence, protein product and expression pattern of this cDNA, termed MRP7, are analyzed. The MRP7 cDNA sequence encodes a 1492 amino acid ABC transporter whose structural architecture resembles that of MRP1, MRP2, MRP3, and MRP6, in that its transmembrane helices are arranged in three membrane spanning domains. However, in contrast to the latter transporters, a conserved N-linked glycosylation site is not found at the N-terminus of MRP7. Comparisons of the MRP7 amino acid sequence indicated that while it is most closely related to other MRP subfamily members, its degree of relatedness is the lowest of any of the known MRP-related transporters. The integrity of the predicted MRP7 coding sequence was confirmed by the synthesis of an approximately 158 kDa protein in reticulocyte lysates programmed with the MRP7 cDNA. While MRP7 transcript was detected in a variety of tissues by RT/PCR, it was not readily detectable by RNA blot analysis, suggesting that it is expressed at low levels in these tissues. Fluorescence in situ hybridization indicated that MRP7 maps to chromosome 6p12-21, in proximity to several genes associated with glutathione conjugation and synthesis. On the basis of these findings and evolutionary cluster analysis, we conclude that MRP7 is a member of the MRP subfamily of amphipathic anion transporters.
Nucleoside-based analogues are mainstays in the treatment of cancer, viral infections, and inflammatory diseases. Recent studies showing that the ATP-binding cassette transporter, multidrug ...resistance protein 4, is able to efflux nucleoside and nucleotide analogues from transfected cells suggests that the pump may affect the efficacy of this class of agents. However, the in vivo pharmacologic functions of the pump are largely unexplored. Here, using Mrp4(-/-) mice as a model system, and the nucleotide analogue, 9'-(2'-phosphonylmethoxyethyl)-adenine (PMEA) as a probe, we investigate the ability of Mrp4 to function in vivo as an endogenous resistance factor. In the absence of alterations in plasma PMEA levels, Mrp4-null mice treated with PMEA exhibit increased lethality associated with marked toxicity in several tissues. Affected tissues include the bone marrow, spleen, thymus, and gastrointestinal tract. In addition, PMEA penetration into the brain is increased in Mrp4(-/-) mice. These findings indicate that Mrp4 is an endogenous resistance factor, and that the pump may be a component of the blood-brain barrier for nucleoside-based analogues. This is the first demonstration that an ATP-binding cassette transporter can affect in vivo tissue sensitivity towards this class of agents.
The majority of gastrointestinal stromal tumors (GISTs) are characterized by oncogenic gain-of-function mutations in the receptor tyrosine kinase (RTK) c-KIT with a minority in PDGFRa. Therapy for ...GISTs has been revolutionized by the use of the selective tyrosine kinase inhibitor imatinib mesylate (IM). For the subset (~10-15%) of GISTs that lack oncogenic mutations in these receptors, the genetic changes driving tumorigenesis are unknown. We recently reported that the gene encoding the insulin-like growth factor 1 receptor (IGF-1R) is amplified in a subset of GISTs, and the IGF-1R protein is over-expressed in WT and pediatric GISTs. In this report we present a more complete picture of the involvement of components of the insulin-like growth factor-signaling pathway in the pathogenesis of GISTs. We also discuss how the IGF pathway may provide additional molecular targets for the treatment of GISTs that respond poorly to IM therapy.
Multidrug resistance-associated protein (MRP)1 and canalicular multispecific organic anion transporter (cMOAT)/MRP2 are ATP-binding cassette (ABC) transporters that confer resistance to natural ...product cytotoxic drugs. We recently described the complete coding sequences of four human MRP/cMOAT subfamily members and found that, among these proteins, MRP3/MOAT-D is most closely related to MRP1 (58% identity; M. G. Belinsky and G. D. Kruh, Br. J. Cancer, 80: 1342-1349, 1999). In the present study, we sought to determine whether MRP3 is capable of conferring resistance to cytotoxic drugs. To address this question, human embryonic kidney 293 cells were transfected with an MRP3 expression vector, and the drug resistance phenotype of the transfected cells was analyzed. The MRP3-transfected cells displayed approximately 4-fold resistance to etoposide and approximately 2-fold resistance to vincristine, compared with control transfected cells. In addition, approximately 1.7-fold resistance was observed for the antimetabolite methotrexate. Increased resistance was not observed for several other natural product agents, including anthracyclines and Taxol. The MRP-transfected cells exhibited reduced accumulation of radiolabeled etoposide, consistent with the operation of a plasma membrane efflux pump. These results indicate that MRP3 confers resistance to some anticancer agents but that its resistance pattern is distinct from the resistance patterns of other ABC transporters involved in resistance to natural product chemotherapeutic agents.
Multidrug resistance-associated protein (MRP) and canalicular multispecific organic anion transporter (cMOAT) are transporter proteins that pump organic anions across cellular membranes and have been ...linked to resistance to cytotoxic drugs. We previously identified MOAT-B, an MRP/cMOAT-related transporter, by use of a polymerase chain reaction approach. However, analysis of expressed sequence tag (EST) databases indicated that there might be additional MRP/cMOAT-related transporters. To further define the MRP/cMOAT subfamily of transporters, we used EST probes to isolate complementary DNAs for two related transporter proteins, MOAT-C and MOAT-D.
MOAT-C and MOAT-D expression patterns in human tissues were determined by RNA blot analysis, and chromosomal localization of the genes was determined by fluorescence in situ hybridization.
MOAT-C is predicted to encode a 1437-amino-acid protein that, among eukaryotic transporters, is most closely related to MRP, cMOAT, and MOAT-B (about 36% identity). However, MOAT-C is less related to MRP and cMOAT than MRP and cMOAT are to each other (about 48% identity). Like MOAT-B, MOAT-C lacks an N-terminal membrane-spanning domain, indicating that the topology of this protein is similarly distinct from that of MRP and cMOAT. MOAT-D is predicted to encode a 1527-amino-acid protein that is the closest known relative of MRP (about 58% identity). MOAT-D is also highly related to cMOAT (about 47% identity). The presence of an N-terminal membrane-spanning domain indicates that the topology of MOAT-D is quite similar to that of MRP and cMOAT. MOAT-C transcripts are widely expressed in human tissues; however, MOAT-D transcript expression is more restricted. The MOAT-C and MOAT-D genes are located at chromosomes 3q27 and 17q21.3, respectively.
On the basis of amino acid identity and protein topology, the MRP/cMOAT transporter subfamily falls into two groups; the first group consists of MRP, cMOAT, and MOAT-D, and the second group consists of MOAT-B and MOAT-C.
The MRP subfamily of ABC transporters from mammals consists of at least seven members, six of which have been implicated in the transport of amphipathic anions. MRP1, MRP2, and MRP3 bear a close ...structural resemblance, confer resistance to a variety of natural products as well as methotrexate, and have the facility for transporting glutathione and glucuronate conjugates. MRP1 is a ubiquitously expressed efflux pump for the products of phase II of xenobiotic detoxification, while MRP2, whose hereditary deficiency results in Dubin-Johnson syndrome, functions to extrude organic anions into the bile. MRP3 is distinguished by its capacity to transport the monoanionic bile constituent glycocholate, and may function as a basolateral back-up system for the detoxification of hepatocytes when the usual canalicular route is impaired by cholestatic conditions. MRP4 and MRP5 resemble each other more closely than they resemble MRPs 1-3 and confer resistance to purine and nucleotide analogs which are either inherently anionic, as in the case of the anti-AIDS drug PMEA, or are phosphorylated and converted to anionic amphiphiles in the cell, as in the case of 6-MP. Given their capacity for transporting cyclic nucleotides, MRP4 and MRP5 have also been implicated in a broad range of cellular signaling processes. The drug resistance activity and physiological substrates of MRP6 are unknown. However, its hereditary deficiency results in pseudoxanthoma elasticum, a multisystem disorder affecting skin, eyes, and blood vessels. It is hoped that elucidation of the resistance profiles and physiological functions of the different members of the MRP subfamily will provide new insights into the molecular basis of clinical drug resistance and spawn new strategies for combating this phenomenon.
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