Dose-dependent disposition of methotrexate in Abcc2 and Abcc3 gene knockout murine models.

Methotrexate (MTX) is a substrate for numerous human ATP-binding cassette (ABC) efflux transporters, yet the impact of these transporters on MTX pharmacokinetics (PK) over a large dose range has not been examined. To investigate the effects of two transporters-ABC subfamily C member 2 (Abcc2; multidrug resistance protein 2) and ABC subfamily C member 3 (Abcc3; multidrug resistance protein 3)-involved in MTX hepatobiliary disposition in vivo, MTX plasma, urine, and feces concentrations were analyzed after 10, 50, and 200 mg/kg i.v. doses to groups of wild type (WT), Abcc2(-/-), and Abcc3(-/-) mice. The absence of Abcc2 caused a decrease in total clearance of MTX relative to WT mice at all dose levels yet was accompanied by compensatory increases in renal excretion and metabolism to 7-hydroxymethotrexate (7OH-MTX). In Abcc3(-/-) mice, total clearance was elevated at the two lower dose levels and was attributed to stimulation of biliary excretion and confirmed by elevated fecal excretion; however, at the high 200 mg/kg dose, clearance was severely retarded and could be attributed to hepatotoxicity because conversion to 7OH-MTX was diminished. The findings confirmed that both Abcc2 and Abcc3 significantly influenced the PK properties of MTX, and depending on the MTX dose and strain, alternate elimination pathways were elicited and saturable.

Contribution of Abcc10 (Mrp7) to in vivo paclitaxel resistance as assessed in Abcc10(-/-) mice.

Recently, we reported that the ATP-binding cassette transporter 10 (ABCC10), also known as multidrug resistance protein 7 (MRP7), is able to confer resistance to a variety of anticancer agents, including taxanes. However, the in vivo functions of the pump have not been determined to any extent. In this study, we generated and analyzed Abcc10(-/-) mice to investigate the ability of Abcc10 to function as an endogenous resistance factor. Mouse embryo fibroblasts derived from Abcc10(-/-) mice were hypersensitive to docetaxel, paclitaxel, vincristine, and cytarabine (Ara-C) and exhibited increased cellular drug accumulation, relative to wild-type controls. Abcc10(-/-) null mice treated with paclitaxel exhibited increased lethality associated with neutropenia and marked bone marrow toxicity. In addition, toxicity in spleen and thymus was evident. These findings indicate that Abcc10 is dispensable for health and viability and that it is an endogenous resistance factor for taxanes, other natural product agents, and nucleoside analogues. This is the first demonstration that an ATP-binding cassette transporter other than P-glycoprotein can affect in vivo tissue sensitivity toward taxanes.

Up-regulation of P-glycoprotein confers acquired resistance to 6-mercaptopurine in human chronic myeloid leukemia cells.

To investigate the mechanisms of cellular resistance to 6-mercaptopurine (6-MP) in chronic myeloid leukemia (CML), a 6-MP resistant cell line (K562-MP5) was established by stepwise selection of the CML cell line (K562). The results of the drug sensitivity analysis of the K562-MP5 cell line revealed the cells to be 339-fold more resistant to 6-MP compared with the parental K562 cells. K562-MP5 cells exhibited decreased accumulation and increased efflux of [(14)C]6-MP and its metabolites. In addition, K562-MP5 cells showed increased [(3)H]MTX transport. K562-MP5 cells over-expressed P-glycoprotein (P-gp) and up-regulated MDR1 mRNA levels. Taken together, these results suggest that the up-regulation of P-gp, which contributes to the decreased accumulation by increasing the efflux of 6-MP and its metabolites, underlies the mechanism of 6-MP resistance in K562 cells.

Influence of blood-brain barrier efflux pumps on the distribution of vincristine in brain and brain tumors.

Vincristine (VCR) is efficacious in some but not all brain cancers and an established substrate of Pgp and Mrp1. However, the extent to which such transporters affect the VCR penetration through the blood-brain barrier (BBB) is poorly understood. To evaluate the role of Pgp and Mrp1 in VCR CNS distribution, VCR concentrations were analyzed under steady-state conditions in normal brain, brain tumor, and bone marrow in wild-type (WT), Mrp1 ko (mrp1-/-), Pgp ko (mdr1a-/-:mdr1b-/-), and TKO (mdr1a-/-:mdr1b-/-:mrp1-/-) mice. VCR normal brain partition coefficients (i.e. tissue/plasma VCR concentrations) in TKO mice were greater than those in WT mice at both targeted 10 and 50 ng/mL plasma VCR concentrations, and ranged from 1.3- to 3.6-fold. VCR brain tumor partition coefficients in Mrp1 mice were greater than WT mice at both doses, being 1.5- and 2.4-fold higher at low and high doses, respectively. TKO mice also showed elevated VCR brain tumor penetration with a brain tumor partition coefficient of 1.9-fold greater than that in WT mice at the high-dose level. The bone marrow partition coefficient in Mrp1 ko mice was 1.65-fold greater than that in WT mice. Within strain comparisons revealed that VCR brain tumor concentrations were significantly greater than normal brain in all strains, ranging from 9- to 40-fold. These findings indicate that disruption of the BBB caused the largest enhancement in VCR tumor concentrations, yet the absence of Mrp1 on the brain tumor vasculature could enhance the penetration compared with that in normal brain.

Expression of ABCC-type nucleotide exporters in blasts of adult acute myeloid leukemia: relation to long-term survival.

PURPOSE: Successful treatment of acute myeloid leukemia (AML) remains a therapeutic challenge, with a high percentage of patients suffering from persistent or relapsed disease. Resistance to drug therapy can develop from increased drug export and/or altered intracellular signaling. Both mechanisms are mediated by the efflux transporters ABCC4 (MRP4), ABCC5 (MRP5), and ABCC11 (MRP8), which are involved in cellular efflux of endogenous signaling molecules (e.g., cyclic adenosine 3', 5'-monophosphate and cyclic guanosine 3',5'-monophosphate) and nucleoside analogues. The nucleoside analogue cytosine arabinoside (AraC) is administered to all patients with AML. EXPERIMENTAL DESIGN: Expression of ABCC transporters MRP4, MRP5, and MRP8 in blast samples from 50 AML patients was investigated by real-time reverse transcription-PCR analysis and correlated with clinical outcome measures. Accumulation of radiolabeled AraC, transport of AraC metabolites, and AraC cytotoxicity were analyzed in MRP8-transfected LLC-PK1 cells. RESULTS: Regression analysis revealed that high expression of MRP8 is associated with a low probability of overall survival assessed over 4 years (P<0.03). MRP8-transfected LLC-PK1 cells accumulated reduced intracellular levels of AraC (63% of the parental vector-transfected LLC-PK1 control cells) as well as AraC metabolites. Furthermore, AraC monophosphate was transported by MRP8-enriched membrane vesicles (116+/-6 versus 65+/-13 pmol/mg/10 minutes by control vesicles), and MRP8-transfected cells were resistant to AraC. CONCLUSION: These data suggest that MRP8 is differentially expressed in AML blasts, that expression of MRP8 serves as a predictive marker for treatment outcome in AML, and that efflux of AraC metabolites by MRP8 is a mechanism that contributes to resistance of AML blasts.

Human multidrug resistance protein 7 (ABCC10) is a resistance factor for nucleoside analogues and epothilone B.

Multidrug resistance protein 7 (MRP7; ABCC10) is an ATP-binding cassette transporter which is able to transport amphipathic anions and confer resistance to docetaxel and, to a lesser extent, vincristine and paclitaxel. Whereas some detail on the resistance profile of MRP7 is known, the activities of the pump have not been completely determined. Here, it is shown by the analysis of MRP7-transfected HEK293 cells that, in addition to natural product agents, MRP7 is also able to confer resistance to nucleoside-based agents, such as the anticancer agents cytarabine (Ara-C) and gemcitabine, and the antiviral agents 2',3'-dideoxycytidine and PMEA. Consistent with the operation of an efflux pump, expression of MRP7 reduced the accumulation of Ara-C and PMEA. In addition, MRP7 is also able to confer resistance to the microtubule-stabilizing agent epothilone B. Ectopic expression of MRP7 in mouse embryo fibroblasts deficient in P-glycoprotein and Mrp1 revealed that MRP7 has a broad resistance profile for natural product agents. In this drug-sensitive cellular background, MRP7 conferred high levels of resistance to docetaxel (46-fold), paclitaxel (116-fold), SN-38 (65-fold), daunorubicin (7.5-fold), etoposide (11-fold), and vincristine (56-fold). Buthionine sulfoximine did not attenuate MRP7-conferred resistance to docetaxel or Ara-C. These experiments indicate that the resistance capabilities of MRP7 include nucleoside-based agents and a range of natural product anticancer agents that includes nontaxane antimicrotubule agents that are not susceptible to P-glycoprotein-mediated transport and that, unlike MRP1 and MRP2, MRP7-mediated drug transport does not involve glutathione.

Cepharanthine is a potent reversal agent for MRP7(ABCC10)-mediated multidrug resistance.

Multidrug resistance protein 7 (MRP7; ABCC10) is an ABC transporter that confers resistance to anticancer agents such as the taxanes. We previously reported that several inhibitors of P-gp and MRP1 were able to inhibit the in vitro transport of E(2)17betaG by MRP7 in membrane vesicles transport assays. However, compounds that are able to reverse MRP7-mediated cellular resistance have not been identified. In this study, we examined the effects of cepharanthine (6',12'-dimethoxy-2,2'-dimethyl-6,7-[methylenebis(oxy)]oxyacanthan), an herbal extract isolated from Stephania cepharantha Hayata, to reverse paclitaxel resistance in MRP7-transfected HEK293 cells. Cepharanthine, at 2microM, completely reversed paclitaxel resistance in MRP7-transfected cells. In contrast, the effect of cepharanthine on the parental transfected cells was significantly less than that on the MRP7-transfected cells. In addition, cepharanthine significantly increased the accumulation of paclitaxel in MRP7-transfected cells almost to the level of control cells in the absence of cepharanthine. The efflux of paclitaxel from MRP7-transfected cells was also significantly inhibited by cepharanthine. The ability of cepharanthine to inhibit MRP7 was analyzed in membrane vesicle assays using E(2)17betaG, an established substrate of MRP7, as a probe. E(2)17betaG transport was competitively inhibited by cepharanthine with a K(i) value of 4.86microM. These findings indicate that cepharanthine reverses MRP7-mediated resistance to paclitaxel in a competitive manner.

The taccalonolides: microtubule stabilizers that circumvent clinically relevant taxane resistance mechanisms.

The taccalonolides are a class of structurally and mechanistically distinct microtubule-stabilizing agents isolated from Tacca chantrieri. A crucial feature of the taxane family of microtubule stabilizers is their susceptibility to cellular resistance mechanisms including overexpression of P-glycoprotein (Pgp), multidrug resistance protein 7 (MRP7), and the betaIII isotype of tubulin. The ability of four taccalonolides, A, E, B, and N, to circumvent these multidrug resistance mechanisms was studied. Taccalonolides A, E, B, and N were effective in vitro against cell lines that overexpress Pgp and MRP7. In addition, taccalonolides A and E were highly active in vivo against a doxorubicin- and paclitaxel-resistant Pgp-expressing tumor, Mam17/ADR. An isogenic HeLa-derived cell line that expresses the betaIII isotype of tubulin was generated to evaluate the effect of betaIII-tubulin on drug sensitivity. When compared with parental HeLa cells, the betaIII-tubulin-overexpressing cell line was less sensitive to paclitaxel, docetaxel, epothilone B, and vinblastine. In striking contrast, the betaIII-tubulin-overexpressing cell line showed greater sensitivity to all four taccalonolides. These data cumulatively suggest that the taccalonolides have advantages over the taxanes in their ability to circumvent multiple drug resistance mechanisms. The ability of the taccalonolides to overcome clinically relevant mechanisms of drug resistance in vitro and in vivo confirms that the taccalonolides represent a valuable addition to the family of microtubule-stabilizing compounds with clinical potential.

ABCC11 expression is regulated by estrogen in MCF7 cells, correlated with estrogen receptor alpha expression in postmenopausal breast tumors and overexpressed in tamoxifen-resistant breast cancer cells.

ABCC11 (Multidrug resistance protein 8; MRP8), a plasma membrane ATP-binding cassette transporter, has been implicated in drug resistance of breast cancer by virtue of its ability to confer resistance to fluoropyrimidines and to efflux methotrexate, and by its expression in this tumor. Expression of ABCC11 in breast, a hormonally regulated tissue, as well as the pump's ability to transport estrogen conjugates, suggest the possibility that expression of ABCC11 may be susceptible to regulation by estrogen. However, nothing is currently known about regulation of this gene. In this study, estradiol (E(2)) treatment reduced expression of ABCC11 mRNA in estrogen receptor (ER)-alpha-positive MCF7 cells, and E(2) antagonists such as ICI 182 780 and tamoxifen (TAM) abrogated E(2)-mediated downregulation. ABCC11 expression was positively correlated with ER-alpha expression in both breast cell lines, and two independent series of tumors from postmenopausal patients. In addition, expression of ABCC11 was upregulated in MCF7 cells exposed to TAM for 72 h, and was overexpressed in TAM-resistant cell lines. Drug sensitivity analysis of the TAM-resistant cells indicated that they were also resistant to 5-fluorouracil (5-FU), consistent with the reported ability of ABCC11 to confer resistance to this agent. These studies indicate that ABCC11 expression is negatively regulated by E(2), but that ABCC11 expression is high in high-expressing ER-alpha breast cancers. Our findings support the notion that expression of ABCC11 in ER-alpha-positive breast cancers may contribute to decreased sensitivity to chemotherapy combinations that include 5-FU. ABCC11 may be a potential predictive tool in the choice of anticancer therapies in ER-positive breast cancers resistant to TAM.

The organic solute transporter alpha-beta, Ostalpha-Ostbeta, is essential for intestinal bile acid transport and homeostasis.

The apical sodium-dependent bile acid transporter (Asbt) is responsible for transport across the intestinal brush border membrane; however, the carrier(s) responsible for basolateral bile acid export into the portal circulation remains to be determined. Although the heteromeric organic solute transporter Ostalpha-Ostbeta exhibits many properties predicted for a candidate intestinal basolateral bile acid transporter, the in vivo functions of Ostalpha-Ostbeta have not been investigated. To determine the role of Ostalpha-Ostbeta in intestinal bile acid absorption, the Ostalpha gene was disrupted by homologous recombination in mice. Ostalpha(-/-) mice were physically indistinguishable from wild-type mice. In everted gut sac experiments, transileal transport of taurocholate was reduced by >80% in Ostalpha(-/-) vs. wild-type mice; the residual taurocholate transport was further reduced to near-background levels in gut sacs prepared from Ostalpha(-/-)Mrp3(-/-) mice. The bile acid pool size was significantly reduced (>65%) in Ostalpha(-/-) mice, but fecal bile acid excretion was not elevated. The decreased pool size in Ostalpha(-/-) mice resulted from reduced hepatic Cyp7a1 expression that was inversely correlated with ileal expression of fibroblast growth factor 15 (FGF15). These data indicate that Ostalpha-Ostbeta is essential for intestinal bile acid transport in mice. Unlike a block in intestinal apical bile acid uptake, genetic ablation of basolateral bile acid export disrupts the classical homeostatic control of hepatic bile acid biosynthesis.

Impact of basolateral multidrug resistance-associated protein (Mrp) 3 and Mrp4 on the hepatobiliary disposition of fexofenadine in perfused mouse livers.

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.

Up-regulation of MRP4 and down-regulation of influx transporters in human leukemic cells with acquired resistance to 6-mercaptopurine.

To investigate the mechanism of cellular resistance to 6-MP, we established a 6-MP resistant cell line (CEM-MP5) by stepwise selection of the human T-lymphoblastic leukemia cell line (CEM). CEM-MP5 cells were about 100-fold resistant to 6-MP compared with parental CEM cells. Western blot analysis demonstrated that multidrug resistant protein 4 (MRP4) was increased in CEM-MP5 cells, whereas the levels of the nucleoside transporters hENT1, hCNT2 and hCNT3 were decreased compared with those of parental CEM cells. Consistent with the operation of an efflux pump, accumulation of [14C]6-MP and/or its metabolites was reduced, and ATP-dependent efflux was increased in CEM-MP5 cells. Taken together these results showed that up-regulation of MRP4 and down-regulation of influx transporters played a major role in 6-MP resistance of CEM-MP5 cells.

Targeted ablation of Abcc1 or Abcc3 in Abcc6(-/-) mice does not modify the ectopic mineralization process.

Pseudoxanthoma elasticum (PXE) is a heritable disorder characterized by ectopic mineralization of connective tissues, with considerable intra- and interfamiliar phenotypic variability. PXE is caused by mutations in the ABCC6 gene, which encodes a transporter protein, MRP6, and targeted ablation of Abcc6 in mice recapitulates the manifestations of PXE. In this study, we examined the hypothesis that the expression of other members of the Abcc family may be altered in Abcc6 null mice, possibly explaining the phenotypic variability because of the functional overlap of these transporters. Analysis of the transcript levels of Abcc1-10 and 12 in the liver of Abcc6 (-/-) mice by quantitative RT-PCR indicated that the levels of other C family mRNAs were not significantly different from wild-type mice. Next, we developed Abcc6/1(-/-) and Abcc6/3(-/-) double null mice and examined them for tissue mineralization. Histopathologic examination, coupled with computerized morphometric analysis, and chemical assay of calcium x phosphate product in the muzzle skin of Abcc1(-/-) and Abcc3(-/-) mice did not reveal evidence of mineralization. Abcc6/1(-/-) and Abcc6/3(-/-) double knock-out mice exhibited connective tissue mineralization similar to that in Abcc6 (-/-) mice. These results emphasize the importance of the Abcc6 gene in the ectopic mineralization process and further suggest that other members of the Abcc family, particularly Abcc1 and Abcc3, do not modulate the effects of Abcc6 in this mouse model.

Predicting human tumor drug concentrations from a preclinical pharmacokinetic model of temozolomide brain disposition.

PURPOSE: Knowledge of drug concentrations in tumors is critical for understanding the determinants of drug accumulation in tumors. Because significant obstacles prevent making these measurements in humans, development of a predictive pharmacokinetic model would be of great value to the translation of preclinical data to the clinic. Our goal was to show how the latter could be achieved for temozolomide, an agent used in the treatment of brain tumors, using an orthotopic brain tumor model in rats. EXPERIMENTAL DESIGN: Rats bearing i.c. tumors received 20 mg/kg i.v. of temozolomide followed by the subsequent measurement of serial plasma, cerebrospinal fluid (CSF), normal brain, and brain tumor temozolomide concentrations. The resultant data provided the framework to develop a hybrid physiologically based pharmacokinetic model for temozolomide in brain. The preclinical pharmacokinetic model was scaled to predict temozolomide concentrations in human CSF, normal brain, and brain tumor, and through a series of Monte Carlo simulations, the accumulation of temozolomide in brain tumors under conditions of altered blood-brain barrier permeability, fractional blood volume, and clinical dosing schedules was evaluated. RESULTS: The developed physiologically based pharmacokinetic model afforded a mechanistic and accurate prediction of temozolomide brain disposition in rats, which through model scale-up procedures accurately predicted the CSF/plasma area under the drug concentration-time curve ratios of 0.2 reported in patients. Through a series of model simulations, it was shown that the brain tumor accumulation of temozolomide varied substantially based on changes in blood-brain barrier permeability and fractional tumor blood volume but minimally based on clinical dosing regimens. CONCLUSIONS: A physiologically based pharmacokinetic modeling approach offers a means to translate preclinical to clinical characteristics of drug disposition in target tissues and, thus, a means to select appropriate drug dosing regimens for achieving optimal target tissue drug concentrations.

Determination of methotrexate and its major metabolite 7-hydroxymethotrexate in mouse plasma and brain tissue by liquid chromatography-tandem mass spectrometry.

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 microl 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.

Physiological and pharmacological functions of Mrp2, Mrp3 and Mrp4 as determined from recent studies on gene-disrupted mice.

The MRP family is composed of nine transporters, at least eight of which are lipophilic anion transporters that are capable of conferring resistance to various anticancer agents. Recently, mice with gene disruptions in Mrp2, Mrp3 and Mrp4 have been developed. This review will discuss insights into the physiological and pharmacological functions of Mrp2, Mrp3 and Mrp4 afforded by investigations of these new mouse models.

Multidrug resistance protein 4 protects bone marrow, thymus, spleen, and intestine from nucleotide analogue-induced damage.

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.

ABCC10, ABCC11, and ABCC12.

Multidrug resistance protein (MRP)7, MRP8, and MRP9 (gene symbols ABCC10, ABCC11, and ABCC12) are recently identified members of the MRP family that are at relatively early stages of investigation. Of these proteins, a physiological function has only been established for MRP8, for which a single nucleotide polymorphism determines wet vs dry earwax type. MRP7 and MRP8 are lipophilic anion pumps that are able to confer resistance to chemotherapeutic agents. MRP7 is competent in the transport of the glucuronide E(2)17betaG, and its resistance profile, which includes several natural product anticancer agents, is distinguished by the taxane docetaxel. MRP8 is able to transport a diverse range of lipophilic anions, including cyclic nucleotides, E(2)17betaG, steroid sulfates such as dehydroepiandrosterone (DHEAS) and E(1)S, glutathione conjugates such as leukotriene C4 and dinitrophenyl-S-glutathione, and monoanionic bile acids. However, the constituent of earwax that is susceptible to transport by MRP8 has not been identified. MRP8 has complex interactions with its substrates, as indicated by the nonreciprocal ability of DHEAS to stimulate E(2)17betaG transport. Similar to the case for other MRPs that possess only two membrane spanning domains (MRP4 and MRP5), MRP8 is a cyclic nucleotide efflux pump that is able to confer resistance to nucleoside-based agents, such as PMEA and 5FU. The functional characteristics of MRP9 are currently unknown.

Evaluation of the role of multidrug resistance-associated protein (Mrp) 3 and Mrp4 in hepatic basolateral excretion of sulfate and glucuronide metabolites of acetaminophen, 4-methylumbelliferone, and harmol in Abcc3-/- and Abcc4-/- mice.

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.