Function Of Multidrug Resistance-associated Protein Research Articles

First-in-human evaluation of 6-bromo-7-[11C]methylpurine, a PET tracer for assessing the function of multidrug resistance-associated proteins in different tissues

PurposeMultidrug resistance-associated protein 1 (MRP1) is a transport protein with a widespread tissue distribution, which has been implicated in the pathophysiology of Alzheimer’s and chronic respiratory disease. PET with 6-bromo-7-[11C]methylpurine ([11C]BMP) has been used to measure MRP1 function in rodents. In this study, [11C]BMP was for the first time characterised in humans to assess the function of MRP1 and other MRP subtypes in different tissues.MethodsThirteen healthy volunteers (7 men, 6 women) underwent dynamic whole-body PET scans on a long axial field-of-view (LAFOV) PET/CT system after intravenous injection of [11C]BMP. Three subjects of each sex were scanned a second time to assess reproducibility. Volumes of interest were outlined for MRP-expressing tissues (cerebral cortex, cerebellum, choroid plexus, retina, lungs, myocardium, kidneys, and liver). From the time-activity curves, the elimination rate constant (kE, h− 1) was derived as a parameter for tissue MRP function and its test-retest variability (TRTV, %) was calculated. Radiation dosimetry was calculated using the Medical Internal Radiation Dose (MIRD) methodology.ResultsMean kE and corresponding TRTV values were: cerebral cortex: 0.055 ± 0.010 h− 1 (− 4 ± 24%), cerebellum: 0.033 ± 0.009 h− 1 (1 ± 39%), choroid plexus: 0.292 ± 0.059 h− 1 (0.1 ± 16%), retina: 0.234 ± 0.045 h− 1 (30 ± 38%), lungs: 0.875 ± 0.095 h− 1 (− 3 ± 11%), myocardium: 0.641 ± 0.105 h− 1 (11 ± 25%), kidneys: 1.378 ± 0.266 h− 1 (14 ± 16%), and liver: 0.685 ± 0.072 h− 1 (7 ± 9%). Significant sex differences were found for kE in the cerebellum, lungs and kidneys. Effective dose was 4.67 ± 0.18 µSv/MBq for men and 4.55 ± 0.18 µSv/MBq for women.ConclusionLAFOV PET/CT with [11C]BMP potentially allows for simultaneous assessment of MRP function in multiple human tissues. Mean TRTV of kE in different tissues was in an acceptable range, except for the retina. The radiation dosimetry of [11C]BMP was in the typical range of 11C-tracers. LAFOV PET/CT holds great potential to assess at a whole-body, multi-tissue level molecular targets relevant for drug disposition in humans.Trial registrationEudraCT 2021-006348-29. Registered 15 December 2021.

CRGD-targeted heparin nanoparticles for effective dual drug treatment of cisplatin-resistant ovarian cancer.

Resistance to the chemotherapeutic agent cisplatin (DDP) is the primary reason for invalid chemotherapy of ovarian cancer. Given the complex mechanisms underlying chemo-resistance, the design of combination therapies based on blocking multiple mechanisms is a rationale to synergistically elevate therapeutic effect for effectively overcoming cancer chemo-resistance. Herein, we demonstrated a multifunctional nanoparticle (DDP-Ola@HR), which could simultaneously co-deliver DDP and Olaparib (Ola, DNA damage repair inhibitor) using targeted ligand cRGD peptide modified with heparin (HR) as nanocarrier, enabling the concurrent tackling of multiple resistance mechanisms to effectively inhibit the growth and metastasis of DDP-resistant ovarian cancer. In combination strategy, heparin could suppress the function of multidrug resistance-associated protein 2 (MRP2) and P-glycoprotein (P-gp) to promote the intracellular accumulation of DDP and Ola by specifically binding with heparanase (HPSE) to down-regulate PI3K/AKT/mTOR signaling pathway, and simultaneously served as a carrier combined with Ola to synergistically enhance the anti-proliferation ability of DDP for resistant ovarian cancer, thus achieving great therapeutic efficacy. Our DDP-Ola@HR could provide a simple and multifunctional combination strategy to trigger an anticipated cascading effect, thus effectively overcoming the chemo-resistance of ovarian cancer.

Deciphering the chemical constituents of Shengjiang Xiexin decoction by ultrahigh-performance liquid chromatography-quadrupole/orbitrap high-resolution mass spectrometry and the impact of 20 characteristic components on multidrug resistance-associated protein 2 in the vesicular transport assay.

Shengjiang Xiexin decoction, a traditional Chinese medical formula, has been utilized to alleviate the delayed-onset diarrhea induced by irino tecan. However, the chemical constituents of this formula and the activities of its constituents remain unclear. In this study, ultrahigh-performance liquid chromatography-quadrupole/orbitrap high-resolution mass spectrometry was employed to comprehensively analyze the chemical constituents of Shengjiang Xiexin decoction. A total of 270 components, including flavonoids, coumarins, triterpenoids, alkaloids, diarylheptanoids and others, were identified or characterized. Multidrug resistance-associated protein 2 is an efflux transporter responsible for regulating drug absorption. A total of 20 characteristic components from the formula were selected to evaluate their effects on the function of multidrug resistance-associated protein 2 using the vesicular transport assay. Glycyrrhizic acid and glycyrrhetinic acid were identified as potential multidrug resistance-associated protein 2 inhibitors, while 9 flavonoid aglycones increased the uptake of the substrate [3 H]-estradiol 17-β-glucuronide in the vesicles. This was the first systematic investigation of the chemical constituents from Shengjiang Xiexin decoction and the effect of its characteristic components on the transporter. The results offered a basis for further exploring the detoxification mechanisms of this formula and its interactions with other drugs.

NHERF1/EBP50 as a Target for Modulation of MRP Function in HepG2 Cells.

As increased expression and activities of efflux transporters (ETs) often cause drug resistance in cancers, we tried modulating ET activity in cancer cells, using scaffold proteins such as ezrin/radixin/moesin (ERM) proteins, and Na+/H+ exchanger regulatory factor-1 (NHERF1)/ERM-binding phosphoprotein of 50 kDa (EBP50). To see whether EBP50 modulated ET activities in human liver cancer HepG2 cells, we used EBP50 siRNA and a designed TAT-PDZ1 peptide. The EBP50 knockdown (EBP50KD) cells had significantly higher intracellular accumulations of Rho123 and carboxy-dichlorofluorescein (CDF), but not H33342 (i.e., the respective substrates of P-glycoprotein (P-gp), multidrug resistance-associated protein (MRP), and breast cancer resistance protein (BCRP)), compared with control HepG2, suggesting that EBP50 knockdown in HepG2 cells decreased activity of P-gp and MRP but not BCRP. Treatment with TAT-PDZ1 peptide (>1 pM) resulted in significantly higher CDF accumulation in HepG2 cells, which persisted for ≥180 min after TAT-PDZ1 peptide treatment. These results imply that EBP50 can modulate ET activities. To our knowledge, this is the first report on using a competitive peptide to modulate interactions between MRP and EBP50.

The Function of Multidrug Resistance-associated Protein 3 in the Transport of Bile Acids under Normal Physiological and Lithocholic Acid-induced Cholestasis Conditions.

The role of multidrug resistance-associated protein 3 (Mrp3) in the transport of bile acid (BA) in drug-induced cholestasis has not been well studied. In this study, wild type and Mrp3 knockout (Mrp3-/-) mice under normal physiological and lithocholic acid (LCA)-induced cholestatic conditions were employed to investigate the role of Mrp3 in BA transport. The levels of BA in serum, liver, gallbladder, intestine, kidney, feces and urine were quantified in both wild type and Mrp3-/- mice via ultra-high performance liquid chromatography triple quadrupole mass spectrometry (UHPLC-MS/MS). Quantitative real-time PCR (RT-PCR) analysis was used to measure the expression of genes related to the transport and synthesis of BA. The results showed that the liver did not suffer more serious damage as a result of cholestasis when Mrp3 was depleted. The level of some individual bile acids changed apparently in the compartments of enterohepatic circulation (EHC) between the two control and model groups, respectively, but the level of serum total bile acid was only slightly reduced for Mrp3-/- groups. In addition, the level of BA-related efflux transporters and synthases increased significantly when Mrp3 was knocked out under normal physiological conditions, but a negligible alteration appeared under cholestatic conditions. Our results indicated that Mrp3 could be responsible for the transport of some specific bile acids, and part of the Mrp3 role could be compensated for by other transporters. Moreover, Mrp3 deficiency has a direct effect on the expression of BA-related synthases and efflux transporters under normal physiological conditions, but this effect could be less prominent under cholestatic conditions. This study could provide much valuable insight into the physiological function of Mrp3 in the transport of bile acids.

Circadian Clock Is Involved in Regulation of Hepatobiliary Transport Mediated by Multidrug Resistance-Associated Protein 2

There has been a growing interest in circadian regulation of the expression and function of drug transporters. In this study, we investigated circadian rhythm in the expression and function of multidrug resistance-associated protein 2 (Mrp2) in mouse liver and involvement of circadian clock in their regulations by using the circadian clock genes (period 1 and period 2) knockout mice. The mRNA and protein expression of Mrp2, P-glycoprotein, and breast cancer resistance protein was measured in the mouse liver at different times of the day. Circadian variation of hepatobiliary excretion of phenolsulfonphthalein, a model substrate of Mrp2, was also investigated in mice. Circadian oscillation of Mrp2 protein expression was clearly observed in the mouse liver with levels down at the light phase and up at the dark phase. The cumulative biliary excretion and biliary clearance of phenolsulfonphthalein from the liver to the bile was 2.37- and 1.74-fold greater in mice administered during the dark phase than in those administered during the light phase, respectively. The circadian oscillation in mRNA expression of Mrp2 disappeared in period 1 and period 2 double knockout mice. These results suggest that the expression and function of Mrp2 show the circadian rhythm, controlled by circadian clock genes.

Modulated function of multidrug resistance-associated proteins in cisplatin-induced acute renal failure rats.

The effect of cisplatin-induced acute renal failure (ARF) on the function and expression of multidrug resistanceassociated proteins (MRPs) was evaluated in rats. Rats received an intraperitoneal injection of cisplatin (9 mg/kg), and the induction of ARF state with high plasma concentrations of indoxyl sulfate and creatinine was observed 72 h after cisplatin treatment. The function of MRPs in the liver, kidney and brain was evaluated by measuring the tissue accumulation and biliary excretion of 2,4-dintrophenyl-S-glutathione (DNP-SG), a substrate for MRPs, after administration of 1-chloro-2,4-dintrobenzene (CDNB), a precursor of DNP-SG, in rats. The levels of MRP1-4 expression were evaluated by Western blot analysis. Effect of ARF plasma components on MRP function was also examined by using calcein acetoxymethyl ester (calcein-AM) in HepG2 cells. In ARF rats (72 h after cisplatin treatment), the accumulation of DNP-SG in the liver, kidney and brain was significantly higher than those in control and cisplatin-treated rats (1 h after treatment). In ARF rats, intrinsic biliary excretion clearance of DNP-SG, estimated by dividing the biliary excretion rate of DNP-SG with the liver concentration, was also significantly reduced, though the expression levels of MRP1-4 in the liver remained unchanged. ARF rat plasma (5%) significantly increased the accumulation of calcein, a MRP substrate, in HepG2 cells after application of calcein-AM. In conclusion, MRP function was found to be suppressed not only in the kidney but also in the liver and brain in cisplatin-induced ARF rats, possibly due to the accumulation of some MRP substrates/inhibitors in plasma.

Tyrosine and aurora kinase inhibitors diminish transport function of multidrug resistance-associated protein (MRP) 4 and breast cancer resistance protein (BCRP)

<p class="PaperKeywordTitle">Tyrosine and aurora kinases are important effectors in signal transduction pathways that are often involved in aberrant cancer cell growth. Tyrosine (TKI) and aurora (AKI) kinase inhibitors are anti-cancer agents specifically designed to target such signaling pathways through TKI/AKI binding to the ATP-binding pocket of kinases thereby leading to diminished kinase activity. Some TKIs have been identified as inhibitors of ATP-binding cassette (ABC) transporters such as P-glycoprotein and breast cancer resistance protein (BCRP), which are commonly upregulated in malignant cells. TKI/AKIs have been investigated as ABC transporter inhibitors in order to facilitate the accumulation of concomitantly administered chemo-therapeutics within cancer cells. However, ABC transporters are prominently expressed in the liver and other eliminating organs, and their inhibition has been linked to intracellular accumulation of drugs, altered disposition, and toxicity. The potential for TKIs/AKIs to inhibit other important hepatic efflux transporters, particularly multidrug resistance-associated proteins (MRPs), remains unknown. The aim of the current study was to compare the inhibitory potency of 20 selected TKI/AKIs against MRP4 and BCRP through the use of inverted membrane vesicle assays. Relative IC<sub>50 </sub>values were estimated by determining TKI/AKI inhibition of MRP4-mediated [<sup>3</sup>H]-dehydroepiandrosterone sulfate uptake and BCRP-mediated [<sup>3</sup>H]-estrone sulfate uptake. To provide insight to the clinical relevance of TKI/AKI inhibition of ABC efflux transporters, the ratio of the steady-state maximum total plasma concentration (C<sub>ss</sub>) to the IC<sub>50</sub> for each compound was calculated with C<sub>ss</sub>/IC<sub>50</sub> ratio >0.1 deemed potentially clinically relevant. Such analysis identified several potentially clinically relevant inhibitors of MRP4: alisertib, danusertib, erlotinib, lapatinib, neratinib, nilotinib, pazopanib, sorafenib, and tozasertib. The potentially clinically relevant inhibition of BCRP was much more extensive and included alisertib, barasertib, danusertib, enzastaurin, erlotinib, gefitinib, imatinib, neratinib, nilotinib, pazopanib, selumetinib, sorafenib, sunitinib, tozasertib, and vandetanib. These findings indicate the significant potential for TKI/AKIs to inhibit multiple ABC efflux transporters. The resulting inhibition data could provide insight regarding the clinical interpretation of pharmacokinetic/pharmacodynamic outcomes when TKI/AKIs are administered concomitantly with additional chemotherapeutic agents.</p>

Modulation of multidrug resistance-associated proteins function in erythrocytes in glycerol-induced acute renal failure rats.

Evaluation of the function of multidrug resistance-associated proteins (MRPs) expressed in erythrocytes and screening of endogenous MRPs modulator(s) in glycerol-induced acute renal failure (ARF) rats. Concentrations of 2,4-dinitrophenyl-S-glutathione (DNP-SG), a substrate for MRPs, in erythrocytes after administration of 1-chloro-2,4-dintrobenzene (CDNB), a precursor of DNP-SG, were determined in control and ARF rats. The screening of endogenous MRPs modulator(s) was performed using washed erythrocytes and inside-out erythrocyte membrane vesicles (IOVs) in vitro. Accumulation of DNP-SG in erythrocytes was observed in ARF rats. Uraemic plasma components exhibited a greater inhibitory effect on DNP-SG uptake by IOVs than control plasma components and increased the DNP-SG accumulation significantly in washed erythrocytes. Several protein-bound uraemic toxins at clinically observed concentrations and bilirubin significantly inhibited DNP-SG uptake by IOVs. In washed erythrocytes, bilirubin (10 μm) and l-kynurenine (100 μm), a precursor of kynurenic acid being MRPs inhibitor, increased DNP-SG accumulation significantly. Glycerol-induced ARF rats contain various MRPs inhibitors in plasma, and membrane-permeable MRP substrates/inhibitors including their precursors inhibit the MRPs function in erythrocytes cooperatively.

Role of ABC Efflux Transporters in the Oral Bioavailability and Drug-induced Intestinal Toxicity

The gastrointestinal tract is the organ that absorbs nutrients and water from foods and drinks. This organ is often exposed to various harmful xenobiotics, and therefore possesses various detoxification/barrier systems, including metabolizing enzymes and efflux transporters. Intestinal epithelial cells express ATP-binding cassette (ABC) efflux transporters such as P-glycoprotein, multidrug resistance-associated proteins (MRPs) and breast cancer resistance protein, in addition to various solute carrier (SLC) influx transporters. These transporters are expressed site- and membrane-specifically in enterocytes, which affects the bioavailability of ingested substrate drugs. Expression and/or function of transporters can be modulated by various compounds, including therapeutic drugs, herbal products, some foods, and by disease states. The modulation of transporters could cause unexpectedly higher or lower blood concentrations, marked inter- and intra-individual variations in pharmacokinetics, and unreliable pharmacological actions in association with toxicities of substrates. Recently, we found that hyperbilirubinemia, which occurs in some disease states, increased intestinal accumulation and toxicity of methotrexate, an MRP substrate, because of the suppression of MRP function by high plasma concentrations of conjugated bilirubin. We also attempted to ameliorate the intestinal toxicity of irinotecan hydrochloride by modulating the hepatic and intestinal functions of MRP2. This review summarizes our findings regarding the role of ABC transporters, especially MRPs, in oral bioavailability and in drug-induced intestinal toxicity. Our approach to treat intestinal toxicity using an MRP2 modulator is also described.

Modulated pharmacokinetics and increased small intestinal toxicity of methotrexate in bilirubin-treated rats

The effect of bilirubin treatment on the pharmacokinetics and small intestinal toxicity of methotrexate was evaluated in rats, since bilirubin and its glucuronide conjugates can suppress multidrug resistance-associated protein-mediated transport. Rats were treated intravenously with bilirubin and the various clearances and tissue distribution of methotrexate were estimated under a steady-state plasma concentration. Intestinal toxicity induced by methotrexate was also evaluated by measuring the leakage of alkaline phosphatase (ALP) activity. Probenecid, an inhibitor for multidrug resistance-associated protein and organic anion transporters, was used for comparison. The treatment with bilirubin increased the steady-state plasma concentration and reduced biliary excretion clearance, urinary excretion clearance and intestinal exsorption clearance of methotrexate, as did treatment with probenecid. The intestinal absorption and jejunum distribution of methotrexate also significantly increased in bilirubin- and probenecid-treated rats. A greater leakage of ALP activity to the luminal fluid, with a lower ALP activity in the intestinal mucosal membrane after intestinal perfusion of methotrexate, was observed in bilirubin- and probenecid-treated rats. Hyperbilirubinemia, which is involved under various disease states, may increase the small intestinal accumulation and toxicities of methotrexate, since high plasma concentrations of conjugated bilirubin can suppress the function of multidrug resistance-associated proteins, which facilitate the efflux of methotrexate out of cells.

Site-specific bidirectional efflux of 2,4-dinitrophenyl-S-glutathione, a substrate of multidrug resistance-associated proteins, in rat intestine and Caco-2 cells

The site-specific function of multidrug-resistance-associated proteins (MRPs), especially MRP2 and MRP3, was examined in rat intestine and human colon adenocarcinoma (Caco-2) cells. The MRP function was evaluated pharmacokinetically by measuring the efflux transport of 2,4-dinitrophenyl-S-glutathione (DNP-SG), an MRP substrate, after application of 1-chloro-2,4-dinitrobenzene (CDNB), a precursor of DNP-SG. The expression of rat and human MRP2 and MRP3 was analysed by Western blotting. The rat jejunum exhibited a higher apical MRP2 and a lower basolateral MRP3 expression than ileum. In accordance with the expression level, DNP-SG efflux to the mucosal surface was significantly greater in jejunum, while serosal efflux was greater in ileum. Site-specific bidirectional efflux of DNP-SG was also observed in in-vivo studies, in which portal and femoral plasma levels and biliary excretion rate of DNP-SG were significantly higher when CDNB was administered to ileum. Caco-2 cells also showed a bidirectional efflux of DNP-SG. Probenecid, an MRP inhibitor, significantly suppressed the mucosal efflux in jejunum and serosal efflux in ileum. In contrast, probenecid significantly suppressed both apical and basolateral efflux of DNP-SG in Caco2 cells, though the inhibition was of small magnitude. In conclusion, the efflux of DNP-SG from enterocytes mediated by MRPs exhibited a significant regional difference in rat intestine, indicating possible variability in intestinal bioavailabilities of MRP substrates, depending on their absorption sites along the intestine.

Reactivity of 6-Halopurine Analogs with Glutathione as a Radiotracer for Assessing Function of Multidrug Resistance-Associated Protein 1

6-Bromo-7-[(11)C]methylpurine is reported to react with glutathione via glutathione S-transferases in the brain and to be converted into a substrate for multidrug resistance-associated protein 1 (MRP1), an efflux pump. The compound with a rapid conversion rate allows quantitative assessment of MRP1 function, but this rate is probably susceptible to interspecies differences. Hence, for application to different species, including humans, it is necessary to adjust the conversion rate by modifying the chemical structure. We therefore designed 6-halo-9-(or 7)-[(14)C]methylpurine (halogen: F, Cl, Br, or I), and evaluated them in vitro with respect to enzymatic reactivity with glutathione using brain homogenates from the mouse, rat, or monkey. There was a marked difference in reactivity between these species. Changes in the position of the methyl group and halogen on N-methyl-6-halopurine provided various compounds possessing wide-ranging reactivity with glutathione. In conclusion, the adjustment of reactivity of 6-bromo-7-[(11)C]methylpurine may allow assessment of MRP1 function in the brain in various species.

Effect of ursodeoxycholic acid treatment on the expression and function of multidrug resistance-associated protein 2 in rat intestine

Effect of ursodeoxycholic acid (UDCA) treatment on the expression and function of intestinal multidrug resistance-associated protein (Mrp) 2 was examined in rats. When rats were orally administered 0.5% UDCA solution for 6 days, mRNA and protein levels of Mrp2 in the intestine were increased about twofold compared with those in untreated rats. In in vitro everted sac study, Mrp2-mediated efflux of 2,4-dinitrophenyl-S-glutathione (DNP-SG) to the mucosal surface was shown to be increased by UDCA treatment. In vivo intestinal exsorption clearance of DNP-SG was also increased by UDCA treatment. In addition, in situ intestinal absorption of methotrexate, a substrate of Mrp2, was decreased by the treatment. These results indicate that the expression and function of intestinal Mrp2 is up-regulated by oral administration of UDCA. © 2008 Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci 98:2822–2831, 2009

MRP- and BCL-2-mediated drug resistance in human SCLC: Effects of apoptotic sphingolipids in vitro

Multidrug-resistance-associated protein (MRP) and BCL-2 contribute to drug resistance expressed in SCLC. To establish whether MRP-mediated drug resistance affects sphingolipid (SL)-induced apoptosis in SCLC, we first examined the human SCLC cell line, UMCC-1, and its MRP over-expressing, drug-resistant subline, UMCC-1/VP. Despite significantly decreased sensitivity to doxorubicin (Dox) and to the etoposide, VP-16, the drug-selected line was essentially equally as sensitive to treatment with exogenous ceramide (Cer), sphingosine (Sp) or dimethyl-sphingosine (DMSP) as the parental line. Next, we observed that high BCL-2-expressing human H69 SCLC cells, that were ∼160-fold more sensitive to Dox than their combined BCL-2 and MRP-over-expressing (H69AR) counterparts, were only ∼5-fold more resistant to DMSP. Time-lapse fluorescence microscopy of either UMCC cell line treated with DMSP-Coumarin revealed comparable extents and kinetics of SL uptake, further ruling out MRP-mediated effects on drug uptake. DMSP potentiated the cytotoxic activity of VP-16 and Taxol, but not Dox, in drug-resistant UMCC-1/VP cells. However, this sensitization did not appear to involve DMSP-mediated effects on the function of MRP in drug export; nor did DMSP strongly shift the balance of pro-apoptotic Sps and anti-apoptotic Sp-1-Ps in these cells. We conclude that SL-induced apoptosis markedly overcomes or bypasses MRP-mediated drug resistance relevant to SCLC and may suggest a novel therapeutic approach to chemotherapy for these tumors.

Naringenin Enhances the Anti-Tumor Effect of Doxorubicin Through Selectively Inhibiting the Activity of Multidrug Resistance-Associated Proteins but not P-glycoprotein

Naringenin has shown paradoxical results to modulate the function of multidrug resistance-associated proteins (MRPs). The aim of this study is to interpret whether naringenin can reverse intrinsic and/or acquired resistance of cancer cells to chemotherapeutic agents. The effects of naringenin on the uptake, retention and cytotoxicity of doxorubicin were investigated in A549, MCF-7, HepG2 and MCF-7/DOX cells. Cellular efflux pathways modulated by naringenin were assessed with their specific substrates and inhibitors. The improved antitumor activity of doxorubicin in combination with naringenin was also investigated in vivo. The IC(50) values of doxorubicin in combination with naringenin in A549 and MCF-7 cells were approximately 2-fold lower than that of doxorubicin alone. The increased sensitivity to doxorubicin by naringenin in HepG2 and MCF-7/DOX cells was not observed. Naringenin increased the cellular doxorubicin accumulation through inhibiting doxorubicin efflux in the cells expressing MRPs but not P-gp. In contrast to doxorubicin alone, doxorubicin in combination with naringenin enhanced antitumor activity in vivo with low systemic toxicity. Naringenin enhances antitumor effect of doxorubicin by selective modulating drug efflux pathways. Naringenin will be a useful adjunct to improve the effectiveness of chemotherapeutic agents in treatment of human cancers.

Noninvasive and Quantitative Assessment of the Function of Multidrug Resistance-Associated Protein 1 in the Living Brain

Multidrug resistance-associated protein 1 (MRP1) acts as a defense mechanism by pumping xenobiotics and endogenous metabolites out of the brain. The currently available techniques for studying brain-to-blood efflux have significant limitations related to either their invasiveness or the qualitative assessment. Here, we describe an in vivo method, which overcomes these limitations for assessing MRP1 function, using positron emission tomography (PET) and a PET probe. 6-Bromo-7-[(11)C]methylpurine was designed to readily enter the brain after intravenous administration and to be efficiently converted to its glutathione conjugate (MRP1 substrate) in situ. Dynamic PET scan provided the brain time-activity curve after injection of 6-bromo-7-[(11)C]methylpurine into mice. The efflux rate of the substrate was kinetically estimated to be 1.4 h(-1) with high precision. Moreover, knockout of Mrp1 gene caused approximately a 90% reduction of the efflux rate, compared with wild-type mice. In conclusion, our method allows noninvasive and quantitative assessment for MRP1 function in the living brain.

Comparison of ABC Transporter Modulation by Atazanavir in Lymphocytes and Human Brain Endothelial Cells: ABC Transporters Are Involved in the Atazanavir-Limited Passage across anin VitroHuman Model of the Blood–Brain Barrier

Efflux pumps, P-glycoprotein (P-gp), multidrug resistance-associated proteins (MRPs), and breast cancer resistance protein (BCRP) have been shown to extrude HIV protease inhibitors from cells. These transporters are present on many barrier sites such as the blood-brain barrier (BBB) and on many circulating cells such as lymphocytes, and could reduce protease inhibitor concentration in sanctuary or HIV-1 target sites. This study compares the potential of the antiretroviral drug atazanavir to modulate P-gp and MRP expression and function in total lymphocytes and in human fetal brain endothelial cells (HBMECs). We address the question of atazanavir transport across the human BBB. Following incubation with atazanavir, P-gp and MRP1 expression was determined by direct immunofluorescence. Transporter function was assessed by measuring fluorescent dye efflux, either with or without specific inhibitors. Atazanavir substrate properties were determined by transport quantification through a validated in vitro human BBB model. Our results show that in contrast to HBMECs, in lymphocytes, atazanavir has no effect on MRP1 and P-gp expression. However, there were overall changes in P-gp function increasing its activity in lymphocytes and HBMECs. Using the in vitro human BBB model, we confirm the interaction of atazanavir with P-gp, MRPs, and BCRP in preventing its passage across this barrier and thus its entry into the central nervous system.

Multidrug resistance-associated proteins are crucial for the viability of activated rat hepatic stellate cells

Hepatic stellate cells (HSCs) survive and proliferate in the chronically injured liver. ATP-binding cassette (ABC) transporters play a crucial role in cell viability by transporting toxic metabolites or xenobiotics out of the cell. ABC transporter expression in HSCs and its relevance to cell viability and/or activation have not been reported so far. The aim of this study was to investigate the expression, regulation, and function of multidrug resistance-associated protein (Mrp)-type and multidrug resistance protein (Mdr)-type ABC transporters in activated rat HSCs. Rat HSCs were exposed to cytokines or oxidative stress. ABC transporter expression was determined by quantitative polymerase chain reaction and immunohistochemistry. HSCs were exposed to the Mdr inhibitors verapamil and PSC-833 and the Mrp inhibitor MK571. Mdr and Mrp transporter function was evaluated with flow cytometry. Apoptosis was determined by activated caspase-3 and acridine orange staining, and necrosis was determined by Sytox green nuclear staining. An in vivo model of carbon tetrachloride (CCl(4))-induced liver fibrosis was used. With respect to hepatocytes, activated HSCs expressed high levels of Mrp1 and comparable levels of Mrp3, Mrp4, Mdr1a, and Mdr1b but not the hepatocyte-specific transporters bile salt export pump, Mrp2, and Mrp6. Mrp1 protein staining correlated with desmin staining in livers from CCl(4)-treated rats. Mrp1 expression increased upon activation of HSCs. Cytokines induced Mdr1b expression only. Oxidative stress was not a major regulator of Mdr and Mrp transporter expression. Activated HSCs became necrotic when exposed to the Mrp inhibitors. Activated HSCs contain relatively high levels of Mrp1. Mrp-type transporters are required for the viability of activated HSCs. Mrp-dependent export of endogenous metabolites is important for the survival of activated HSCs in chronic liver diseases.

Modulation of function of multidrug resistance associated-proteins by Kaempferia parviflora extracts and their components

In this study, the effects of extracts and flavone derivatives from the rhizome of Kaempferia parviflora on multidrug resistance associated-proteins (MRP)-mediated transport in A549 cells were examined. The cells employed express MRP1 and MRP2, but not P-glycoprotein. The cellular accumulation of calcein, an MRP substrate, was significantly increased by various MRP inhibitors without being affected by verapamil, a typical P-glycoprotein inhibitor. Ethanol and aqueous extracts from K. parviflora rhizome increased the accumulation of calcein and doxorubicin in A549 cells in a concentration-dependent manner. The inhibitory potency of the ethanol extract for MRP function was greater than that of the aqueous extract. Among six flavone derivatives isolated from K. parviflora rhizome, 5,7-dimethoxyflavone exhibited a maximal stimulatory effect on the accumulation of doxorubicin in A549 cells. The accumulation of doxorubicin was increased by four flavone derivatives without 5-hydroxy group, but not by the other two flavone derivatives with 5-hydroxy group. In addition, 5,7-dimethoxyflavone and 3,5,7,3′,4′-pentamethoxyflavone decreased resistance to doxorubicin in A549 cells. These findings indicate that extracts and flavone derivatives from the rhizome of K. parviflora suppress MRP function, and therefore may be useful as modulators of multidrug resistance in cancer cells.