Multidrug Resistance-associated Protein Inhibitor Research Articles

Biological Distribution after Oral Administration of Radioiodine-Labeled Acetaminophen to Estimate Gastrointestinal Absorption Function via OATPs, OATs, and/or MRPs

We evaluated the whole-body distribution of orally-administered radioiodine-125 labeled acetaminophen (125I-AP) to estimate gastrointestinal absorption of anionic drugs. 125I-AP was added to human embryonic kidney (HEK)293 and Flp293 cells expressing human organic anion transporting polypeptide (OATP)1B1/3, OATP2B1, organic anion transporter (OAT)1/2/3, or carnitine/organic cation transporter (OCTN)2, with and without bromosulfalein (OATP and multidrug resistance-associated protein (MRP) inhibitor) and probenecid (OAT and MRP inhibitor). The biological distribution in mice was determined by oral administration of 125I-AP with and without bromosulfalein and by intravenous administration of 125I-AP. The uptake of 125I-AP was significantly higher in HEK293/OATP1B1, OATP1B3, OATP2B1, OAT1, and OAT2 cells than that in mock cells. Bromosulfalein and probenecid inhibited OATP- and OAT-mediated uptake, respectively. Moreover, 125I-AP was easily excreted in the urine when administered intravenously. The accumulation of 125I-AP was significantly lower in the blood and urinary bladder of mice receiving oral administration of both 125I-AP and bromosulfalein than those receiving only 125I-AP, but significantly higher in the small intestine due to inhibition of OATPs and/or MRPs. This study indicates that whole-body distribution after oral 125I-AP administration can be used to estimate gastrointestinal absorption in the small intestine via OATPs, OATs, and/or MRPs by measuring radioactivity in the urinary bladder.

Targeting multidrug resistance-associated protein 1 (MRP1)-expressing cancers: Beyond pharmacological inhibition.

Resistance to chemotherapy remains one of the most significant obstacles to successful cancer treatment. While inhibiting drug efflux mediated by ATP-binding cassette (ABC) transporters is a seemingly attractive and logical approach to combat multidrug resistance (MDR), small molecule inhibition of ABC transporters has so far failed to confer clinical benefit, despite considerable efforts by medicinal chemists, biologists, and clinicians. The long-sought treatment to eradicate cancers displaying ABC transporter overexpression may therefore lie within alternative targeting strategies. When aberrantly expressed, the ABC transporter multidrug resistance-associated protein 1 (MRP1, ABCC1) confers MDR, but can also shift cellular redox balance, leaving the cell vulnerable to select agents. Here, we explore the physiological roles of MRP1, the rational for targeting this transporter in cancer, the development of small molecule MRP1 inhibitors, and the most recent developments in alternative therapeutic approaches for targeting cancers with MRP1 overexpression. We discuss approaches that extend beyond simple MRP1 inhibition by exploiting the collateral sensitivity to glutathione depletion and ferroptosis, the rationale for targeting the shared transcriptional regulators of both MRP1 and glutathione biosynthesis, advances in gene silencing, and new molecules that modulate transporter activity to the detriment of the cancer cell. These strategies illustrate promising new approaches to address multidrug resistant disease that extend beyond the simple reversal of MDR and offer exciting routes for further research.

Transport Characteristics of 6-Mercaptopurine in Brain Microvascular Endothelial Cells Derived From Human Induced Pluripotent Stem Cells.

The likelihood of reoccurrence of acute lymphoblastic leukemia is influenced by the cerebral concentration of the therapeutic agent 6-mercaptopurine (6-MP) during treatment. Therefore, it is important to understand the blood-brain barrier (BBB) transport mechanism of 6-MP. The purpose of this study was to characterize this mechanism using human induced pluripotent stem cell-derived microvascular endothelial cells (hiPS-BMECs). The permeability coefficient of 6-MP across hiPS-BMECs monolayer in the basal-to-apical direction (B-to-A) was significantly greater than that in the opposite direction (A-to-B). The inhibition profiles of 6-MP transport in the A-to-B direction were different from those in the B-to-A direction. Transport in the A-to-B direction was mainly inhibited by adenine (an inhibitor of equilibrative nucleobase transporter 1; ENBT1), while transport in the B-to-A direction was significantly reduced by inhibitors of multidrug resistance-associated proteins (MRPs), especially zaprinast (an MRP5 inhibitor). Immunocytochemical analyses demonstrated the expression of ENBT1 and MRP5 proteins in hiPS-BMECs. We confirmed that the cellular uptake of 6-MP is decreased by ENBT1 inhibitors in hiPS-BMECs and by knockdown of ENBT1 in hCMEC/D3 cells. These results suggest that ENBT1 and MRP5 make substantial contributions to the transport of 6-MP in hiPS-BMECs and hCMEC/D3 cells.

Suppression of Proliferation of Human Glioblastoma Cells by Combined Phosphodiesterase and Multidrug Resistance-Associated Protein 1 Inhibition.

The paucity of currently available therapies for glioblastoma multiforme requires novel approaches to the treatment of this brain tumour. Disrupting cyclic nucleotide-signalling through phosphodiesterase (PDE) inhibition may be a promising way of suppressing glioblastoma growth. Here, we examined the effects of 28 PDE inhibitors, covering all the major PDE classes, on the proliferation of the human U87MG, A172 and T98G glioblastoma cells. The PDE10A inhibitors PF-2545920, PQ10 and papaverine, the PDE3/4 inhibitor trequinsin and the putative PDE5 inhibitor MY-5445 potently decreased glioblastoma cell proliferation. The synergistic suppression of glioblastoma cell proliferation was achieved by combining PF-2545920 and MY-5445. Furthermore, a co-incubation with drugs that block the activity of the multidrug resistance-associated protein 1 (MRP1) augmented these effects. In particular, a combination comprising the MRP1 inhibitor reversan, PF-2545920 and MY-5445, all at low micromolar concentrations, afforded nearly complete inhibition of glioblastoma cell growth. Thus, the potent suppression of glioblastoma cell viability may be achieved by combining MRP1 inhibitors with PDE inhibitors at a lower toxicity than that of the standard chemotherapeutic agents, thereby providing a new combination therapy for this challenging malignancy.

Van der Waals force-driven indomethacin-ss-paclitaxel nanodrugs for reversing multidrug resistance and enhancing NSCLC therapy

The high expression of multidrug resistance-associated protein 1 (MRP1) in cancer cells caused serious multidrug resistance (MDR), which limited the effectiveness of paclitaxel (PTX) in non-small cell lung cancer (NSCLC) chemotherapy. Indomethacin (IND), a kind of non-steroidal anti-inflammatory drugs (NSAIDs), which has been confirmed to be a potential MRP1 inhibitor. Taking into account the advantages of old drugs without extra controversial biosafety issue, in this manuscript, the disulfide bond (-S-S-) was employed for connecting IND and PTX to construct conjugate IND-S-S-PTX, which was further self-assembled and formed nanodrug (IND-S-S-PTX NPs). The particle size of IND-S-S-PTX NPs was ~160 nm with a narrow PDI value of 0.099, which distributed well in water and also exhibited a stable characteristic. Moreover, due to the existence of disulfide bond, the NPs were sensitive to the high level of glutathione (GSH) in tumor microenvironment. Molecular dynamics (MD) simulation presented the process of self-assembly in detail. Density functional theory (DFT) calculations revealed that the main driving force in self-assembly process was originated from the van der waals force. In addition, this carrier-free nano drug delivery systems (nDDs) could reverse the MDR by downregulating the expression of MRP1 protein in A549/taxol.

A multidrug resistance‐associated protein inhibitor is a potential enhancer of the benzyl isothiocyanate‐induced apoptosis induction in human colorectal cancer cells

The increasing drug efflux through the ATP-binding cassette (ABC) transporters is the most plausible mechanism that mediates resistance to the anticancer phytochemicals, such as benzyl isothiocyanate (BITC), as well as chemotherapy drugs. To identify a potential component to overcome this resistance by combinatory utilization, we focused on multidrug resistance-associated proteins (MRPs) pumping various drug metabolites with glutathione as well as the organic anions. The pharmacological treatment of an MRP inhibitor, MK571, significantly potentiated the BITC-induced antiproliferation, coincided with the enhanced accumulation of BITC and glutathione in human colorectal cancer HCT-116 cells. MK571 also enhanced the apoptosis induction as well as activation of the mitogen-activated protein kinases and caspase-3, whereas it did not affect their basal levels. These results suggested that, since MRPs might play a pivotal role in the BITC efflux, MK571 potentiates the BITC-induced antiproliferation in human colorectal cancer cells through inhibition of the glutathione-dependent BITC efflux.

131I]MIBG exports via MRP transporters and inhibition of the MRP transporters improves accumulation of [131I]MIBG in neuroblastoma

Introduction131I-labeled m-iodobenzylguanidine ([131I]MIBG) has been used to treat neuroblastoma patients, but [131I]MIBG may be immediately excreted from the cancer cells by the adenosine triphosphate binding cassette transporters, similar to anticancer drugs. The purpose of this study was to clarify the efflux mechanism of [131I]MIBG in neuroblastomas and improve accumulation by inhibition of the transporter in neuroblastomas. Methods[131I]MIBG was incubated in human embryonic kidney (HEK)293 cells expressing human organic anion transporting polypeptide (OATP)1B1, OATP1B3, OATP2B1, organic anion transporter (OAT)1 and OAT2, organic cation transporter (OCT)1 and OCT2, and sodium taurocholate cotransporting polypeptide, and in vesicles expressing P-glycoprotein (MDR1), multidrug resistance associated protein (MRP)1–4, or breast cancer resistance protein with and without MK-571 and probenecid (MRP inhibitors). Time activity curves of [131I]MIBG with and without MK-571 and probenecid were established using an SK-N-SH neuroblastoma cell line, and transporter expression of multiple drug resistance was measured. Biodistribution and SPECT imaging examinations were conducted using [123I]MIBG with and without probenecid in SK-N-SH-bearing mice. Results[131I]MIBG uptake was significantly higher in OAT1, OAT2, OCT1, and OCT2 than in mock cells. Uptake via OCT1 and OCT2 was little inhibited by MK-571 and probenecid. [131I]MIBG uptake into vesicles that highly expressed MRP1 or MRP4 was significantly higher in ATP than in AMP, and these inhibitors restored uptake to levels similar to that in AMP. Examining the time activity curves for [131I]MIBG in SK-N-SH cells, higher expressions of MDR1, MRP1, MRP4, and MK-571, or probenecid loading produced significantly higher uptake than in control at most incubation times. The ratios of tumors to blood or muscle in SK-N-SH-bearing mice were significantly increased by probenecid loading in comparison with normal mice. Conclusions[131I]MIBG exports via MRP1 and MRP4 in neuroblastoma. The accumulation and tumor-to-blood or muscle ratios of [131I]MIBG are improved by inhibition of MRPs with probenecid in neuroblastoma. Advances in knowledge[131I]MIBG, widely used for treatment of neuroendocrine tumors including neuroblastoma, is excreted via MRP1 and MRP4 in neuroblastoma. Implications for patient careLoading with probenecid, OAT, and MRP inhibitors improves [131I]MIBG accumulation.

Transport mechanism and affinity of [99mTc]Tc-mercaptoacetyltriglycine ([99mTc]MAG3) on the apical membrane of renal proximal tubule cells

Technetium-99m-labeled mercaptoacetyltriglycine ([99mTc]MAG3) is widely used for evaluation of transplanted kidneys, diagnosis of tubular necrosis, and scintigraphic studies of tubular function. [99mTc]MAG3 is a substrate for organic anion transporter (OAT)1 and OAT3 on the basolateral membrane side for renal secretion. We investigated the transport mechanism and affinity of [99mTc]MAG3 on the apical membrane of renal proximal tubule cells for renal secretion. Adenosine triphosphate-binding cassette (ABC) transporters for renal secretion of [99mTc]MAG3 were examined using ABC transporter vesicles expressing multiple drug resistance 1 (MDR1), breast cancer resistance protein (BCRP), multidrug resistance-associated protein (MRP)2, and MRP4. MK-571, a MRP inhibitor, was applied to measure the Km and Vmax of MRP2 and MRP4 in a vesicle transport assay. Single photon emission computed tomography (SPECT) was performed in normal rats and MRP2-deficient Eisai hyperbilirubinuria rats (EHBR) using [99mTc]MAG3 with and without MK-571. [99mTc]MAG3 uptake in adenosine triphosphate was significantly higher than that in adenosine monophosphate in vesicles that highly expressed MRP2 and MRP4. The affinity of [99mTc]MAG3 for MRP4 was higher than that for MRP2. Renal secretion via MRP2 and MRP4 was identified by comparing normal and EHBR rats with and without MK-571 on SPECT. [99mTc]MAG3 is transported via MRP2 and MRP4 on the apical membrane of renal proximal tubule cells. The affinity of MRP4 is higher than that of MRP2. Significance statement[99mTc]MAG3, widely used for evaluation of transplanted kidneys, diagnosis of tubular necrosis, and scintigraphic studies of tubular function, is transported via MRP2 and MRP4 on the apical membrane of renal proximal tubule cells. The affinity of MRP4 is higher than that of MRP2.

Leflunomide increased the renal exposure of acyclovir by inhibiting OAT1/3 and MRP2.

Rheumatoid arthritis patients can be prescribed a combination of immunosuppressive drug leflunomide (LEF) and the antiviral drug acyclovir to reduce the high risk of infection. Acyclovir is a substrate of organic anion transporter (OAT) 1/3 and multidrug resistance-associated protein (MRP) 2. Considering the extraordinarily long half-life of LEF’s active metabolite teriflunomide (TER) and the kidney injury risk of acyclovir, it is necessary to elucidate the potential impact of LEF on the disposition of acyclovir. Here we used a specific MRP inhibitor MK571 and probenecid (OAT1/3 and MRP2 inhibitor) to assess the effects of MRP2 and OAT1/3 on the pharmacokinetics and tissue distribution of acyclovir in rats. We showed that LEF and probenecid, but not MK571 significantly increased the plasma concentration of acyclovir. However, kidney and liver exposures of acyclovir were increased when coadministered with LEF, probenecid or MK571. The kidney/plasma ratio of acyclovir was increased to approximately 2-fold by LEF or probenecid, whereas it was increased to as much as 14.5-fold by MK571. Consistently, these drugs markedly decreased the urinary excretion of acyclovir. TER (0.5−100 μmol/L) dose-dependently increased the accumulation of acyclovir in MRP2-MDCK cells with an IC50 value of 4.91 μmol/L. TER (5 μmol/L) significantly inhibited the uptake of acyclovir in hOAT1/3-HEK293 cells. These results suggest that LEF/TER increased the kidney accumulation of acyclovir by inhibiting the efflux transporter MRP2, which increased its kidney/plasma ratio and renal injury risk. However, the inhibitory effects of LEF/TER on OAT1/3 reduced the tubular cells’ uptake of acyclovir and increased the plasma concentration.

ATP-binding Cassette Transporters Substantially Reduce Estimates of ALDH-positive Cancer Cells based on Aldefluor and AldeRed588 Assays

Aldehyde dehydrogenase (ALDH) assays measure the accumulated fluorescence of enzyme products. However, cancer cells frequently co-express ALDH and ATP-binding cassette (ABC) transporters, which might mediate efflux of ALDH assay reagents. We demonstrate expression of active multidrug resistance protein1 (MDR1), multidrug resistance-associated protein (MRP), and breast cancer resistance protein (BCRP) in CT26 cancer cells as well as expression of MRP and BCRP in HT29 cancer cells. Without transporter inhibition, only small portions of both cell types were estimated to be ALDH-positive based on Aldefluor and AldeRed588 assays. However, MK-571 (MRP inhibitor) and novobiocin (BCRP inhibitor) substantially increased the rate of ALDH-positive CT26 cells based on either Aldefluor or AldeRed588 assays. Verapamil (MDR inhibitor) did not influence assay results. MK-571 also substantially increased the rate of ALDH-positive HT29 cells. Limiting dilution assays demonstrated greater numbers of tumor-spheres formed by Aldefluor-positive compared to -negative CT26 cells selected in the presence of MK-571 or novobiocin but not in their absence. These results reveal that Aldefluor and AldeRed588 products are efficient substrates for MRP- and BCRP-mediated efflux and substantially reduce estimated ALDH positivity rates in cancer cells. These findings demonstrate that complete blockade of these transporters is important to ensure accurate ALDH assay results and to develop newer assay techniques.

NOX1/NADPH oxidase regulates the expression of multidrug resistance-associated protein 1 and maintains intracellular glutathione levels.

The involvement of superoxide-generating NADPH oxidase (NOX) in the cytotoxic effects of cigarette smoke extracts has been documented. However, the underlying molecular mechanisms and NOX isoform involved have not been fully clarified. Among the different NADPH oxidase isoforms identified so far, NOX1 and NOX4 were found to be expressed in rat H9c2 cardiomyocytes. When H9c2 cells were exposed to acrolein or methyl vinyl ketone (MVK), major toxic components of cigarette smoke extracts, a dose-dependentdecline in cell viability was observed. Unexpectedly, disruption of Nox1 as well as Nox4 significantly exacerbated cytotoxicity induced by acrolein or MVK. Compared with Nox4-disrupted cells, Nox1-disrupted cells were more vulnerable to acrolein and MVK at lower concentrations. Disruption of Nox1 markedly attenuated the levels of total and reduced glutathione (GSH) in H9c2 clones. Reduction in the cystine level in the culture medium to deplete intracellular GSH significantly exacerbated acrolein or MVK-induced cytotoxicity. Nox1 disruption neither attenuated the level of glutamate-cystine antiporter protein nor the activity of glutamate-cysteine ligase, both rate-limiting factors for GSH synthesis. On the other hand, increased expression of multidrug resistance-associated protein 1 (MRP1), which mediates glutathione efflux, was demonstrated in Nox1-disrupted cells. The augmented toxicity of acrolein and MVK in these cells was partially but significantly blunted in the presence of an MRP1 inhibitor, reversan. Taken together, these results show that NOX1/NADPH oxidase regulates the expression of MRP1 to maintain intracellular GSH levels in cardiomyocytes and protect against cytotoxic components of cigarette smoke extracts. A novel crosstalk between NOX1 and MRP1 was demonstrated in this study.

Different Efflux Transporter Affinity and Metabolism of 99mTc-2-Methoxyisobutylisonitrile and 99mTc-Tetrofosmin for Multidrug Resistance Monitoring in Cancer.

Little is known about the affinity and stability of 99mTc-labeled 2-methoxyisobutylisonitrile (99mTc-MIBI) and tetrofosmin (99mTc-TF) for imaging of multiple drug resistance transporters in cancer. We examined the affinity of 99mTc-labeled compounds for these transporters and their stability. 99mTc-MIBI and 99mTc-TF were incubated in vesicles expressing P-glycoprotein (MDR1), multidrug resistance-associated protein (MRP)1-4, or breast cancer resistance protein with and without verapamil (MDR1 inhibitor) or MK-571 (MRP inhibitor). Time activity curves of 99mTc-labeled compounds were established using SK-N-SH neuroblastoma, SK-MEL-28 melanoma, and PC-3 prostate adenocarcinoma cell lines, and transporter expression of multiple drug resistance was measured in these cells. The stability was evaluated. In vesicles, 99mTc-labeled compounds had affinity for MDR1 and MRP1. 99mTc-TF had additional affinity for MRP2 and MRP3. In SK-N-SH cells expressing MDR1 and MRP1, MK-571 produced the highest uptake of both 99mTc-labeled compounds. 99mTc-MIBI uptake with inhibitors was higher than 99mTc-TF uptake with inhibitors. 99mTc-TF was taken up more in SK-MEL-28 cells expressing MRP1 and MRP2 than PC-3 cells expressing MRP1 and MRP3. 99mTc-MIBI was metabolized, whereas 99mTc-TF had high stability. 99mTc-MIBI is exported via MDR1 and MRP1 (MRP1 > MDR1) at greater levels and more quickly compared to 99mTc-TF, which is exported via MDR1 and MRP1-3 (MRP1 > MDR1; MRP1, 2 > MRP3). Because 99mTc-MIBI is metabolized, clinical imaging for monitoring MDR and shorter examination times may be possible with an earlier scanning time on late phase imaging. 99mTc-TF has high stability and accurately reflects the function of MDR1 and MRP1-3.

In vitro evaluation of the hepatic disposition of colistin

ObjectiveColistin (polymyxin E) is a polycation antibiotic which is increasingly used (administered as Colistin Methane Sulfonate ‐ CMS) as a salvage therapy in (critically ill) patients with multidrug resistant gram‐negative infection[1]. Because colistin was introduced into clinical practice over 50 years ago, detailed trials on efficacy, safety and pharmacokinetics including underlying mechanisms are lacking. For instance, the metabolic fate of colistin is still largely unknown. Colistin is handled by glomerular filtration in the kidney, but almost completely reabsorbed in the renal tubuli. Less than 1% is eventually cleared by the kidney[2]. The purpose of the present study was to characterize the hepatic disposition of colistin and the role of transporters.MethodsTo characterize the metabolism of colistin, initial incubations were performed using colistin sulphate with rat liver microsomes (up to 1 mg protein/ml). Subsequently, rat and human hepatocytes in suspension (8 million/ml) were used as more complete in vitro drug metabolism tools. To measure hepatic uptake rates of colistin, the oil‐spin method was also applied to suspended hepatocytes (1 million cells/ml). Organic Anion Transporting Polypeptide (OATP) inhibitors (atazanavir and rifampicin) and Multidrug Resistance‐associated Protein (MRP) inhibitors (MK571 and benzbromarone) were used to evaluate the role of these uptake and efflux transporters in the disposition of colistin in rat and human hepatocytes. All samples were analysed by a validated LC‐MSMS method.ResultsColistin, when incubated at 2 mg/L, which is the PKPD target correlating with a high probability of clinical efficacy, was stable up to 4 hr during the incubation with rat liver microsomes. However, a slow time‐dependent decrease in colistin concentration (C0= 2 μg/mL) was found in suspended rat and human hepatocytes, with half‐life values of 9.9 h and 18 h, respectively. Based on these in vitro metabolism data, the predicted human in vivo blood clearance, assuming the well‐stirred model (fu=0.5), was 0.10 ml/min.kg body weight, explaining only a minor part of the total blood clearance based on clinical data [0.67 ml/min.kg body weight]. Both a saturable (transporter‐mediated) and non‐saturable (passive diffusion) component were found to contribute to colistin uptake by hepatocytes. When incubated at the clinically relevant concentration, the intracellular accumulation of colistin in rat and human hepatocytes was 1.9 and 6.2‐fold higher, respectively, in the presence of the MRP inhibitor benzbromarone (30 μM).ConclusionColistin was slowly metabolised in (rat > human) hepatocytes, apparently without involvement of CYP450 enzymes. MRP‐mediated efflux dominates the hepatic disposition of colistin, while one or more uptake transporters may also be involved. Transporter mediated uptake will be explored using Chinese hamster ovary (CHO) cell lines. These in vitro results will be used to simulate the in vivo disposition of colistin using Physiology‐based Pharmacokinetic modelling in Simcyp®.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Elucidation of the Intestinal Absorption Mechanism of Loganin in the Human Intestinal Caco-2 Cell Model.

Loganin, iridoid glycosides, is the main bioactive ingredients in the plant Strychnos nux-vomica L. and demonstrates various pharmacological effects, though poor oral bioavailability in rats. In this study, the intestinal absorption mechanism of loganin was investigated using the human intestinal Caco-2 cell monolayer model in both the apical-to-basolateral (A-B) and the basolateral-to-apical (B-A) direction; additionally, transport characteristics were systematically investigated at different concentrations, pHs, temperatures, and potential transporters. The absorption permeability (PappAB) of loganin, which ranged from 12.17 to 14.78 × 10−6cm/s, was high at four tested concentrations (5, 20, 40, and 80μM), while the major permeation mechanism of loganin was found to be passive diffusion with active efflux mediated by multidrug resistance-associated protein (MRP) and breast cancer resistance protein (BCRP). In addition, it was found that loganin was not the substrate of efflux transporter P-glycoprotein (P-gp) since the selective inhibitor (verapamil) of the efflux transporter exhibited little effects on the transport of loganin in the human intestinal Caco-2 cells. Meanwhile, transport from the apical to the basolateral side increased 2.09-fold after addition of a MRP inhibitor and 2.32-fold after addition of a BCRP inhibitor. In summary, our results clearly demonstrate, for the first time, a good permeability of loganin in the human intestinal Caco-2 cell model and elucidate, in detail, the intestinal absorption mechanism and the effects of transporters on iridoid glycosides compounds.

Severe hypokalemia due to a possible drug–drug interaction between vinblastine and antiretrovirals in a HIV-infected patient with Hodgkin’s lymphoma

A 60-year-old HIV-1 infected woman on antiretroviral therapy (emtricitabine/tenofovir, and ritonavir-boosted atazanavir) developed Hodgkin's lymphoma. The patient initiated ABVD (doxorubicin, bleomycin, vinblastine and dacarbazine) chemotherapy and presented with neutropenia and severe hypokalemia. Hypokalemia was considered as part of a proximal tubular renal dysfunction, and other causes of hypokalemia were excluded. Due to suspicion of drug--drug interactions between antiretrovirals and vinblastine, ritonavir-boosted atazanavir was switched to dolutegravir and the patient continued emtricitabine/tenofovir. In the subsequent ABVD cycles, no neutropenia or hypokalemia were observed. Vinblastine is metabolized by the hepatic P450 cytochrome isoenzyme CYP3A4, therefore, concomitant administration with protease inhibitors may increase plasma levels of vinblastine. Vinblastine is also a substrate and inhibitor of multidrug resistance-associated protein 2 (MRP2) transporter in the proximal renal tubule. Inhibition of this renal transporter could increase tenofovir renal toxicity. Our hypothesis is that the hypokalemia could be a result of a tenofovir-mediated tubular damage triggered by the increased vinblastine serum levels secondary to a CYP3A4 inhibition by ritonavir. To the best of our knowledge, this is the first report of severe hypokalemia and proximal tubular renal dysfunction as a result of a possible drug-drug interaction between vinblastine, tenofovir and ritonavir-boosted atazanavir.

Effect of Efflux Transporter Inhibition on the Distribution of Fluconazole in the Rat Brain.

Multidrug resistance-associated proteins (MRPs) and organic anion transporters (OATs) are expressed on the blood-brain barrier (BBB) and blood-cerebrospinal fluid barrier (BCSFB), preventing the entry of or the pumping out of numerous molecules. Fluconazole is widely used to treat fungal meningoencephalitis. The effect of these transporters on the distribution of fluconazole in the brain is unclear. We used microdialysis to compare the distribution of fluconazole in the rat brain with and without co-administration of probenecid, a MRP and OAT inhibitor. Additionally, we also observed the difference in fluconazole distribution between the two barriers. The results showed that probenecid increased the penetration of fluconazole into the BBB but did not alter the penetration of fluconazole into the BCSFB of rats. The penetration of the BBB and BCSFB by fluconazole did not statistically differ according to physiological condition. These results demonstrate that transporters that can be inhibited by probenecid may be involved in fluconazole resistance at the BBB and provide a laboratory basis for predicting brain extracellular fluid (ECF) concentration using the cerebrospinal fluid (CSF) concentration of fluconazole.

Transfer of Multidrug Resistance Among Acute Myeloid Leukemia Cells Via Extracellular Vesicles and Their Micrornas Cargo

Resistance of cancer cells to chemotherapy is a major issue in acute myeloid leukemia (AML) and is usually due to a clonal selection of resistant leukemic cells. Recently, a horizontal transfer of chemoresistance among tumor cells has been reported via extracellular vesicles (EVs). EVs are vesicles ranging from 0.03 to 1µm generated by almost all cell types. They carry cellular components such as nucleic acids, membrane and cytosolic proteins. By interacting with cells and by transferring their content, these vesicles could modify target cells’ phenotype. Because of their presence in biological fluids, a lot of studies have tried to use EVs as biomarkers.The aim of this research is to investigate the role of EVs in chemoresistance in AML. For this purpose, the sensitive strain of the promyelocytic leukemia HL60 cell line was compared to its multiresistant strain: HL60/AR. The latter overexpresses multidrug resistance-associated protein 1 (MRP1), an efflux pump conferring resistance to anthracyclines. The transfer of this resistance between the two strains is obtained by treating HL60 with EVs generated by HL60/AR.The production of EVs by HL60/AR was confirmed by transmission electron microscopy. EVs from around 100 to 800 nm were highlighted. EVs from HL60/AR were then isolated by ultracentrifugation and their interaction with HL60 was investigated by labeling EVs with a fluorescent lipid membrane linker. The transmission of this fluorescence to HL60 was monitored by flow cytometry and demonstrated a binding or an incorporation of EVs.In order to check if this interaction has an impact on HL60 resistance to daunorubicin, HL60 were treated with EVs, once for 20 hours or four consecutive times for 48 hours each. The resistance to daunorubicin of EVs-treated HL60 was then assessed by a MTT cytotoxicity assay and revealed that EVs increase survival of cells to daunorubicin. The resistant profile between the cells treated once or four times with EVs were somewhat different and highlighted a potential implication of nucleic acids cargo in this resistance transfer. Indeed, nucleic acids transferred by EVs would need more than 20 hours to impact target cells.Afterwards, we investigated the changes in phenotype that could be responsible for this increased resistance of EVs-treated cells. First, the expression of MRP1 was evaluated by a flow cytometry drug retention assay based on the fluorescence exhibited by daunorubicin and consisting in analysis by flow cytometry of the daunorubicin retention of cells with or without the presence of a specific MRP1 inhibitor. This experiment emphasized an expression of MRP1 after EVs treatment. Oxidative stress also plays an important role in daunorubicin cytotoxicity. Thus reactive oxygen species (ROS) generation assays were performed on EVs-treated cells. These cells showed a decreased production of daunorubicin-induced ROS.We finally analyzed the microRNAs content of isolated EVs. This was performed by TaqMan® Human MicroRNA Array on EVs from HL60/AR but also on EVs generated by HL60. This experiment aimed at highlighting the microRNAs cargo potentially involved in this chemoresistance transfer but also at identifying microRNAs differentially expressed between the two strains to serve as biomarker of chemoresistance in vivo. Out of the 380 microRNAs tested, 32 presented a differential expression between HL60 and HL60/AR. Among them, 5 microRNAs presented a more than 8-fold change between the two strains: miR-19b, miR-20a, miR-488, miR-519a and miR-523. miR-519a was the only microRNA more expressed in EVs produced by HL60, compared to EVs from HL60/AR.In conclusion, this study demonstrates the ability of resistant leukemic cells to transfer, at least partially, their chemoresistance via EVs. This could be due to a direct transfer of MRP1 transported by EVs but also to a nucleic acid transfer. In this context, some microRNAs have been highlighted for their high differential expression in EVs between sensitive and chemoresistant cells. These results bring out a potential clinical use of these circulating microRNAs as chemoresistance biomarkers in AML. DisclosuresNo relevant conflicts of interest to declare.

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.

MiR-145 sensitizes breast cancer to doxorubicin by targeting multidrug resistance-associated protein-1.

Multidrug resistance-associated protein 1 (MRP1) is an important efflux transporter and overexpression of MRP1 usually leads to chemoresistance in breast cancer. Here, we found MRP1 overexpressed in human breast cancer tissues and breast cancer cell lines (compared with normal breast tissues and cell line, respectively). And MRP1 level increased in doxorubicin resistant MCF-7 cells compared with parental MCF-7 cells. Increasing evidences suggest microRNAs (miRNAs) influence chemotherapy response. We found miR-145 level decreased in human breast cancer tissues, breast cancer cell lines and doxorubicin resistant MCF-7 cells, and inversely correlated with MRP1 expression level. In the process of constructing MCF-7 doxorubicin resistant cell line, escalating doxorubicin markedly decreased miR-145 level, following by increased MRP1 level. Further study showed, miR-145 suppressed MRP1 expression by directly targeting MRP1 3′-untranslated regions. Overexpression of miR-145 sensitized breast cancer cells to doxorubicin in vitro and enhanced to doxorubicin chemotherapy in vivo through inducing intracellular doxorubicin accumulation via inhibiting MRP1. Taken together, our study revealed miR-145 sensitizes breast cancer to doxorubicin by targeting MRP1 and indicated the potential application in developing MRP1 inhibitor.

Inhibition of multixenobiotic resistance transporters (MXR) by silver nanoparticles and ions in vitro and in Daphnia magna

The P-glycoprotein (P-gp, ABCB1) and multidrug resistance associated protein 1 (MRP1), important members of the ABC (ATP-binding cassette) transporters, protect cells and organisms via efflux of xenobiotics and are responsible for the phenomenon of multidrug or multixenobiotic resistance (MXR). In this study we first evaluated, in vitro, the interaction of silver nanoparticles (Ag NPs, 20, 23 and 27nm), Ag 200nm particles and Ag ions (AgNO3) with MXR efflux transporters using MDCKII and the P-gp over-expressing MDCKII-MDR1 cells and calcein-AM as a substrate of the transporters. Next the in vivo modulation of MXR activity was studied in Daphnia magna juveniles with the model P-gp and MRP1 inhibitors verapamil-HCl and MK571, respectively. The common environmental contaminants perfluorooctane sulfonate and bisphenol A, previously observed to interfere with the P-gp in vitro, also inhibited the efflux of calcein in vivo. Small-sized Ag NPs (with biomolecules present on the surface) and AgNO3 inhibited the MXR activity in daphnids and MDCKII-MDR1 cells, but abcb1 gene expression remained unchanged. Both Ag NPs and dissolved ions contributed to the effects. This study provides evidence of the interference of Ag NPs and AgNO3 with the MXR activity both in vitro and in D. magna, and should be taken into account when Ag NP toxicity is assessed.