Mrp2 Substrate Research Articles

Flurbiprofen inhibits cAMP transport by MRP4/ABCC4 increasing the potency of gemcitabine treatment in PDAC cell models

Pancreatic ductal adenocarcinoma (PDAC) remains a highly malignant cancer with a grim prognosis due to its early metastasis and resistance to current chemotherapies, such as Gemcitabine (GEM). We have previously demonstrated that cAMP exclusion by MRP4 is critical for PDAC cell proliferation, establishing this transporter as a promising prognostic marker and therapeutic target. In search for novel therapeutic options to improve GEM efficacy, we conducted a drug repositioning screening to identify potential inhibitors of cAMP transport by MRP4. Several non-steroidal anti-inflammatory drugs (NSAIDs) can inhibit the transport of certain MRP4 substrates. In this study, we assessed the efficacy of sixteen NSAIDs in inhibiting cAMP transport mediated by MRP4, identifying seven potent inhibitors based on their IC50 values. The most potent inhibitors were further tested for their effect on cell proliferation and migration. Flurbiprofen emerged as the most potent inhibitor of both MRP4-mediated cAMP transport and cell proliferation. Overexpression of MRP4 in BxPC-3 cells significantly increased GEM resistance, and co-administration of flurbiprofen with GEM markedly enhanced the latter's potency inhibiting PDAC cells proliferation. These findings position flurbiprofen as a potent inhibitor of cAMP transport by MRP4 and a promising adjunctive therapy to enhance GEM effectiveness in PDAC treatment.

Distinguishing Molecular Properties of OAT, OATP, and MRP Drug Substrates by Machine Learning.

The movement of organic anionic drugs across cell membranes is partly governed by interactions with SLC and ABC transporters in the intestine, liver, kidney, blood-brain barrier, placenta, breast, and other tissues. Major transporters involved include organic anion transporters (OATs, SLC22 family), organic anion transporting polypeptides (OATPs, SLCO family), and multidrug resistance proteins (MRPs, ABCC family). However, the sets of molecular properties of drugs that are necessary for interactions with OATs (OAT1, OAT3) vs. OATPs (OATP1B1, OATP1B3) vs. MRPs (MRP2, MRP4) are not well-understood. Defining these molecular properties is necessary for a better understanding of drug and metabolite handling across the gut-liver-kidney axis, gut-brain axis, and other multi-organ axes. It is also useful for tissue targeting of small molecule drugs and predicting drug-drug interactions and drug-metabolite interactions. Here, we curated a database of drugs shown to interact with these transporters in vitro and used chemoinformatic approaches to describe their molecular properties. We then sought to define sets of molecular properties that distinguish drugs interacting with OATs, OATPs, and MRPs in binary classifications using machine learning and artificial intelligence approaches. We identified sets of key molecular properties (e.g., rotatable bond count, lipophilicity, number of ringed structures) for classifying OATs vs. MRPs and OATs vs. OATPs. However, sets of molecular properties differentiating OATP vs. MRP substrates were less evident, as drugs interacting with MRP2 and MRP4 do not form a tight group owing to differing hydrophobicity and molecular complexity for interactions with the two transporters. If the results also hold for endogenous metabolites, they may deepen our knowledge of organ crosstalk, as described in the Remote Sensing and Signaling Theory. The results also provide a molecular basis for understanding how small organic molecules differentially interact with OATs, OATPs, and MRPs.

Natural Product Baohuoside I Impairs the Stability and Membrane Location of MRP2 Reciprocally Regulated by SUMOylation and Ubiquitination in Hepatocytes.

Epimedii Folium (EF) is a botanical dietary supplement to benefit immunity. Baohuoside I (BI), a prenylated flavonoid derived from EF, has exhibited the cholestatic risk before. Here, the mechanism of BI on the stability and membrane localization of liver MRP2, a bile acid exporter in the canalicular membrane of hepatocytes, was investigated. The fluorescent substrate of MRP2, CMFDA was accumulated in sandwich-cultured primary mouse hepatocytes (SCH) under BI stimulation, followed by reduced membrane MRP2 expression. BI triggered MRP2 endocytosis associated with oxidative stress via inhibition of the NRF2 signaling pathway. Meanwhile, BI promoted the degradation of MRP2 by reducing its SUMOylation and enhancing its ubiquitination level. Co-IP and fluorescence colocalization experiments all proved that MRP2 was a substrate protein for SUMOylation for SUMO proteins. CHX assays showed that SUMO1 prolonged the half-life of MRP2 and further increased its membrane expression, which could be reversed by UBC9 knockdown. Correspondingly, MRP2 accumulated in the cytoplasm by GP78 knockdown or under MG132 treatment. Additionally, the SUMOylation sites of MRP2 were predicted by the algorithm, and a conversion of lysines to arginines at positions 940 and 953 of human MRP2 caused its decreased stability and membrane location. K940 was further identified as the essential ubiquitination site for MRP2 by an in vitro ubiquitination assay. Moreover, the decreased ubiquitination of MRP2 enhanced the SUMOylation MRP2 and vice versa, and the crosstalk of these two modifiers could be disrupted by BI. Collectively, our findings indicated the process of MRP2 turnover from the membrane to cytoplasm at the post-translational level and further elucidated the novel toxicological mechanism of BI.

Screening of photosensitizers-ATP binding cassette (ABC) transporter interactions in vitro.

Aim: ATP-binding cassette (ABC) transporters are proteins responsible for the efflux of drug molecules from cancer cells, reducing the efficacy of anti-cancer treatments. This study assesses the susceptibility of a panel of clinically used photosensitizers to be transported by ABC transporters in vitro. Methods: The involvement of P-glycoprotein (P-gp/ABCB1), breast cancer resistance protein (BCRP/ABCG2), and multidrug resistance-associated protein 1 (MRP1/ABCC1) in the transport of 7 clinically utilized photosensitizers [benzoporphyrin derivative (BPD), temoporfin, redaporfin, talaporfin sodium, rose bengal, methylene blue, and indocyanine green] were investigated using human breast cancer cell lines following well-established protocols. Briefly, parental MCF-7 cells and sublines that overexpress P-gp (MCF-7 TX400), ABCG2 (MCF-7 MX100), or MRP1 (MCF-7/VP) were treated with photosensitizers with and without ABC transporter inhibitors. Intracellular levels of photosensitizers were measured using extraction method and flow cytometry to determine whether the ABC transporters are associated with efflux or uptake of photosensitizers. Results: The ABCG2 inhibitor (fumitremorgin C) and P-gp inhibitor (valspodar) effectively blocked the transport mediated by ABCG2 and P-gp of rose bengal and BPD. Redaporfin showed increased accumulation in the presence of valspodar with flow cytometry. Interestingly, MCF-7/VP cells were found to have reduced intracellular accumulation of rose bengal, which was restored with MRP1 inhibitor (MK571). The cell viability assay showed photodynamic therapy (PDT) resistance with Redaporfin in P-gp-overexpressing cells, BPD in ABCG2- and P-gp-overexpressing cells, and with Rose bengal in ABCG2-, P-gp- and MRP1-overexpressing cells, respectively. However, no change in intracellular retention was observed for other photosensitizers. Conclusion: In summary, our study provided new knowledge that temoporfin, talaporfin sodium, methylene blue, and indocyanine green are not substrates of ABCG2, P-gp, or MRP1. Redaporfin is a substrate for P-gp. BPD is a known substrate of ABCG2 and P-gp. Rose bengal is a substrate of ABCG2, P-gp, and MRP1. The results presented here indicate ABC transporter substrate status as a possible cause for cellular resistance to photodynamic therapy with rose bengal, redaporfin, and BPD.

Abstract A139: Preclinical characterization and prediction of human pharmacokinetics and efficacious dose for VIP236, a novel alpha V beta 3 binding small molecule-drug conjugate (SMDC)

Abstract VIP236 is a small molecule drug conjugate (SMDC) consisting of an alpha v beta 3 (αvβ3) integrin binder linked to an optimized camptothecin (optCPT) payload via a protease sensitive peptide sequence. The overall drug design strategy for VIP236 is targeted delivery to the tumor microenvironment (TME) with extracellular release of the opCPT by neutrophil elastase (NE) present in the TME. VIP236 is anticipated to kill tumor cells while reducing effects to normal tissue. VIP236 was characterized in preclinical pharmacokinetic (PK) and efficacy studies to predict its human PK and efficacious dose. Briefly, VIP236 was administered intravenously (IV) to mice, rats, and dogs to characterize both its in vivo PK (1-2 mg/kg) and its routes of excretion (bile-duct cannulated [BDC] rat at 10 mg/kg). VIP236 was evaluated in vitro for its protein binding characteristics and as a substrate of human drug transporters. In addition, VIP236 was dosed to MX-1 (breast cancer) xenografted mice at doses ranging from 26-40 mg/kg IV on a 3 days on/4 days off regimen to characterize its antitumor activity. VIP236 had low plasma clearance in mice, rats, and dogs (CL=2.7-4.4 mL/min/kg) and low volume of distribution (Vss = 0.11 to 0.25 L/kg). Terminal half-life ranged from 0.93 hour in mice to 3.07 hours in dogs. In BDC rats, VIP236 was primarily excreted in bile with biliary clearance accounting for essentially all the total body CL. There was minimal to no metabolites in rat bile and urine. In vitro VIP236 exhibited low permeability in MDCKII cell monolayers and was identified as a substrate of BSEP and MRP2. VIP236 was highly protein bound to plasma protein with fraction unbound being <5% in all species tested. VIP236 showed robust tumor regression in MX-1 xenografted mice at all doses tested. Allometric methods were used to predict a low human VIP236 CL (0.87-2.68 mL/min/kg) and Vss (0.10 L/kg). Translational pharmacokinetic/pharmacodynamic modeling was used to predict human efficacious doses and exposures by benchmarking VIP236 against irinotecan antitumor activity in MX-1 xenograft mice after 5 cycles of dosing. Based on this analysis, predicted human doses ranged from 1-5 mg/kg IV on a 2 days on/5 days off regimen. VIP236 is currently being evaluated in a first-in-human study in patients with advanced or metastatic solid tumors (NCT05712889). Citation Format: Harvey Wong, Beatrix Stelte-Ludwig, Hans-Georg Lerchen, Amy J Johnson, Melanie M Frigault, Raquel Izumi, Ahmed Hamdy. Preclinical characterization and prediction of human pharmacokinetics and efficacious dose for VIP236, a novel alpha V beta 3 binding small molecule-drug conjugate (SMDC) [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr A139.

Modulation of Multidrug Resistance Protein 1-mediated Transport Processes by the Antiviral Drug Ritonavir in Cultured Primary Astrocytes

The Multidrug Resistance Protein 1 (Mrp1) is an ATP-dependent efflux transporter and a major facilitator of drug resistance in mammalian cells during cancer and HIV therapy. In brain, Mrp1-mediated GSH export from astrocytes is the first step in the supply of GSH precursors to neurons. To reveal potential mechanisms underlying the drug-induced modulation of Mrp1-mediated transport processes, we investigated the effects of the antiviral drug ritonavir on cultured rat primary astrocytes. Ritonavir strongly stimulated the Mrp1-mediated export of glutathione (GSH) by decreasing the Km value from 200 nmol/mg to 28 nmol/mg. In contrast, ritonavir decreased the export of the other Mrp1 substrates glutathione disulfide (GSSG) and bimane-glutathione. To give explanation for these apparently contradictory observations, we performed in silico docking analysis and molecular dynamics simulations using a homology model of rat Mrp1 to predict the binding modes of ritonavir, GSH and GSSG to Mrp1. The results suggest that ritonavir binds to the hydrophilic part of the bipartite binding site of Mrp1 and thereby differently affects the binding and transport of the Mrp1 substrates. These new insights into the modulation of Mrp1-mediated export processes by ritonavir provide a new model to better understand GSH-dependent detoxification processes in brain cells.

Effect of budesonide on pulmonary activity of multidrug resistance-associated protein 1 assessed with PET imaging in rats

Multidrug resistance-associated protein 1 (MRP1/ABCC1) is a highly abundant efflux transporter in the lungs, which protects cells from toxins and oxidative stress and has been implicated in the pathophysiology of chronic obstructive pulmonary disease and cystic fibrosis. There is evidence from in vitro studies that the inhaled glucocorticoid budesonide can inhibit MRP1 activity. We used positron emission tomography (PET) imaging with 6-bromo-7-[11C]methylpurine ([11C]BMP), which is transformed in vivo into a radiolabeled MRP1 substrate, to assess whether intratracheally (i.t.) aerosolized budesonide affects pulmonary MRP1 activity in rats. Three groups of rats (n = 5–6 each) underwent dynamic PET scans of the lungs after i.t. aerosolization of either [11C]BMP alone, or [11C]BMP mixed with either budesonide (0.04 mg, corresponding to the maximum soluble dose) or the model MRP1 inhibitor MK571 (2 mg). From PET-measured radioactivity concentration-time curves, the rate constant describing radioactivity elimination from the right lung (kE,lung) and the area under the curve (AUClung) were calculated from 0 to 5 min after start of the PET scan as measures of pulmonary MRP1 activity. Co-administration of MK571 resulted in a pronounced decrease in kE,lung (25-fold, p < 0.0001) and an increase in AUClung (5.3-fold, p < 0.0001) when compared with vehicle-treated animals. In contrast, in budesonide-treated animals kE,lung and AUClung were not significantly different from the vehicle group. Our results show that i.t. aerosolized budesonide at an approximately 5 times higher dose than the maximum clinical dose leads to no change in pulmonary MRP1 activity, suggesting a lack of an effect of inhaled budesonide treatment on the MRP1-mediated cellular detoxifying capacity of the lungs. However, the strong effect observed for MK571 raises the possibility for the occurrence of transporter-mediated drug-drug interactions at the pulmonary epithelium with inhaled medicines.

Sinusoidal Organic Anion-Transporting Polypeptide 1B1/1B3 and Bile Canalicular Multidrug Resistance-Associated Protein 2 Play an Essential Role in the Hepatobiliary Disposition of a Synthetic Cyclic Dinucleotide (STING Agonist)

The liver is central to the elimination of many drugs from the body involving multiple processes and understanding of these processes is important to quantitively assess hepatic clearance of drugs. The synthetic STING (STimulator of INterferon Genes protein) agonist is a new class of drugs currently being evaluated in clinical trials as a potential anticancer therapy. In this study, we used ML00960317 (synthetic STING agonist) to investigate the hepatobiliary disposition of this novel molecular entity. A bile-duct cannulated (BDC) rat study indicated that biliary excretion is the major route of elimination for ML00960317 (84% of parent dose in bile). The human biliary clearance using in vitro sandwich cultured human hepatocyte model predicted significant biliary excretion of ML00960317 (biliary excretion index (BEI) of 47%). Moreover, the transport studies using transporter expressing cell lines, hepatocytes, and membrane vesicles indicated that ML00960317 is a robust substrate of OATP1B1, OATP1B3, and MRP2. Using relative expression factor approach, the combined contribution of OATP1B1 (fraction transported (ft) = 0.62) and OATP1B3 (ft = 0.31) was found to be 93% of the active uptake clearance of ML00960317 into the liver. Furthermore, OATP1B1 and OATP1B3-mediated uptake of ML00960317 was inhibited by rifampicin with IC50 of 6.5 and 2.3 μM, respectively indicating an in vivo DDI risk (R value of 1.5 and 2.5 for OATP1B1 and OATP1B3, respectively). These results highlighted an important role of OATP1B1, OATP1B3, and MRP2 in the hepatobiliary disposition of ML00960317. These pathways may act as rate-determining steps in the hepatic clearance of ML00960317 thus presenting clinical DDI risk.Graphical

Transport and Permeation Properties of Dapivirine: Understanding Potential Drug-Drug Interactions.

The dapivirine (DPV) vaginal ring was developed by the nonprofit International Partnership for Microbicides (IPM) for reducing the risk of HIV infection. A clinical study (IPM 028) showed that concomitant use of the DPV ring and miconazole (MIC) altered DPV pharmacokinetic profile. In this work, we investigated whether or not DPV transport and permeation contributed to the observed DPV-MIC interaction. Our study evaluated the interaction between DPV and several transporters that are highly expressed in the human female reproductive tract, including MRP1, MRP4, P-gp, BCRP, and ENT1, using vesicular and cellular systems. We also evaluated the impact of DPV/MIC on cellular tight junctions by monitoring transepithelial electrical resistance with the Ussing chamber. Lastly, we evaluated the effect of MIC on DPV permeability across human cervical tissue. Our findings showed that DPV was not a substrate of MRP1, MRP4, P-gp, BCRP, or ENT1 transporters. Additionally, DPV did not inhibit the activity of these transporters. DPV, MIC, and their combination also did not disrupt cellular tight junctions. MIC did not affect DPV tissue permeability but significantly reduced DPV tissue levels. Therefore, our results suggest that the DPV-MIC interaction is not due to these five transporters, altered tight junction integrity, or altered tissue permeability.

Slug Mediates MRP2 Expression in Non-Small Cell Lung Cancer Cells.

Transcriptional factors, such as Snail, Slug, and Smuc, that cause epithelial-mesenchymal transition are thought to regulate the expression of Ezrin, Radixin, and Moesin (ERM proteins), which serve as anchors for efflux transporters on the plasma membrane surface. Our previous results using lung cancer clinical samples indicated a correlation between Slug and efflux transporter MRP2. In the current study, we aimed to evaluate the relationships between MRP2, ERM proteins, and Slug in lung cancer cells. HCC827 cells were transfected by Mock and Slug plasmid. Both mRNA expression levels and protein expression levels were measured. Then, the activity of MRP2 was evaluated using CDCF and SN-38 (MRP2 substrates). HCC827 cells transfected with the Slug plasmid showed significantly higher mRNA expression levels of MRP2 than the Mock-transfected cells. However, the mRNA expression levels of ERM proteins did not show a significant difference between Slug-transfected cells and Mock-transfected cells. Protein expression of MRP2 was increased in Slug-transfected cells. The uptake of both CDCF and SN-38 was significantly decreased after transfection with Slug. This change was abrogated by treatment with MK571, an MRP2 inhibitor. The viability of Slug-transfected cells, compared to Mock cells, significantly increased after incubation with SN-38. Thus, Slug may increase the mRNA and protein expression of MRP2 without regulation by ERM proteins in HCC827 cells, thereby enhancing MRP2 activity. Inhibition of Slug may reduce the efficacy of multidrug resistance in lung cancer.

A novel experimental approach for the selective isolation and characterization of human RNase MRP

ABSTRACT RNase MRP is a ribonucleoprotein complex involved in the endoribonucleolytic cleavage of different RNAs. Mutations in the RNA component of the RNP are the cause of cartilage hair hypoplasia. Patients with cartilage hair hypoplasia are characterized by skeletal dysplasia. Biochemical purification of RNase MRP is desired to be able to study its biochemical function, composition and activity in both healthy and disease situations. Due to the high similarity with RNase P, a method to specifically isolate the RNase MRP complex is currently lacking. By fusing a streptavidin-binding RNA aptamer, the S1m-aptamer, to the RNase MRP RNA we have been able to compare the relative expression levels of wildtype and mutant MRP RNAs. Moreover, we were able to isolate active RNase MRP complexes. We observed that mutant MRP RNAs are expressed at lower levels and have lower catalytic activity compared to the wildtype RNA. The observation that a single nucleotide substitution at position 40 in the P3 domain but not in other domains of RNase MRP RNA severely reduced the binding of the Rpp25 protein subunit confirmed that the P3 region harbours the main binding site for this protein. Altogether, this study shows that the RNA aptamer tagging approach can be used to identify RNase MRP substrates, but also to study the effect of mutations on MRP RNA expression levels and RNase MRP composition and endoribonuclease activity.

Freshly isolated retinal capillaries to determine efflux transporter function at the inner BRB

Since it has been known that in vitro cell lines for analyzing drug transport at the inner blood-retinal barrier (BRB) do not completely retain several in vivo functions, new ex vivo/in vitro methods to evaluate drug transport across the inner BRB help us understand the role of this barrier in maintaining the homeostasis of vision and regulating drug distribution to the retina. To expand the limitations of existing in vitro approaches, we established a protocol to isolate fresh rat retinal capillaries as ex vivo model of the inner BRB. Fresh retinal capillaries were prepared by applying serial filtration steps and using density gradient centrifugation. We performed mRNA and protein analyses by reverse transcription-polymerase chain reaction and immunostaining that indicated expression of marker proteins such as facilitative glucose transporter 1 and claudin-5 in freshly isolated rat retinal capillaries. We also used fluorescent transporter substrates to characterize functional activity of organic anion transporter (Oat) 3, P-glycoprotein (P-gp), breast cancer resistance protein (Bcrp), and multidrug resistance-associated protein (Mrp) 4 in isolated retinal capillaries. Capillary luminal accumulation of fluorescent substrates of P-glycoprotein and Bcrp was decreased in the presence of transporter inhibitors. Moreover, luminal accumulation of the Oat3 and Mrp4 substrate, 8-(2-[fluoresceinyl]aminoethylthio) adenosine-3′,5′-cyclic monophosphate (8-[fluo]-cAMP), was reduced by substrates/inhibitors of Oat3 and Mrp4. In conclusion, our study shows that freshly isolated retinal capillaries retain marker protein expression and transporter functional activity. It is suggested that isolated retinal capillaries are a useful tool to study transport across the inner BRB. Using freshly isolated retinal capillaries, we anticipate applying this approach to determine the role of transporters at the inner BRB during pathophysiological states of the eye and evaluate the drug delivery to the retina.

Abstract 1125: Altered therapeutic response to metronomic chemotherapy in a metabolic syndrome model of mice bearing a mammary adenocarcinoma: Implication of intestinal ABC transporters

Abstract Breast cancer is one of the most prevalent and deadly cancers worldwide. Its prognosis depends on the cell type, as well as the age and comorbidities of each patient. A novel therapeutic approach is metronomic chemotherapy (MCT) characterized by chronic, low dose drug administration with therapeutic efficacy andlow or null toxicity as majoradvantages. The comorbidity that worsens cancer prognosis is metabolic syndrome (MS) characterized by obesity, insulinresistance, hyperglycemia, hypertriglyceridemia and hypercholesterolemia, and a chronic systemic inflammatory state. These conditions influence the intestinal biochemical barrier, altering the multidrug resistance-associated protein 2 (Mrp2) and P-glycoprotein (P-gp activities. Significantly, these two efflux pumps transport chemotherapeutics drugs such as cyclophosphamide (Cy). Our aim was to evaluate the effectiveness and toxicity of MCT with Cy in mice with metabolic syndrome bearing a mammary adenocarcinoma. Simultaneously, the activities of the intestinal efflux transporters were assessed. CBi male mice (5 weeks n=20/group), were fed with a chow diet (C) and a diet with 40% calories of fat (HFD) throughout the experiment. At 16 weeks, the development of MS was confirmed by biochemical and morphological parameters. The Mrp2 and P-gp activities were evaluated using the in vitro model of everted intestinal sacs. Once the MS features were settled, mice werechallenged s.c. with triple negative M-406 mammary adenocarcinoma (day 0); when the tumor was palpable, mice were distributed into 4 groups (n=8/group):GI: C no treatment, GII:C+Cy (30 mg/kg/day in drinking water), GIII: HFD no treatment and GIV: HFD+Cy. Efflux of Mrp2 substrate DNP-SG decreased 64% in HFD respect to C (P&amp;lt;0.05); transport rate of rhodamine 123 by P-gp decreased 55% in HFD vs C (P&amp;lt;0.05). At the end of the experiment (day 22), the tumor volume was lower in GIIvsGI and in GIVvsGIII (P&amp;lt;0.0001).The % inhibition of tumor growth in GII was higher than thatof GIV (P=0.052). The activity of Mrp2 or P-gpit was not modified by the tumor or MCT and remained decreased. It was observed a 30% decrease in body weight and leukopenia in GIV, indicating toxicity. We conclude that the induction of MS impairs the intestinal Mrp2 and P-gp activities bringing about differences in Cy absorption, leading to toxicity; in addition, MCT has lower antitumor effectiveness in animals with MS. The age-related changes and metabolic syndrome comorbid conditions urge to reconsider the therapeutics. Citation Format: María Manuela Barranco, Martín Habib, Bianca Di Carlo, Nicolás Sigal, María Sylvestre Begnis, Felipe Zechinatti, Antonela Del Giudice, María Jose Rico, Viviana Rosa Rosados, O. Graciela Scharovsky, Silvina Stella Maris Villanueva, Fabiana García, Leandro E Mainetti. Altered therapeutic response to metronomic chemotherapy in a metabolic syndrome model of mice bearing a mammary adenocarcinoma: Implication of intestinal ABC transporters [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1125.

Novel Symmetrical Cage Compounds as Inhibitors of the Symmetrical MRP4-Efflux Pump for Anticancer Therapy.

Within the last decades cancer treatment improved by the availability of more specifically acting drugs that address molecular target structures in cancer cells. However, those target-sensitive drugs suffer from ongoing resistances resulting from mutations and moreover they are affected by the cancer phenomenon of multidrug resistance. A multidrug resistant cancer can hardly be treated with the common drugs, so that there have been long efforts to develop drugs to combat that resistance. Transmembrane efflux pumps are the main cause of the multidrug resistance in cancer. Early inhibitors disappointed in cancer treatment without a proof of expression of a respective efflux pump. Recent studies in efflux pump expressing cancer show convincing effects of those inhibitors. Based on the molecular symmetry of the efflux pump multidrug resistant protein (MRP) 4 we synthesized symmetric inhibitors with varied substitution patterns. They were evaluated in a MRP4-overexpressing cancer cell line model to prove structure-dependent effects on the inhibition of the efflux pump activity in an uptake assay of a fluorescent MRP4 substrate. The most active compound was tested to resentisize the MRP4-overexpressing cell line towards a clinically relevant anticancer drug as proof-of-principle to encourage for further preclinical studies.

Carbamazepine Shows Plasma and Tissue Pharmacokinetic Interactions with Ajuga bracteosa Extract in Rats

AbstractCarbamazepine (CBZ) is the first-line anticonvulsant drug with a narrow therapeutic index (NTI) and is a substrate for CYP3A4 and MRP-2. Ajuga bracteosa (AB), family Lamiaceae is widely used to treat epilepsy, gastric diseases, and protects against liver damage in folk. It contains bioactive metabolites, which are powerful inhibitors of CYP3A4, CYP3A5, CYP19, CYP2C19 enzymes and P-gp transporter. Concomitant use of NTI drugs with herbs, like AB increase the chances of herb-drug interactions (HDIs). This study was aimed to analyze the Ajuga bracteosa crude extract (ABCE) and to investigate its effect on the pharmacokinetics of CBZ in rats. In the pre-treatment study, rats received ABCE (1000 mg/kg) orally for 14 days, followed by a single dose of CBZ (80 mg/kg) on the 15th day. In the co-administration study, single doses of ABCE and CBZ were administered concomitantly in one session. All the doses were administered in 0.5% carboxymethylcellulose (CMC) as a vehicle. HPLC analysis showed that extract contained 1.3 mg/g ursolic acid, 2.1 mg/g sitosterol and 2.9 mg/g stigmasterol. Non-compartmental pharmacokinetic analysis showed an increase in Cmax, AUC0-∞, MRT, and t1/2 with a decrease in tmax, Vd and Cl of CBZ in both, pre-treated and co-administered groups vs controls. An increase in CBZ concentration in liver tissue of both pre-treated as well as co-administered animals was observed as compared to control. The above results suggested possible HDIs between AB and CBZ thus, may warrant CBZ dose adjustment in epileptic patients with simultaneous administration of AB or its products.

Functional Alterations of Multidrug Resistance-Associated Proteins 2 and 5, and Breast Cancer Resistance Protein upon Snail-Induced Epithelial-Mesenchymal Transition in HCC827 Cells.

Our previous report indicated that Snail-induced epithelial-mesenchymal transition (EMT) enhanced P-glycoprotein (P-gp) function and drug resistance to P-gp substrate anticancer drug in a human non-small cell lung cancer (NSCLC) cell line, HCC827. Our objective is to evaluate the changes in the mRNA and protein expression levels and the functions of multidrug resistance-associated protein (MRP) 2, MRP5 and breast cancer resistance protein (BCRP). Snail-expressing HCC827 cells showed increased mRNA levels of Snail and a mesenchymal marker vimentin, and decreased mRNA levels of an epithelial marker E-cadherin after transduction, indicating that Snail had induced EMT consistent with our previous reports. The mRNA level of MRP2 was significantly decreased, while that of MRP5 remained unchanged, in Snail-expressing cells. The expression levels of MRP2 and MRP5 proteins in whole-cell homogenate were unchanged in Snail-expressing cells, but MRP5 protein showed significantly increased membrane localization. Snail-transduction increased the efflux transport of 5-(and-6)-carboxy-2',7'-dichlorofluorescein (CDCF), a substrate of MRP2, 3 and 5. This increase was blocked by MK571, which inhibits MRP1, 2, and 5. Toxicity of cisplatin, a substrate of MRP2 and 5, was significantly decreased in Snail-expressing cells. BCRP mRNA and protein levels were both decreased in Snail-expressing cells, which showed an increase in the intracellular accumulation of 7-ethyl-10-hydroxycamptothecin (SN-38), a BCRP substrate, resulting in reduced viability. These results suggested that MRP5 function appears to be increased via an increase in membrane localization, whereas the BCRP function is decreased via a decrease in the expression level in HCC827 cells with Snail-induced EMT.

Contribution of Uptake and Efflux Transporters to Oral Pharmacokinetics of Furosemide.

Furosemide is a widely used diuretic for treating excessive fluid accumulation caused by disease conditions like heart failure and liver cirrhosis. Furosemide tablet formulation exhibits variable pharmacokinetics (PK) with bioavailability ranging from 10 to almost 100%. To explain the variable absorption, we integrated the physicochemical, in vitro dissolution, permeability, distribution, and the elimination parameters of furosemide in a physiologically-based pharmacokinetic (PBPK) model. Although the intravenous PBPK model reasonably described the observed in vivo PK data, the reported low passive permeability failed to capture the observed data after oral administration. To mechanistically justify this discrepancy, we hypothesized that transporter-mediated uptake contributes to the oral absorption of furosemide in conjunction with passive permeability. Our in vitro results confirmed that furosemide is a substrate of intestinal breast cancer resistance protein (BCRP), multidrug resistance-associated protein 4 (MRP4), and organic anion transporting polypeptide 2B1 (OATP2B1), but it is not a substrate of P-glycoprotein (P-gp) and MRP2. We then estimated the net transporter-mediated intestinal uptake and integrated it into the PBPK model under both fasting and fed conditions. Our in vitro data and PBPK model suggest that the absorption of furosemide is permeability-limited, and OATP2B1 and MRP4 are important for its permeability across intestinal membrane. Further, as furosemide has been proposed as a probe substrate of renal organic anion transporters (OATs) for assessing clinical drug–drug interactions (DDIs) during drug development, the confounding effects of intestinal transporters identified in this study on furosemide PK should be considered in the clinical transporter DDI studies.

Tobacco Smoke and Inhaled Drugs Alter Expression and Activity of Multidrug Resistance-Associated Protein-1 (MRP1) in Human Distal Lung Epithelial Cells in vitro

Multidrug resistance-associated protein-1 (MRP1/ABCC1) is highly expressed in human lung tissues. Recent studies suggest that it significantly affects the pulmonary disposition of its substrates, both after pulmonary and systemic administration. To better understand the molecular mechanisms involved, we studied the expression, subcellular localization and activity of MRP1 in freshly isolated human alveolar epithelial type 2 (AT2) and type 1-like (AT1-like) cells in primary culture, and in the NCI-H441 cell line. Moreover, the effect of cigarette smoke extract (CSE) and a series of inhaled drugs on MRP1 abundance and activity was investigated in vitro. MRP1 expression levels were measured by q-PCR and immunoblot in AT2 and AT1-like cells from different donors and in several passages of the NCI-H441 cell line. The subcellular localization of the transporter was studied by confocal laser scanning microscopy and cell surface protein biotinylation. MRP1 activity was assessed by bidirectional transport and efflux experiments using the MRP1 substrate, 5(6)-carboxyfluorescein [CF; formed intracellularly from 5(6)-carboxyfluorescein-diacetate (CFDA)] in AT1-like and NCI-H441 cell monolayers. Furthermore, the effect of CSE as well as several bronchodilators and inhaled corticosteroids on MRP1 abundance and CF efflux was investigated. MRP1 protein abundance increased upon differentiation from AT2 to AT1-like phenotype, however, ABCC1 gene levels remained unchanged. MRP1 abundance in NCI-H441 cells were comparable to those found in AT1-like cells. The transporter was detected primarily in basolateral membranes of both cell types which was consistent with net basolateral efflux of CF. Likewise, bidirectional transport studies showed net apical-to-basolateral transport of CF which was sensitive to the MRP1 inhibitor MK-571. Budesonide, beclomethasone dipropionate, salbutamol sulfate, and CSE decreased CF efflux in a concentration-dependent manner. Interestingly, CSE increased MRP1 abundance, whereas budesonide, beclomethasone dipropionate, salbutamol sulfate did not have such effect. CSE and inhaled drugs can reduce MRP1 activity in vitro, which implies the transporter being a potential drug target in the treatment of chronic obstructive pulmonary disease (COPD). Moreover, MRP1 expression level, localization and activity were comparable in human AT1-like and NCI-H441 cells. Therefore, the cell line can be a useful alternative in vitro model to study MRP1 in distal lung epithelium.

MRP4 is responsible for the efflux transport of mycophenolic acid β-d glucuronide (MPAG) from hepatocytes to blood

Mycophenolic acid (MPA) has become a cornerstone of immunosuppressive therapy, in particular for transplant patients. In the gastrointestinal tract, the liver and the kidney, MPA is mainly metabolized into phenyl-β-d glucuronide (MPAG). Knowledge about the interactions between MPA/MPAG and membrane transporters is still fragmented. The aim of the present study was to explore these interactions with the basolateral hepatic MRP4 transporter. The inhibition of the MRP4-driven transport by various drugs which can be concomitantly prescribed was also evaluated. In vitro experiments using vesicles overexpressing MRP4 showed an ATP-dependent transport of MPAG driven by MRP4 (Michaelis–Menten constant of 233.9 ± 32.8 µM). MPA was not effluxed by MRP4. MRP4-mediated transport of MPAG was inhibited (from −43% to −84%) by ibuprofen, cefazolin, cefotaxime and micafungin. An in silico approach based on molecular docking and molecular dynamics simulations rationalized the mode of binding of MPAG to MRP4. The presence of the glucuronide moiety in MPAG was highlighted as key, being prone to make electrostatic and H-bond interactions with specific residues of the MRP4 protein chamber. This explains why MPAG is a substrate of MRP4 whereas MPA is not.

Human multidrug resistance protein 4 (MRP4) is a cellular efflux transporter for paracetamol glutathione and cysteine conjugates

Paracetamol (acetaminophen, APAP) overdose is a leading cause of acute drug-induced liver failure. APAP hepatotoxicity is mediated by the reactive metabolite N-acetyl-p-benzoquinone imine (NAPQI). NAPQI is inactivated by conjugation with glutathione (GSH) to APAP-GSH, which is further converted into its cysteine derivative APAP-CYS. Before necrosis of hepatocytes occurs, APAP-CYS is measurable in plasma of the affected patient and it has been proposed as an early biomarker of acetaminophen toxicity. APAP-GSH and APAP-CYS can be extruded by hepatocytes, but the transporters involved are unknown. In this study we examined whether ATP-binding cassette (ABC) transporters play a role in the cellular efflux of APAP, APAP-GSH, and APAP-CYS. The ABC transport proteins P-gp/ABCB1, BSEP/ABCB11, BCRP/ABCG2, and MRP/ABCC1-5 were overexpressed in HEK293 cells and membrane vesicles were produced. Whereas P-gp, BSEP, MRP3, MRP5, and BCRP did not transport any of the compounds, uptake of APAP-GSH was found for MRP1, MRP2 and MRP4. APAP-CYS appeared to be a substrate of MRP4 and none of the ABC proteins transported APAP. The results suggest that the NAPQI metabolite APAP-CYS can be excreted into plasma by MRP4, where it could be a useful biomarker for APAP exposure and toxicity. Characterization of the cellular efflux of APAP-CYS is important for its development as a biomarker, because plasma concentrations might be influenced by drug-transporter interactions and upregulation of MRP4.