MRP Inhibitor Research Articles

The multidrug resistance protein 4 is expressed and functionally active in isolated bladder from pig.

Multidrug resistance proteins type 4 (MRP4) and 5 (MRP5) play pivotal roles in the transport of cyclic nucleotides in various tissues. However, their specific functions within the lower urinary tract remain relatively unexplored. This study aimed to investigate the effect of pharmacological inhibition of MRPs on cyclic nucleotide signaling in isolated pig bladder. The relaxation responses of the bladder were assessed in the presence of the MRP inhibitor, MK571. The temporal changes in intra- and extracellular levels of cAMP and cGMP in stimulated tissues were determined by mass spectrometry. The gene (ABCC4) and protein (MRP4) expression were also determined. MK571 administration resulted in a modest relaxation effect of approximately 26% in carbachol-precontracted bladders. The relaxation induced by phosphodiesterase inhibitors such as cilostazol, tadalafil, and sildenafil was significantly potentiated in the presence of MK571. In contrast, no significant potentiation was observed in the relaxation induced by substances elevating cAMP levels or stimulators of soluble guanylate cyclase. Following forskolin stimulation, both intracellular and extracellular cAMP concentrations increased by approximately 15.8-fold and 12-fold, respectively. Similarly, stimulation with tadalafil + BAY 41-2272 resulted in roughly 8.2-fold and 3.4-fold increases in intracellular and extracellular cGMP concentrations, respectively. The presence of MK571 reduced only the extracellular levels of cGMP. This study reveals the presence and function of MRP4 transporters within the porcine bladder and paves the way for future research exploring the role of this transporter in both underactive and overactive bladder disorders.NEW & NOTEWORTHY This study investigates the impact of pharmacological inhibition of MRP4 and MRP5 transporters on cyclic nucleotide signaling in isolated pig bladders. MK571 administration led to modest relaxation, with enhanced effects observed in the presence of phosphodiesterase inhibitors. However, substances elevating cAMP levels remained unaffected. MK571 selectively reduced extracellular cGMP levels. These findings shed light on the role of MRP4 transporters in the porcine bladder, opening avenues for further research into bladder disorders.

Evaluation of Novel Diaza Cage Compounds as MRP Modulators in Cancer Cells.

Novel MRP modulators are needed to combat MRP-mediated multidrug resistance (MDR) in cancer cells. Anticancer drug resistance is the main problem in cancer therapy. Causative multidrug efflux pumps are attractive target structures for the development of inhibitors of their activity. We synthesized novel cage dimeric 1,4-dihydropyridines to evaluate them as MRP modulators in cancer cells targeting MRP1, MRP2, and MRP4. Cage compounds were synthesized by solution dimerization of monomeric 1,4-dihydropyridines and a final functionalization reaction. The MRP modulation was determined in cellular efflux assays by the use of the flow cytometry technique as well as cellular fluorescent measurements with each fluorescent substrate of the efflux pumps. Difluoro phenyl and methoxy or dimethoxy benzyl substitutions were most favourable for the MRP1 and MRP2 inhibition, whereas monofluor phenyl and dimethoxy benzyl substitutions were most favourable for the MRP4 inhibition. Effective inhibitors were identified that were demonstrated to restore the respective cancer cell line sensitivity for the anticancer drug as a proof-of-concept that encourages further preclinical studies.</p>.

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.

Cytotoxicity and reversal effect of sertraline, fluoxetine, and citalopram on MRP1- and MRP7-mediated MDR.

Cancer is one of the leading causes of death worldwide, and the development of resistance to chemotherapy drugs is a major challenge in treating malignancies. In recent years, researchers have focused on understanding the mechanisms of multidrug resistance (MDR) in cancer cells and have identified the overexpression of ATP-binding cassette (ABC) transporters, including ABCC1/MRP1 and ABCC10/MRP7, as a key factor in the development of MDR. In this study, we aimed to investigate whether three drugs (sertraline, fluoxetine, and citalopram) from the selective serotonin reuptake inhibitor (SSRI) family, commonly used as antidepressants, could be repurposed as inhibitors of MRP1 and MRP7 transporters and reverse MDR in cancer cells. Using a combination of in silico predictions and in vitro validations, we analyzed the interaction of MRP1 and MRP7 with the drugs and evaluated their ability to hinder cell resistance. We used computational tools to identify and analyze the binding site of these three molecules and determine their binding energy. Subsequently, we conducted experimental assays to assess cell viability when treated with various standard chemotherapies, both with and without the presence of SSRI inhibitors. Our results show that all three SSRI drugs exhibited inhibitory/reversal effects in the presence of chemotherapies on both MRP1-overexpressed cells and MRP7-overexpressed cells, suggesting that these medications have the potential to be repurposed to target MDR in cancer cells. These findings may open the door to using FDA-approved medications in combination therapy protocols to treat highly resistant malignancies and improve the efficacy of chemotherapy treatment. Our research highlights the importance of investigating and repurposing existing drugs to overcome MDR in cancer treatment.

Potential suppression of multidrug-resistance-associated protein 1 by coumarin derivatives: an insight from molecular docking and MD simulation studies

Human MRP1 protein plays a vital role in cancer multidrug resistance. Coumarins show promising pharmacological properties. Virtual screening, ADMET, molecular docking and molecular dynamics (MD) simulations were utilized as pharmacoinformatic tools to identify potential MRP1 inhibitors among coumarin derivatives. Using in silico ADMET, 50 hits were further investigated for their selectivity toward the nucleotide-binding domains (NBDs) of MRP1 using molecular docking. Accordingly, coumarin, its symmetrical ketone derivative Lig. No. 4, and Reversan were candidates for focused docking study with the NBDs domains compared with ATP. The result indicates that Lig. No. 4, with the best binding score, interacts with NBDs via hydrogen bonds with residues: GLN713, LYS684, GLY683, CYS682 in NBD1, and GLY1432, GLY771, SER769 and GLN1374 in NBD2, which mostly overlap with ATP binding residues. Moreover, doxorubicin (Doxo) was docked to the transmembrane domains (TMDs) active site of MRP1. Doxo interaction with TMDs was subjected to MD simulation in the NBDs free and occupied with Lig. No. 4 states. The results showed that Doxo interacts more strongly with TMD residues in inward facing feature of TMDs helices. However, when Lig. No. 4 exists in NBDs, Doxo interactions are different, and TMD helices show more outward-facing conformation. This result may suggest a partial competitive inhibition mechanism for the Lig. No. 4 on MRP1 compared with ATP. So, it may inhibit active complex formation by interfering with ATP entrance to NBDs and locking MRP1 conformation in outward-facing mode. This study suggests a valuable coumarin derivative that can be further investigated for potent MRP1 inhibitors. Communicated by Ramaswamy H. Sarma

Transport and interaction mechanism of four pesticide residues from Chaenomeles speciosa across Caco-2 cells

The presence of multiple pesticide residues in agricultural production highlights the need for studying mixture interaction during transepithelial transport. This study applied the Caco-2 cell model to investigate the interaction of four pesticide residues (carbendazim, epoxiconazole, phoxim, and chlorpyrifos) in Chaenomeles speciosa during transepithelial transport. Results demonstrated that co-treatment with pesticide mixtures generally increased the cumulative transport amount of carbendazim and epoxiconazole by 0.32–1.60 times and 0.32–0.98 times, respectively, compared to individual treatments. Notably, the combination of carbendazim and epoxiconazole displayed a significant synergistic effect. The use of transporter inhibitors and molecular docking analysis provided insights into the interaction mechanism, suggesting that the competitive inhibition of MRP2 and/or BCRP binding via π-bonds contributed to the inhibition of BL-to-AP efflux and a significant increase in AP-to-BL influx of carbendazim and epoxiconazole. The results are of great theoretical significance and practical value for risk assessment of multiple pesticide residues in agricultural products.

Establishment and Molecular Characterization of an In Vitro Model for PARPi-Resistant Ovarian Cancer.

Overcoming PARPi resistance is a high clinical priority. We established and characterized comparative in vitro models of acquired PARPi resistance, derived from either a BRCA1-proficient or BRCA1-deficient isogenic background by long-term exposure to olaparib. While parental cell lines already exhibited a certain level of intrinsic activity of multidrug resistance (MDR) proteins, resulting PARPi-resistant cells from both models further converted toward MDR. In both models, the PARPi-resistant phenotype was shaped by (i) cross-resistance to other PARPis (ii) impaired susceptibility toward the formation of DNA-platinum adducts upon exposure to cisplatin, which could be reverted by the drug efflux inhibitors verapamil or diphenhydramine, and (iii) reduced PARP-trapping activity. However, the signature and activity of ABC-transporter expression and the cross-resistance spectra to other chemotherapeutic drugs considerably diverged between the BRCA1-proficient vs. BRCA1-deficient models. Using dual-fluorescence co-culture experiments, we observed that PARPi-resistant cells had a competitive disadvantage over PARPi-sensitive cells in a drug-free medium. However, they rapidly gained clonal dominance under olaparib selection pressure, which could be mitigated by the MRP1 inhibitor MK-751. Conclusively, we present a well-characterized in vitro model, which could be instrumental in dissecting mechanisms of PARPi resistance from HR-proficient vs. HR-deficient background and in studying clonal dynamics of PARPi-resistant cells in response to experimental drugs, such as novel olaparib-sensitizers.

Abstracts from The Aerosol Society Drug Delivery to the Lungs 33.

Abstracts from The Aerosol Society Drug Delivery to the Lungs 33.

APC Loss Prevents Doxorubicin-Induced Cell Death by Increasing Drug Efflux and a Chemoresistant Cell Population in Breast Cancer.

Chemoresistance is a major health concern affecting cancer patients. Resistance is multifactorial, with one mechanism being the increased expression of ABC transporters (such as MDR1 and MRP1), which are drug efflux transporters capable of preventing intracellular accumulation of drugs and cell death. Our lab showed that the loss of Adenomatous Polyposis Coli (APC) caused an intrinsic resistance to doxorubicin (DOX), potentially through an enhanced tumor-initiating cell (TIC) population and the increased activation of STAT3 mediating the expression of MDR1 in the absence of WNT being activated. Here, in primary mouse mammary tumor cells, the loss of APC decreased the accumulation of DOX while increasing the protein levels of MDR1 and MRP1. We demonstrated decreased APC mRNA and protein levels in breast cancer patients compared with normal tissue. Using patient samples and a panel of human breast cancer cell lines, we found no significant trend between APC and either MDR1 or MRP1. Since the protein expression patterns did not show a correlation between the ABC transporters and the expression of APC, we evaluated the drug transporter activity. In mouse mammary tumor cells, the pharmacological inhibition or genetic silencing of MDR1 or MRP1, respectively, decreased the TIC population and increased DOX-induced apoptosis, supporting the use of ABC transporter inhibitors as therapeutic targets in APC-deficient tumors.

Modulatory Activity of the Copper Chelate, Copper N-(2-Hydroxy Acetophenone) Glycinate, in ABC-transporter-expressing Cell Lines.

Metal-containing compounds (e.g., platinum complexes) belong to the standard armamentarium of cancer chemotherapy. Copper N-(2-hydroxy acetophenone) glycinate (CuNG) exerts anticancer activity in vitro and in vivo and modulates drug resistance related to glutathione or P-glycoprotein. The potential of CuNG to interact with ATP-binding cassette (ABC) transporters has not been fully explored yet. This study focused on the modulatory effects of CuNG on four ABC transporters (MRP1, MRP1, BCRP, and P-glycoprotein). Cell viability, drug uptake and ABC transporter expression were measured by resazurin assays, flow cytometry, and ELISA in HL60AR, MDCKII-hBCRP, and Caco-2 cells. CuNG increased doxorubicin sensitivity of MRP1-over-expressing HL60AR with a similar efficacy as the control MRP1 inhibitor MK571. CuNG also increased MRP1's efflux activity. Comparable results were obtained with MDCKII cells over-expressing hBCRP. ELISA assays revealed that the expression of MRP1 in HL60AR cells and BCRP in MDCKII- cells was predominant but other ABC-transporters were also expressed at lower levels. Caco-2 cells expressed high levels of MRP2, but MRP1, BCRP, and P-glycoprotein were also expressed. In contrast to the two former cell lines, CuNG increased doxorubicin resistance and decreased efflux activity in Caco-2 cells. CuNG exerted different modulatory activities towards ABC-transporter-expressing cells. While CuNG-mediated ABC-transporter inhibition may improve tumor chemotherapy (like in HL60AR and MDCKII-hBCRP cells), CuNG-mediated enhanced ABC-transport (like in Caco-2 cells) may be a new strategy to ameliorate inflammatory diseases associated with decreased ABC-transporter expression such as ulcerative colitis.

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.

Studies on the interaction of five triazole fungicides with human renal transporters in cells

The widespread use of triazole fungicides in agricultural production poses a potential risk to human health. This study investigates the interaction of five triazole fungicides, i.e., tebuconazole, triticonazole, hexaconazole, penconazole, and uniconazole with human renal transporters, including OAT1, OAT3, OCT2, OCTN1, OCTN2, MATE1, MATE2-K, MRP2, MDR1, and BCRP, using transgenic cell models. For uptake transporters, triticonazole was the substrate of OAT1 and OAT3 and the inhibitor of OCT2. Tebuconazole and penconazole inhibited OCTN2 (100 μM), while tebuconazole, triticonazole, hexaconazole, penconazole, and uniconazole inhibited MATE1 (100 μM). Tebuconazole and hexaconazole inhibited MATE2-K (100 μM). All five triazole fungicides were not substrates or strong inhibitors of MRP2, MDR1, and BCRP efflux transporters. Penconazole inhibited OCT2 with IC50 = 1.12 μM. Penconazole and uniconazole inhibited MATE1 with IC50 = 0.94 μM and 0.87 μM. Tebuconazole and hexaconazole inhibited MATE2-K with IC50 = 0.96 μM and 1.04 μM, indicating that triazole fungicides may inhibit renal drug transporter activity at low concentrations. Triticonazole was transported by OAT1 and OAT3, and the Km values of triticonazole were 5.81 ± 1.75 and 47.35 ± 14.27, respectively. Tebuconazole and uniconazole were transported by OAT3, and the Km values of tebuconazole and uniconazole were 30.28 ± 7.18 and 87.61 ± 31.70, respectively, which may induce nephrotoxicity.

The inhibitory effect and mechanism of Yi-qi-hua-yu-jie-du decoction on the drug resistance of gastric cancer stem cells based on ABC transporters

BackgroundThe drug resistance of tumor stem cells is an obstacle in gastric cancer (GC) treatment and the high expression of ABC transporters is a classic reason for drug resistance. This study aimed to construct a reliable GC drug-resistant stem cell model and explore the inhibitory effect and mechanism of Yi-qi-hua-yu-jie-du medicated serum (YQHY) on the drug resistance of GC stem cells based on ABC transporters.MethodsThe tumor stemness biomarker CD44 was primary identification from WGCNA. The magnetic-activated cell sorting (MACS) method was used to separate CD44( +)BGC823/5-Fu (BGC823/5–Fu-CSCs) cells and the stemness characteristics were verified from multiple dimensions. Then, the drug resistance index and expression of ABC transporter genes MDR1 and MRP1 were detected in CD44(−)/CD44(+) cells. The inhibition and apoptosis rates of the cells administrated with YQHY or/and 5-Fu were calculated to confirm that YQHY can suppress the drug resistance of BGC823/5-Fu-CSCs. Afterwards, the effects of YQHY on the expression of MDR1 and MRP1 and the activation of the PI3K/Akt/Nrf2 pathway were observed. Finally, under the administration of IGF-1 (the activator of PI3K/Akt pathway) and Nrf2 siRNA, the mechanism of YQHY on reversing the drug resistance of BGC823/5–Fu-CSCs through inhibiting the expression of MDR1 and MRP1 via PI3K/Akt/Nrf2 was verified.ResultsCD44 was a reliable GC stemness biomarker and can be applied to construct the drug-resistant GC stem cell model CD44(+)BGC823/5-Fu. The growth rate, cell proliferation index, soft agar colony formation, expression of stemness specific genes and tumorigenesis ability of CD44(+)BGC823/5-Fu cells were significantly higher than those of CD44(−)BGC823/5-Fu cells. BGC823/5–Fu-CSCs exhibited strong drug resistance to 5-Fu and high expression of ABC transporter genes MDR1 and MRP1 compared to CD44(-) cells. YQHY increased the inhibition and apoptosis rates to efficiently inhibit the drug resistance of BGC823/5–Fu-CSCs. Meanwhile, it suppressed the expression of MDR1 and MRP1 and restrained the activation of PI3K/Akt/Nrf2 signaling pathway. Finally, it was found that IGF-1 partially restored the activation of PI3K/Akt/Nrf2 pathway, alleviated the inhibition of MDR1 and MRP1, blocked the proliferation-inhibitory and apoptosis-promotion effects. YQHY and si-Nrf2 synergistically suppressed the MDR1/MRP1 expression and the drug resistance of BGC823/5–Fu-CSCs.ConclusionsCD44 was a reliable GC stemness biomarker, and the high expression of ABC transporter genes MDR1 and MRP1 was an important feature of drug-resistant stem cells. YQHY inhibited the MDR1 and MRP1 expression via PI3K/Akt/Nrf2 pathway, thus reversing the drug resistance of BGC823/5–Fu-CSCs.

Transporters in Drug Development: International Transporter Consortium Update on Emerging Transporters of Clinical Importance.

During its fourth transporter workshop in 2021, the International Transporter Consortium (ITC) provided updates on emerging clinically relevant transporters for drug development. Previously highlighted and new transporters were considered based on up-to-date clinical evidence of their importance in drug-drug interactions and potential for altered drug efficacy and safety, including drug-nutrient interactions leading to nutrient deficiencies. For the first time, folate transport pathways (PCFT, RFC, and FRα) were examined in-depth as a potential mechanism of drug-induced folate deficiency and related toxicities (e.g., neural tube defects and megaloblastic anemia). However, routine toxicology studies conducted in support of drug development appear sufficient to flag such folate deficiency toxicities, whereas prospective prediction from in vitro folate metabolism and transport inhibition is not well enough established to inform drug development. Previous suggestion of a retrospective study of intestinal OATP2B1 inhibition to explain unexpected decreases in drug exposure were updated. Furthermore, when the absorption of a new molecular entity is more rapid and extensive than can be explained by passive permeability, evaluation of the OATP2B1 transport may be considered. Emerging research on hepatic and renal OAT2 is summarized, but current understanding of the importance of OAT2 was deemed insufficient to justify specific consideration for drug development. Hepatic, renal, and intestinal MRPs (MRP2, MRP3, and MRP4) were revisited. MRPs may be considered when they are suspected to be the major determinant of drug disposition (e.g., direct glucuronide conjugates); MRP2 inhibition as a mechanistic explanation for drug-induced hyperbilirubinemia remains justified. There were no major changes in recommendations from previous ITC whitepapers.

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.

Acyclovir Brain Disposition: Interactions with P-gp, Bcrp, Mrp2, and Oat3 at the Blood-Brain Barrier.

Acyclovir is effective in treating herpes simplex virus infections of the central nervous system. The purpose of this study was to investigate the interactions between acyclovir and the efflux pumps P-glycoprotein (P-gp), breast cancer resistance protein (Bcrp), multidrug resistance protein 2 (Mrp2), and organic anion transporter 3 (Oat3) at the blood-brain barrier (BBB). Acyclovir concentrations in the blood and brain were evaluated by microdialysis and high-performance liquid chromatography. Acyclovir pharmacokinetic parameters, including the area under the unbound blood concentration-time curve (AUCu,blood), the area under the unbound brain concentration-time curve (AUCu,brain), and the ratio of AUCu,brain to AUCu,blood (Kp.uu.brain), were evaluated in the presence and absence of elacridar (P-gp/Bcrp inhibitor, 7.5 mg/kg), tariquidar (P-gp/Bcrp inhibitor, 7.5 mg/kg), MK571 (Mrp2 inhibitor, 7.5 mg/kg), cyclosporine (P-gp/Bcrp/Mrp2 inhibitor, 25 mg/kg), and probenecid (Oat3 inhibitor, 50 mg/kg). The average AUCu,blood, AUCu,brain, and Kp.uu.brain in rats who received acyclovir (25 mg/kg, intravenous) alone were 1377.7 min·μg/ml, 435.4 min·μg/ml, and 31.6%, respectively. Probenecid drastically increased the AUCu,blood of acyclovir 1.73-fold, whereas coadministration with elacridar, tariquidar, MK571, and cyclosporine did not alter the blood pharmacokinetic parameters of acyclovir. Elacridar, tariquidar, MK571, cyclosporine, and probenecid significantly increased the AUCu,brain of acyclovir 1.51-, 1.54-, 1.47-, 1.95-, and 2.34-fold, respectively. Additionally, the Kp.uu.brain of acyclovir markedly increased 1.48-, 1.63-, 1.39-, 1.90-, and 1.35-fold following elacridar, tariquidar, MK571, cyclosporine, and probenecid administration, respectively. The present study demonstrated that P-gp, Bcrp, Mrp2, and Oat3 inhibition increased the penetration of acyclovir across the BBB, supporting the hypothesis that these efflux pumps restrict the distribution of acyclovir in the brain.

Inhibitory Effect of 5,7-Dimethoxyfl avone On Rosuvastatin Uptake From The Apical Membrane Of Caco-2 Cells

5,7-Dimethoxyfl avone (5,7-DMF) is a natural polymethoxyfl avone, and acts as an inhibitor of ABC efflux transporters (BCRP, MRP2 and/ or P-gp), and rosuvastatin is taken up via OATP2B1 and secreted by BCRP and MRP2. In this study, we investigated the effect of 5,7-DMF on the transport of rosuvastatin through the apical membrane and the accumulation in Caco-2 cells. Furthermore, we investigated whether the rosuvastatin accumulation is mediated by monocarboxylate transporter 1 (MCT1), in addition to OATP2B1. Coincubation with 5,7- DMF significantly increased the cellular accumulation of rosuvastatin from the apical membranes of Caco-2 cells cultured on the plastic dish. Coincubation with Ko-143 (a BCRP inhibitor) or MK-571 (an MRP inhibitor) significantly increased the rosuvastatin accumulation, whereas coincubation with verapamil (a P-gp inhibitor) did not. Coincubation with benzoic acid or pravastatin, which are substrates of both OATP2B1 and MCT1, signifi cantly decreased the rosuvastatin accumulation, whereas coincubation with estron-3-sulfate or sulfobromophthalein, which are substrates of both OATP2B1 and ABC efflux transporter, did not decrease and increased the rosuvastatin accumulation, respectively. On the other hand, the transcellular transport of rosuvastatin from basolateral to apical side (B-to-A transport) was markedly higher than that from apical to basolateral side (A-to-B transport). Coincubation with 5,7-DMF from the apical side significantly increased the A-to-B transport and the accumulation of rosuvastatin, whereas that from the basolateral side significantly decreased the B-to-A transport of rosuvastatin with increases in the accumulation. These results suggest that 5,7-DMF may increase the rosuvastatin accumulation as a result of the inhibition of rosuvastatin efflux mediated by BCRP and MRP2, and the rosuvastatin transport through the apical membrane may be mediated by not only OATB2B1 but also MCT1.

The hepatocyte export carrier inhibition assay improves the separation of hepatotoxic from non-hepatotoxic compounds

An in vitro/in silico method that determines the risk of human drug induced liver injury in relation to oral doses and blood concentrations of drugs was recently introduced. This method utilizes information on the maximal blood concentration (Cmax) for a specific dose of a test compound, which can be estimated using physiologically-based pharmacokinetic modelling, and a cytotoxicity test in cultured human hepatocytes. In the present study, we analyzed if the addition of an assay that measures the inhibition of bile acid export carriers, like BSEP and/or MRP2, to the existing method improves the differentiation of hepatotoxic and non-hepatotoxic compounds. Therefore, an export assay for 5-chloromethylfluorescein diacetate (CMFDA) was established. We tested 36 compounds in a concentration-dependent manner for which the risk of hepatotoxicity for specific oral doses and the capacity to inhibit hepatocyte export carriers are known. Compared to the CTB cytotoxicity test, substantially lower EC10 values were obtained using the CMFDA assay for several known BSEP and/or MRP2 inhibitors. To quantify if the addition of the CMFDA assay to our test system improves the overall separation of hepatotoxic from non-hepatotoxic compounds, the toxicity separation index (TSI) was calculated. We obtained a better TSI using the lower alert concentration from either the CMFDA or the CTB test (TSI: 0.886) compared to considering the CTB test alone (TSI: 0.775). In conclusion, the data show that integration of the CMFDA assay with an in vitro test battery improves the differentiation of hepatotoxic and non-hepatotoxic compounds in a set of compounds that includes bile acid export carrier inhibitors.

Aidi injection, a traditional Chinese medicine extract, reverses Gefitinib resistance in non-small cell lung cancer cells

IntroductionAidi injection is a traditional Chinese medicine containing multiple anti-tumour and immunomodulatory phytochemicals. While it synergistically enhances the efficacy of conventional chemotherapy in patients with non-small cell lung cancer (NSCLC), its effect on epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor (TKI) sensitivity in NSCLC remains unclear. This study aimed to investigate the effect and mechanisms of Aidi injection on the sensitivity of human NSCLC cell lines to gefitinib. MethodsEffect of Aidi injection on gefitinib sensitivity was assessed by MTT, colony formation and apoptosis assays in three NSCLC cell lines (A549, HCC827 and H1975). The association between the expression of genes and the overall survival was analysed by accessing TCGA lung adenocarcinoma datasets. The effect of Aidi injection on multidrug resistance-associated protein 2 (MRP2, encoded by ABCC2 gene) function and gefitinib sensitivity was compared between parental HEK293 cell and HEK293 overexpressing MRP2 cells (HEK/MRP2). The principle components of Aidi injection were determined by LC-MS/MS and the interaction of Aidi components with MRP2 protein explored using molecular docking. ResultsAidi injection enhanced gefitinib sensitivity (P < 0.05) and increased gefitinib-induced apoptosis rate (P < 0.05) in three NSCLC cell lines. Analysis of TCGA lung adenocarcinoma dataset showed that patients with a high expression of ABCC2 had significantly poorer survival (P = 0.007546). Aidi injection inhibited MRP2 activity in a concentration-dependant manner in HEK/MRP2 cells (P < 0.05). The combination of gefitinib with Aidi injection gave additive or weakly synergistic growth inhibition in HEK/MRP2 cells but exhibited antagonistic cytotoxicity in HEK293 cells. There were 11 main chemical components contained in Aidi injection, including astragalosides II and IV, cantharidin, etheutheroside E, ginsenosides Rb1, Rc, Rd, Re and Rg1, isofraxidin, and syringin. Docking studies showed strong affinity of Ginsenoside_Re towards MRP2. ConclusionsAidi injection may have the potential to be an adjuvant regimen to prevent and/or reverse common gefitinib resistance in NSCLC. The in silico and principle component analyses gives insight on ginsenoside_Re being a potential MRP2 inhibitor in Aidi injection.

In Vitro Assessment of the Drug-Drug Interaction Potential of Verinurad and Its Metabolites as Substrates and Inhibitors of Metabolizing Enzymes and Drug Transporters.

Verinurad is a selective uric acid transporter 1 (URAT1) inhibitor in development for the treatment of chronic kidney disease and heart failure. In humans, two major acyl glucuronide metabolites have been identified: direct glucuronide M1 and N-oxide glucuronide M8. Using in vitro systems recommended by regulatory agencies, we evaluated the interactions of verinurad, M1, and M8 with major drug-metabolizing enzymes and transporters and the potential for clinically relevant drug-drug interactions (DDIs). The IC50 for inhibition of CYP2C8, CYP2C9, and CYP3A4/5 for verinurad was ≥14.5 µM, and maximum free plasma concentration (Iu,max)/IC50 was <0.02 at the anticipated therapeutic Cmax and therefore not considered a DDI risk. Verinurad was not an inducer of CYP1A2, CYP2B6, or CYP3A4/5. Verinurad was identified as a substrate of the hepatic uptake transporter organic anion-transporting polypeptide (OATP) 1B3. Since verinurad hepatic uptake involved both active and passive transport, there is a low risk of clinically relevant DDIs with OATP, and further study is warranted. Verinurad was a substrate of the efflux transporters P-glycoprotein (P-gp) and breast cancer resistance protein (BCRP), and renal transporter organic anion transporter 1 (OAT1), although it is not considered a DDI risk in vivo because of dose-proportional pharmacokinetics (P-gp and BCRP) and limited renal excretion of verinurad (OAT1). M1 and M8 were substrates of multidrug resistance-associated protein (MRP) 2 and MRP4 and inhibitors of MRP2. Apart from verinurad being a substrate of OATP1B3 in vitro, the potential for clinically relevant DDIs involving verinurad and its metabolites as victims or perpetrators of metabolizing enzymes or drug transporters is considered low. SIGNIFICANCE STATEMENT: Drug transporters and metabolizing enzymes have an important role in the absorption and disposition of a drug and its metabolites. Using in vitro systems recommended by regulatory agencies, we determined that, apart from verinurad being a substrate of organic anion-transporting polypeptide 1B3, the potential for clinically relevant drug-drug interactions involving verinurad and its metabolites M1 and M8 as victims or perpetrators of metabolizing enzymes or drug transporters is considered low.