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1
Michaelis, Martin, Florian Rothweiler, Thomas Nerreter, Marijke Van Rikxoort, Mohsen Sharifi, Michael Wiese, Taravat Ghafourian, and Jindrich Cinatl. "Differential Effects of the Oncogenic BRAF Inhibitor PLX4032 (Vemurafenib) and its Progenitor PLX4720 on ABCB1 Function." Journal of Pharmacy & Pharmaceutical Sciences 17, no. 1 (April 6, 2014): 154. http://dx.doi.org/10.18433/j3tw24.
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PURPOSE: The clinically approved oncogenic BRAF inhibitor PLX4032 (vemurafenib) was shown to be a substrate of the ATP-binding cassette (ABC) transporter ABCB1. Here, we compared PLX4032 and its structurally closely related precursor compound PLX4720 for their interference with ABCB1 and the ABCB1-mediated compound transport using docking and cell culture experiments. METHODS: For the docking study of PLX4032 and PLX4720 with ABCB1, we analysed binding of both compounds to mouse Abcb1a and to human ABCB1 using a homology model of human ABCB1 based on the 3D structure of Abcb1a. Naturally ABCB1 expressing cells including V600E BRAF-mutated and BRAF wild-type melanoma cells and cells transduced with a lentiviral vector encoding for ABCB1 were used as cell culture models. ABCB1 expression and function were studied by the use of fluorescent and cytotoxic ABCB1 substrates in combination with ABCB1 inhibitors. RESULTS: Docking experiments predicted PLX4032 to interact stronger with ABCB1 than PLX4720. Experimental studies using different cellular models and structurally different ABCB1 substrates confirmed that PLX4032 interfered stronger with ABCB1 function than PLX4720. For example, PLX4032 (20µM) induced a 4-fold enhanced rhodamine 123 accumulation compared to PLX4720 (20µM) in ABCB1-transduced UKF-NB-3 cells and reduced the IC50 for the cytotoxic ABCB1 substrate vincristine in this model by 21-fold in contrast to a 9-fold decrease induced by PLX4720. CONCLUSIONS: PLX4032 exerted stronger effects on ABCB1-mediated drug transport than PLX4720. This indicates that small changes in a molecule can substantially modify its interaction with ABCB1, a promiscuous transporter that transports structurally different compounds.This article is open to POST-PUBLICATION REVIEW. Registered readers (see “For Readers”) may comment by clicking on ABSTRACT on the issue’s contents page.PURPOSE: The clinically approved oncogenic BRAF inhibitor PLX4032 (vemurafenib) was shown to be a substrate of the ATP-binding cassette (ABC) transporter ABCB1. Here, we compared PLX4032 and its structurally closely related precursor compound PLX4720 for their interference with ABCB1 and the ABCB1-mediated compound transport using docking and cell culture experiments. METHODS: For the docking study of PLX4032 and PLX4720 with ABCB1, we analysed binding of both compounds to mouse Abcb1a and to human ABCB1 using a homology model of human ABCB1 based on the 3D structure of Abcb1a. Naturally ABCB1 expressing cells including V600E BRAF-mutated and BRAF wild-type melanoma cells and cells transduced with a lentiviral vector encoding for ABCB1 were used as cell culture models. ABCB1 expression and function were studied by the use of fluorescent and cytotoxic ABCB1 substrates in combination with ABCB1 inhibitors. RESULTS: Docking experiments predicted PLX4032 to interact stronger with ABCB1 than PLX4720. Experimental studies using different cellular models and structurally different ABCB1 substrates confirmed that PLX4032 interfered stronger with ABCB1 function than PLX4720. For example, PLX4032 (20µM) induced a 4-fold enhanced rhodamine 123 accumulation compared to PLX4720 (20µM) in ABCB1-transduced UKF-NB-3 cells and reduced the IC50 for the cytotoxic ABCB1 substrate vincristine in this model by 21-fold in contrast to a 9-fold decrease induced by PLX4720. CONCLUSIONS: PLX4032 exerted stronger effects on ABCB1-mediated drug transport than PLX4720. This indicates that small changes in a molecule can substantially modify its interaction with ABCB1, a promiscuous transporter that transports structurally different compounds. This article is open to POST-PUBLICATION REVIEW. Registered readers (see “For Readers”) may comment by clicking on ABSTRACT on the issue’s contents page.
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Michaelis, Martin, Yvonne Voges, Florian Rothweiler, Fabian Weipert, Amara Zia-Ahmad, Jaroslav Cinatl, Andreas von Deimling, et al. "Testing of the Survivin Suppressant YM155 in a Large Panel of Drug-Resistant Neuroblastoma Cell Lines." Cancers 12, no. 3 (March 2, 2020): 577. http://dx.doi.org/10.3390/cancers12030577.
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The survivin suppressant YM155 is a drug candidate for neuroblastoma. Here, we tested YM155 in 101 neuroblastoma cell lines (19 parental cell lines, 82 drug-adapted sublines). Seventy seven (77) cell lines displayed YM155 IC50s in the range of clinical YM155 concentrations. ABCB1 was an important determinant of YM155 resistance. The activity of the ABCB1 inhibitor zosuquidar ranged from being similar to that of the structurally different ABCB1 inhibitor verapamil to being 65-fold higher. ABCB1 sequence variations may be responsible for this, suggesting that the design of variant-specific ABCB1 inhibitors may be possible. Further, we showed that ABCC1 confers YM155 resistance. Previously, p53 depletion had resulted in decreased YM155 sensitivity. However, TP53-mutant cells were not generally less sensitive to YM155 than TP53 wild-type cells in this study. Finally, YM155 cross-resistance profiles differed between cells adapted to drugs as similar as cisplatin and carboplatin. In conclusion, the large cell line panel was necessary to reveal an unanticipated complexity of the YM155 response in neuroblastoma cell lines with acquired drug resistance. Novel findings include that ABCC1 mediates YM155 resistance and that YM155 cross-resistance profiles differ between cell lines adapted to drugs as similar as cisplatin and carboplatin.
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Michaelis, Martin, Florian Rothweiler, Thomas Nerreter, Mohsen Sharifi, Taravat Ghafourian, and Jindrich Cinatl. "Karanjin interferes with ABCB1, ABCC1, and ABCG2." Journal of Pharmacy & Pharmaceutical Sciences 17, no. 1 (March 10, 2014): 92. http://dx.doi.org/10.18433/j3bw2s.
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PURPOSE: The prominent ATP-binding cassette (ABC) transporters ABCB1, ABCC1, and ABCG2 are involved in substance transport across physiological barriers and therefore in drug absorption, distribution, and elimination. They also mediate multi-drug resistance in cancer cells. Different flavonoids are known to interfere with different ABC transporters. Here, the effect of the furanoflavonol karanjin, a potential drug with antiglycaemic, gastroprotective, antifungal, and antibacterial effects, was investigated on ABCB1, ABCC1, and ABCG2-mediated drug transport in comparison to the flavonoids apigenin, genistein, and naringenin. METHODS: Cells expressing the relevant transporters (ABCB1: UKF-NB-3ABCB1, UKF-NB-3rVCR10; ABCC1: G62, PC-3rVCR20; ABCG2: UKF-NB-3ABCG2) were used in combination with specific fluorescent and cytotoxic ABC transporter substrates and ABC transporter inhibitors to study ABC transporter function. Moreover, the effects of the investigated flavonoids were determined on the ABC transporter ATPase activities. RESULTS: Karanjin interfered with drug efflux mediated by ABCB1, ABCC1, and ABCG2 and enhanced the ATPase activity of all three transporters. Moreover, karanjin exerted more pronounced effects than the control flavonoids apigenin, genistein, and naringenin on all three transporters. Most notably, karanjin interfered with ABCB1 at low concentrations being about 1µM. CONCLUSIONS: Taken together, these findings should be taken into account during further consideration of karanjin as a potential drug for different therapeutic indications. The effects on ABCB1, ABCC1, and ABCG2 may affect the pharmacokinetics of co-administered drugs. This article is open to POST-PUBLICATION REVIEW. Registered readers (see “For Readers”) may comment by clicking on ABSTRACT on the issue’s contents page.METHODS: Cells expressing the relevant transporters (ABCB1: UKF-NB-3ABCB1, UKF-NB-3rVCR10; ABCC1: G62, PC-3rVCR20; ABCG2: UKF-NB-3ABCG2) were used in combination with specific fluorescent and cytotoxic ABC transporter substrates and ABC transporter inhibitors to study ABC transporter function. Moreover, the effects of the investigated flavonoids were determined on the ABC transporter ATPase activities.RESULTS: Karanjin interfered with drug efflux mediated by ABCB1, ABCC1, and ABCG2 and enhanced the ATPase activity of all three transporters. Moreover, karanjin exerted more pronounced effects than the control flavonoids apigenin, genistein, and naringenin on all three transporters. Most notably, karanjin interfered with ABCB1 at low concentrations being about 1µM.CONCLUSIONS: Taken together, these findings should be taken into account during further consideration of karanjin as a potential drug for different therapeutic indications. The effects on ABCB1, ABCC1, and ABCG2 may affect the pharmacokinetics of co-administered drugs.
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Yin, Wei, Jianfeng Xu, and Yanjiao Mao. "Synergistic effects of autophagy inhibitors combined with cisplatin against cisplatin-resistant nasopharyngeal cancer cells." Biochemistry and Cell Biology 99, no. 3 (June 2021): 322–29. http://dx.doi.org/10.1139/bcb-2020-0283.
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This study explored the synergistic effects of autophagy inhibitors combined with cisplatin against cisplatin-resistant nasopharyngeal cancer cells by treating HNE-1 and cisplatin (diamminedichloroplatinum; DDP)-resistant HNE1/DDP nasopharyngeal cancer cell lines with DDP, autophagy inhibitors, or a combination of autophagy inhibitors and DDP. Cell viability was determined via MTT (colorimetric) and colony-forming assays, and the rate of apoptosis was determined using propidium iodide (PI) and annexin V double-staining. The expressions of proteins were determined by Western blotting. For our in-vivo studies, a murine xenograft model was established to evaluate the anti-tumor effects of the combination of autophagy inhibitor and DDP. The results showed that treatment with DDP increased the expressions of ATP-binding cassette sub-family B member 1 (ABCB1), ATP Binding Cassette Subfamily C Member 1 (ABCC1), and P-glycoprotein 1 (P-gp) in the HNE1/DDP cell lines. Treatment with chloroquine decreased the expression levels of ABCB1, ABCC1, and P-gp, and increased the formation of LC3-II and the expression levels of p62 in the HNE1/DDP cells. Additionally, the combination of autophagy inhibitors and DDP produced a synergistic effect on DDP-induced cell death and apoptosis. Furthermore, the combination of the autophagy inhibitor and DDP showed significant anti-tumor effects in the xenograft mouse model. In summary, autophagy inhibitors show synergistic anti-tumor effects with DDP in vitro against DDP-resistant nasopharyngeal cancer cells and in vivo in our xenograft murine model.
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Ibrahim, Mahmoud A. A., Khlood A. A. Abdeljawaad, Alaa H. M. Abdelrahman, Laila A. Jaragh-Alhadad, Hesham Farouk Oraby, Eslam B. Elkaeed, Gamal A. H. Mekhemer, et al. "Exploring Natural Product Activity and Species Source Candidates for Hunting ABCB1 Transporter Inhibitors: An In Silico Drug Discovery Study." Molecules 27, no. 10 (May 12, 2022): 3104. http://dx.doi.org/10.3390/molecules27103104.
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The P-glycoprotein (P-gp/ABCB1) is responsible for a xenobiotic efflux pump that shackles intracellular drug accumulation. Additionally, it is included in the dud of considerable antiviral and anticancer chemotherapies because of the multidrug resistance (MDR) phenomenon. In the search for prospective anticancer drugs that inhibit the ABCB1 transporter, the Natural Product Activity and Species Source (NPASS) database, containing >35,000 molecules, was explored for identifying ABCB1 inhibitors. The performance of AutoDock4.2.6 software to anticipate ABCB1 docking score and pose was first assessed according to available experimental data. The docking scores of the NPASS molecules were predicted against the ABCB1 transporter. Molecular dynamics (MD) simulations were conducted for molecules with docking scores lower than taxol, a reference inhibitor, pursued by molecular mechanics-generalized Born surface area (MM-GBSA) binding energy estimations. On the basis of MM-GBSA calculations, five compounds revealed promising binding affinities as ABCB1 inhibitors with ΔGbinding < −105.0 kcal/mol. The binding affinity and stability of the identified inhibitors were compared to the chemotherapeutic agent. Structural and energetical analyses unveiled great steadiness of the investigated inhibitors within the ABCB1 active site throughout 100 ns MD simulations. Conclusively, these findings point out that NPC104372, NPC475164, NPC2313, NPC197736, and NPC477344 hold guarantees as potential ABCB1 drug candidates and warrant further in vitro/in vivo tests.
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Natarajan, Karthika, Jasjeet Bhullar, Suneet Shukla, Mehmet Burcu, Suresh V. Ambudkar, and Maria R. Baer. "The Pim Kinase Inhibitor SGI-1776 Chemosensitizes Multidrug Resistant Cells by Both Inhibiting Drug Transport by ABCB1 and ABCG2 and Decreasing ABCB1 and ABCG2 Surface Expression On Cells That Overexpress Pim-1." Blood 120, no. 21 (November 16, 2012): 2462. http://dx.doi.org/10.1182/blood.v120.21.2462.2462.
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Abstract Abstract 2462 Overexpression of the ATP-binding cassette (ABC) cellular drug efflux proteins ABCB1 and ABCG2 on acute myeloid leukemia (AML) cells is associated with inferior chemotherapy outcomes. Nevertheless, inhibitors of drug transport have not improved treatment outcomes in clinical trials. The serine/threonine kinase Pim-1, encoded by a proto-oncogene originally identified as the proviral integration site in Moloney murine leukemia virus lymphomagenesis, is expressed in AML and is implicated in regulation of multiple key cellular processes, as well as drug resistance. Our group has shown that Pim-1 phosphorylates ABCB1 and ABCG2 and promotes their translocation to the cell surface, where they mediate drug efflux. The imidazo[1,2-b]pyridazine small molecule SGI-1776 (Tolero Pharmaceuticals, Inc. Salt Lake City, UT) is the first Pim kinase inhibitor to have entered clinical testing. SGI-1776 has been shown to sensitize ABCB1-overexpressing drug-resistant cells to ABCB1 substrate cancer chemotherapy drugs, but chemosensitization was found to be associated with direct inhibition of drug transport mediated by ABCB1. Moreover, while silencing of Pim-1 expression with siRNA was found to sensitize ABCG2-overexpressing cells to ABCG2 substrate chemotherapy drugs, the effects of SGI-1776 on resistance mediated by ABCG2 have not been studied. Therefore we studied the Pim-1-dependent and -independent effects of SGI-1776 on chemosensitivity of cells overexpressing ABCB1 and ABCG2. SGI-1776 at the Pim-1-inhibitory and non-cytotoxic concentration of 1 μM decreased the IC50s of ABCB1 and ABCG2 substrate drugs including daunorubicin and mitoxantrone 2- to 4-fold in leukemia and myeloma cell lines overexpressing ABCB1 and ABCG2, but had no effect on the IC50 of the non-substrate drug cytarabine, and no effect in parental cells. SGI-1776 also increased apoptosis of ABCB1- and ABCG2-overexpressing leukemia and myeloma cells exposed to ABCB1 and ABCG2 substrate chemotherapy drugs, respectively, and decreased their colony formation in the presence of substrate, but not non-substrate, chemotherapy drugs, with no effect on parental cells. We found that SGI-1776 decreased ABCB1 and ABCG2 surface expression, measured by flow cytometry, on K562/ABCB1 (p=0.013) and K562/ABCG2 (p=0.0038) leukemia cells, respectively, both of which express Pim-1 at high levels, without decrease in total cellular ABCB1 and ABCG2 expression, measured by Western blot analysis. In contrast, SGI-1776 had no effect on ABCB1 and ABCG2 surface expression on HL60/VCR leukemia and 8226/MR20 myeloma cells, which express ABCB1 and ABCG2, respectively, but express Pim-1 at lower levels. Thus SGI-1776 decreased ABCB1 and ABCG2 surface expression on cells that overexpress Pim-1, consistent with decreased cell surface translocation of ABCB1 and ABCG2 as a result of inhibition of Pim-1, but also chemosensitized cells expressing ABCB1 and ABCG2 in the absence of effects on ABCB1 and ABCG2 cell surface expression. We found that SGI-1776 indeed inhibited uptake of fluorescent substrates of both ABCB1 and ABCG2, measured by flow cytometry, in a concentration-dependent manner. We further determined that SGI-1776 inhibited ABCB1 and ABCG2 photoaffinity labeling with the transport substrate [125I]-IAAP and stimulated ABCB1 and ABCG2 ATPase activity, consistent with binding to drug-binding sites of ABCB1 and ABCG2 and inhibition of substrate transport by both proteins. Thus SGI-1776 both inhibits drug transport by ABCB1 and ABCG2 and decreases ABCB1 and ABCG2 surface expression on cells that overexpress Pim-1. Pim-1 is thought to be a clinically promising therapeutic target in AML and other malignancies, and other Pim kinase inhibitors are in preclinical and clinical development. Subsequent clinically applicable Pim kinase inhibitors should be characterized with regard to interactions with ABCB1 and ABCG2. In particular, while therapeutic strategies based on inhibition of drug transport mediated by ABCB1 with competitive inhibitors including PSC-833, zosuquidar and cyclosporin A have largely been clinically unsuccessful, inhibition of ABCB1 and ABCG2 cell surface translocation by Pim kinase inhibitors may have therapeutic implications. Disclosures: No relevant conflicts of interest to declare.
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Nosol, Kamil, Ksenija Romane, Rossitza N. Irobalieva, Amer Alam, Julia Kowal, Naoya Fujita, and Kaspar P. Locher. "Cryo-EM structures reveal distinct mechanisms of inhibition of the human multidrug transporter ABCB1." Proceedings of the National Academy of Sciences 117, no. 42 (October 5, 2020): 26245–53. http://dx.doi.org/10.1073/pnas.2010264117.
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ABCB1 detoxifies cells by exporting diverse xenobiotic compounds, thereby limiting drug disposition and contributing to multidrug resistance in cancer cells. Multiple small-molecule inhibitors and inhibitory antibodies have been developed for therapeutic applications, but the structural basis of their activity is insufficiently understood. We determined cryo-EM structures of nanodisc-reconstituted, human ABCB1 in complex with the Fab fragment of the inhibitory, monoclonal antibody MRK16 and bound to a substrate (the antitumor drug vincristine) or to the potent inhibitors elacridar, tariquidar, or zosuquidar. We found that inhibitors bound in pairs, with one molecule lodged in the central drug-binding pocket and a second extending into a phenylalanine-rich cavity that we termed the “access tunnel.” This finding explains how inhibitors can act as substrates at low concentration, but interfere with the early steps of the peristaltic extrusion mechanism at higher concentration. Our structural data will also help the development of more potent and selective ABCB1 inhibitors.
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Winter, Stuart S., Debbie M. Lovato, Hadya M. Khawaja, Bruce S. Edwards, Irena D. Steele, Susan M. Young, Tudor I. Oprea, Larry A. Sklar, and Richard S. Larson. "Identification of Off-Patent Drugs That Reverse Daunorubicin Efflux Mediated by ABCB1 in T-ALL Cells." Blood 108, no. 11 (November 16, 2006): 2603. http://dx.doi.org/10.1182/blood.v108.11.2603.2603.
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Abstract In acute leukemias, the overexpression of P-glycoprotein, encoded by the ATP Binding Cassette B1 (ABCB1) gene contributes to multidrug resistance (MDR), and is considered one of the major obstacles to successful cancer chemotherapy. The identification of small molecules that reverse ABCB1 efflux activity is critical for the successful treatment of cancers, including relapsed T-lineage acute lymphoblastic leukemia (T-ALL). However, the dose-limiting toxicities of many MDR reversal agents have restricted their use in clinical trials, and more effective and clinically applicable reversal agents remain to be identified. We have recently developed a T-ALL cell line that overexpresses ABCB1 and exhibits multiple drug resistance (MDR) to daunorubicin, prednisolone, and vincristine, but not L-asparaginase. The MDR can be reversed by suppression of ABCB1 expresssion with siRNA or 5 μM cyclosporine (CSA). Using this cell line, we developed a flow cytometry based, high-throughput screening assay that quantifies ABCB1 efflux using the fluorescent probe JC-1 (Swerts, et al Leuk Lymphoma 45:2221–8, 2004). We screened a library of 880 off-patent drugs for their ability to inhibit ABCB1 efflux at concentrations of 4 μM, and 19 compounds were identified, including CSA. Based on a published record of safe internal use in humans, 12 compounds were retained for further analysis (3 compounds were originally described as calcium channel blockers, 1 as sodium channel blocker, 1 as ACE inhibitor, 1 as dopamine uptake inhibitor, 1 as Topoisomerase II inhibitor, 2 as steroids, 2 as antifungal and 1 as immunosuppressant). We determined the 50% inhibitory concentration (IC50) of drug for ABCB1 efflux, the efflux reversal concentration of drug that rescued DNR-induced T-ALL cell death (ECrev50), and the corresponding in vitro toxic dose in 50% of treated T-ALL cells (TD50). Due to space limitations, detailed data could not be presented in the abstract. To our knowledge, 8 of the 12 compounds have not been previously described as ABCB1 inhibitors. These compounds may be useful as chemosensitizers or as lead compounds for development of improved ABCB1 inhibitors in T-ALL as well as other cancers.
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Wang, Jingqiu, Dong-Hua Yang, Yuqi Yang, Jing-Quan Wang, Chao-Yun Cai, Zi-Ning Lei, Qiu-Xu Teng, Zhuo-Xun Wu, Linguo Zhao, and Zhe-Sheng Chen. "Overexpression of ABCB1 Transporter Confers Resistance to mTOR Inhibitor WYE-354 in Cancer Cells." International Journal of Molecular Sciences 21, no. 4 (February 19, 2020): 1387. http://dx.doi.org/10.3390/ijms21041387.
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The overexpressing ABCB1 transporter is one of the key factors leading to multidrug resistance (MDR). Thus, many ABCB1 inhibitors have been found to be able to overcome ABCB1-mediated MDR. However, some inhibitors also work as a substrate of ABCB1, which indicates that in order to achieve an effective reversal dosage, a higher concentration is needed to overcome the pumped function of ABCB1, which may concurrently increase the toxicity. WYE-354 is an effective and specific mTOR (mammalian target of rapamycin) inhibitor, which recently has been reported to reverse ABCB1-mediated MDR. In the current study, 3-(4,5-dimethylthiazolyl)-2,5-diphenyltetrazolium bromide (MTT) assay was carried out to determine the cell viability and reversal effect of WYE-354 in parental and drug-resistant cells. Drug accumulation was performed to examine the effect of WYE-354 on the cellular accumulation of chemotherapeutic drugs. The ATPase (adenosine triphosphatase) activity of the ABCB1 transporter in the presence or absence of WYE-354 was conducted in order to determine the impact of WYE-354 on ATP hydrolysis. Western blot analysis and immunofluorescence assay were used to investigate the protein molecules related to MDR. In addition, the interaction between the WYE-354 and ABCB1 transporter was investigated via in silico analysis. We demonstrated that WYE-354 is a substrate of ABCB1, that the overexpression of the ABCB1 transporter decreases the efficacy of WYE-354, and that the resistant WYE-354 can be reversed by an ABCB1 inhibitor at a pharmacological achievable concentration. Furthermore, WYE-354 increased the intracellular accumulation of paclitaxel in the ABCB1-mediated MDR cell line, without affecting the corresponding parental cell line, which indicated that WYE-354 could compete with other chemotherapeutic drugs for the ABCB1 transporter substrate binding site. In addition, WYE-354 received a high score in the docking analysis, indicating a strong interaction between WYE-354 and the ABCB1 transporter. The results of the ATPase analysis showed that WYE-354 could stimulate ABCB1 ATPase activity. Treatment with WYE-354 did not affect the protein expression or subcellular localization of the ABCB1. This study provides evidence that WYE-354 is a substrate of the ABCB1 transporter, implicating that WYE-354 should be avoided for use in ABCB1-mediated MDR cancer.
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Dohse, Marius, Robert W. Robey, Cornelia Brendel, Susan Bates, Andreas Neubauer, and Christian Scharenberg. "Efflux of the Tyrosine Kinase Inhibitors Imatinib and Nilotinib (AMN107) Is Mediated by ABCB1 (MDR1)-Type P-Glycoprotein." Blood 108, no. 11 (November 16, 2006): 1367. http://dx.doi.org/10.1182/blood.v108.11.1367.1367.
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Abstract Chronic myeloid leukemia (CML) is a cancer affecting the hematopoietic stem cell (HSC) and is considered to be caused by the unregulated activity of the BCR-ABL tyrosine kinase. The development of tyrosine kinase inhibitors (TKIs) such as imatinib has revolutionized CML-therapy. However, resistance to imatinib has become a clinical reality and several potential mechanisms have been postulated to explain the imatinib resistance observed in CML cells. Among these, inherent protective mechanisms, such as overexpression of ATP-binding cassette (ABC) transporters, may lead to relapse or drug-resistance in CML patients receiving imatinib. Imatinib has previously been suggested to be a substrate for ABCB1, but conflicting data have been reported regarding this issue. However, whether the novel second-generation TKI nilotinib is a substrate for ABCB1 has not been investigated previously. Thus, we sought to characterize the interactions between ABCB1 and imatinib and nilotinib. We report that the TKIs imatinib and nilotinib show a reversible inhibition of ABCB1-mediated Rhodamine efflux in murine HSCs at clinically achieved concentrations. Imatinib abrogates Rhodamine efflux in HSC at 5 μM while nilotinib had a similar effect at a concentration of 0.2 μM. Additional studies with ABCB1-transfected HEK293 cells confirm nilotinib as a more potent inhibitor of ABCB1 than imatinib. Cytotoxicity studies using ABCB1-transfected HEK293 cells with Doxorubicine demonstrated inhibition of ABCB1-mediated efflux of Doxorubicine with increasing TKI concentration. In order to determine whether imatinib and nilotinib are in fact substrates and function not only as inhibitors of ABCB1, we performed experiments with various concentrations of radiolabeled imatinib and nilotinib. When ABCB1-transfected cells were incubated with 0.2 μM 14C-imatinib, intracellular concentrations were significantly lower compared to cells incubated with 14C-imatinib in the presence of different established ABCB1 inhibitors. However, transfected cells that were incubated in the presence of 14C-Imatinib at 1 μM or higher did not display reduced intracellular drug levels. In studies with the novel TKI nilotinib, ABCB1-expressing cells retained significantly less 14C-nilotinib compared to cells incubated with nilotinib in the presence of ABCB1 inhibitors, even at micromolar concentrations (33 % at 1 μM). However, similar to high concentrations with imatinib, the accumulation defect was not observed at supraphysiological concentrations of nilotinib. These experiments demonstrate both TKIs to be substrates for ABCB1 and indicate that TKI-efflux has a threshold and that TKIs at higher concentrations overwhelm the extrusion capacity of ABC transporters, offering an explanation for the conflicting reports as to whether TKIs are indeed substrates or only inhibitors. Since ABCB1 is known to be expressed on HSCs, we speculate that ABCB1 expression could mediate resistance in CML stem cells against imatinib and to the novel second-generation TKI nilotinib. Moreover, with ABCB1 being typically active at the Blood-Brain Barrier, decreased cerebrospinal levels of TKIs may have important clinical impact for the treatment of BCR-ABL positive B-ALL.
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Davies, Andrea, Athina Giannoudis, Claire M. Lucas, Robert J. Harris, Paul W. Manley, Munir Pirmohamed, and Richard E. Clark. "Characterisation of Nilotinib Transport in Chronic Myeloid Leukaemia Cells." Blood 110, no. 11 (November 16, 2007): 2364. http://dx.doi.org/10.1182/blood.v110.11.2364.2364.
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Abstract We have previously shown that imatinib is a substrate for both the efflux transporter P-glycoprotein (ABCB1; MDR-1 gene product) and the influx transporter human Organic Cation Transporter 1 (hOCT1; SLCA22). High hOCT1 and low ABCB1 expression levels in chronic myeloid leukaemia (CML) patients correlate with improved clinical outcome (Wang L. et al. Clinical Pharmacology and Therapeutics; epub June 13th 2007). The second generation tyrosine kinase inhibitor nilotinib is 30-fold more potent than imatinib and is effective in imatinib-resistant patients. However, its cellular transport has not been characterised. The CML cell line KCL22 was used as it has low basal hOCT1 expression. Drug transport studies of 14C-radiolabelled nilotinib (kind gift from Novartis) were performed using high expressing hOCT1 transfected KCL22 cells, with or without OCT1 and ABCB1 inhibitors. Further vectorial ABCB1 efflux studies were performed on confluent monolayers of ABCB1 transfected Type II Madin Darby canine kidney (MDCKII) cells. Nilotinib efflux was not modulated in the presence of the ABCB1 inhibitors PSC-833 (10μM), tariquidar (500nM) and verapamil (500μM), and no evidence of transport via ABCB1 was seen in the transfected ABCB1 MDCKII cells. Nilotinib influx was not modulated in hOCT1 over-expressing cells, or in the presence of OCT1 inhibitors prazosin (100μM) and amantadine (500μM). Nilotinib uptake was also not modulated by the hOCT1 substrate tetraethylamine (TEA) (5μM), however nilotinib inhibited TEA uptake. The distribution coefficient (logD) of nilotinib was 2.1, compared with 0.81 for imatinib, demonstrating high lipophilicity for nilotinib. Flow cytometry revealed greater suppression of phospho-CrKL (a surrogate marker for BCR-ABL blockade) with nilotinib after 1 hour when compared with imatinib, regardless of hOCT1 expression, indicating that nilotinib is effective at BCR-ABL suppression even at low hOCT1 levels, in contrast to imatinib. Nilotinib was not a substrate for the ABCB1 and hOCT1 transporters; however it may be a potential inhibitor of hOCT1. Therefore the level of expression of ABCB1 and hOCT1 in CML is unlikely to be an indicator of the clinical outcome for patients receiving nilotinib, in contrast to what we have shown for imatinib. Nilotinib may therefore be effective in patients who have low hOCT1 expression before start of therapy.
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van Tellingen, Olaf, Mark C. de Gooijer, Stefanie Zuidema, Amber Meurs, Ceren H. Çitirikkaya, Nikkie Venekamp, and Jos H. Beijnen. "EXTH-72. CONTINUOUS INFUSION STUDIES REVEAL THE POTENCY OF ELACRIDAR TO ACT AS A PHARMACO-ENHANCER FOR TREATMENT OF INTRACRANIAL DISEASES BY INHIBITING ABCB1 AND ABCG2 AT THE BLOOD-BRAIN BARRIER." Neuro-Oncology 22, Supplement_2 (November 2020): ii103. http://dx.doi.org/10.1093/neuonc/noaa215.426.
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Abstract The blood-brain barrier (BBB) is a formidable hurdle to successful pharmacotherapy of intracranial diseases. ABCB1 and ABCG2 are efflux transporters that play an important role in the BBB, keeping substances out of the brain. Elacridar and tariquidar are third-generation ABCB1-inhibitors developed for treatment of multidrug-resistant tumors. Later, they were shown to also inhibit ABCG2. We aim to improve pharmacotherapy of brain cancer by concomitant use of potentially effective drugs with elacridar. This study was undertaken to determine the relationship between the plasma concentration of the inhibitor and the brain-to-plasma (B/P) ratio of (model) substrate drugs in order to assess which type of drug may best qualify taking into account clinically achievable plasma levels of the inhibitor. We used Abcg2;Abcb1a/b double knockout (DKO), Abcb1a/b KO, Abcg2 KO and wild-type mice receiving a cocktail of 9 drugs at a fixed low dose plus a range of doses of inhibitor by 3-h intraperitoneal infusion to achieve steady-state conditions. DKO mice are the reference for complete inhibition, while single KO mice allow interrogation of the other transporter when using dual substrate drugs. Complete inhibition of Abcb1 by elacridar requires plasma levels of about 1000 nM. Inhibition of Abcg2 is more difficult. For erlotinib and palbociclib about 1000 nM of elacridar is sufficient, but other more profound substrate drugs (e.g. vemurafenib and afatinib) do not reach the B/P ratios achieved in DKO mice, even at 4000 nM. The improvement in B/P ratio that can be reached differs per substrate. Compounds like palbociclib benefit markedly from elacridar with B/P ratios rising from 0.25 to 7, whereas others (e.g. erlotinib and dasatinib) increase from about 0.10 to only 0.40. Thus, elacridar is an efficient pharmaco-enhancer of ABCB1 substrates and weaker ABCG2 substrates, but is not able to improve brain delivery of drugs that are profound ABCG2 substrates.
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Wenzel, Tim, Thomas Büch, Nicole Urban, Ulrike Weirauch, Katrin Schierle, Achim Aigner, Michael Schaefer, and Hermann Kalwa. "Restoration of MARCK enhances chemosensitivity in cancer." Journal of Cancer Research and Clinical Oncology 146, no. 4 (February 13, 2020): 843–58. http://dx.doi.org/10.1007/s00432-020-03149-2.
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Abstract Purpose Increased ATP-binding-cassette (ABC) transporter activity is a major cause of chemotherapy resistance in cancer. The ABC transporter family member ABCB1 is often overexpressed in colorectal cancer (CRC). Phosphatidylinositol-4,5-bisphosphat (PI(4,5)P2)-dependent pathways are involved in the regulation of ABCB1 function. The protein Myristoylated Alanine-Rich C-Kinase Substrate (MARCKS) is a pivotal regulator of PI(4,5)P2 and inactivated in many CRC cancers via genetic deletion or hyperphosphorylation. Therefore, MARCKS may critically impact ABCB1. Methods CRC samples as well as CRC cell lines were tested for a connection between MARCKS and ABCB1 via immunofluorescence and Western-blot analysis. ABCB1 function was studied via calcein influx assay under treatment with known ABCB1 inhibitors (verapamil, tariquidar) as well as the kinase inhibitor bosutinib. ABCB1 internalization and MARCKS translocation was analyzed via confocal microscopy exploiting the endocytosis inhibitors chlorpromazine and dynasore. Abundance of PI(4,5)P2 was monitored by intramolecular fluorescence resonance energy transfer (FRET). Reproductive cell survival was studied via colorimetric WST-1 and clonogenic assays in combination with exposure to the chemotherapeutics doxorubicin and 5-fuorouracil (5-FU). Results We found increased ABCB1 expression in MARCKS negative CRC patient tumor samples and established CRC cell lines. Mechanistically, the reconstitution of MARCKS function via recombinant expression or the pharmacological inhibition of MARCKS phosphorylation led to a substantial decrease in ABCB1 activity. In CRC cells, bosutinib treatment resulted in a MARCKS translocation from the cytosol to the plasma membrane, while simultaneously, ABCB1 was relocated to intracellular compartments. Inhibition of MARCKS phosphorylation via bosutinib rendered cells more sensitive to the chemotherapeutics doxorubicin and 5-FU. Conclusions Cells devoid of MARCKS function showed incomplete ABCB1 internalization, leading to higher ABCB1 activity enhancing chemoresistance. Vice versa our data suggest the prevention of MARCKS inhibition by reversing hyperphosphorylation or genomic restoration after deletion as two promising approaches to overcome tumor cell resistance towards chemotherapeutic ABCB1 substrates.
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Sorf, Ales, Simona Sucha, Anselm Morell, Eva Novotna, Frantisek Staud, Alzbeta Zavrelova, Benjamin Visek, Vladimir Wsol, and Martina Ceckova. "Targeting Pharmacokinetic Drug Resistance in Acute Myeloid Leukemia Cells with CDK4/6 Inhibitors." Cancers 12, no. 6 (June 16, 2020): 1596. http://dx.doi.org/10.3390/cancers12061596.
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Pharmacotherapy of acute myeloid leukemia (AML) remains challenging, and the disease has one of the lowest curability rates among hematological malignancies. The therapy outcomes are often compromised by the existence of a resistant AML phenotype associated with overexpression of ABCB1 and ABCG2 transporters. Because AML induction therapy frequently consists of anthracycline-like drugs, their efficiency may also be diminished by drug biotransformation via carbonyl reducing enzymes (CRE). In this study, we investigated the modulatory potential of the CDK4/6 inhibitors abemaciclib, palbociclib, and ribociclib on AML resistance using peripheral blood mononuclear cells (PBMC) isolated from patients with de novo diagnosed AML. We first confirmed inhibitory effect of the tested drugs on ABCB1 and ABCG2 in ABC transporter-expressing resistant HL-60 cells while also showing the ability to sensitize the cells to cytotoxic drugs even as no effect on AML-relevant CRE isoforms was observed. All tested CDK4/6 inhibitors elevated mitoxantrone accumulations in CD34+ PBMC and enhanced accumulation of mitoxantrone was found with abemaciclib and ribociclib in PBMC of FLT3-ITD- patients. Importantly, the accumulation rate in the presence of CDK4/6 inhibitors positively correlated with ABCB1 expression in CD34+ patients and led to enhanced apoptosis of PBMC in contrast to CD34− samples. In summary, combination therapy involving CDK4/6 inhibitors could favorably target multidrug resistance, especially when personalized based on CD34− and ABCB1-related markers.
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Linke, Dinah, Lukas Donix, Claudia Peitzsch, Holger H. H. Erb, Anna Dubrovska, Manuel Pfeifer, Christian Thomas, Susanne Fuessel, and Kati Erdmann. "Comprehensive Evaluation of Multiple Approaches Targeting ABCB1 to Resensitize Docetaxel-Resistant Prostate Cancer Cell Lines." International Journal of Molecular Sciences 24, no. 1 (December 30, 2022): 666. http://dx.doi.org/10.3390/ijms24010666.
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Docetaxel (DTX) is a mainstay in the treatment of metastatic prostate cancer. Failure of DTX therapy is often associated with multidrug resistance caused by overexpression of efflux membrane transporters of the ABC family such as the glycoprotein ABCB1. This study investigated multiple approaches targeting ABCB1 to resensitize DTX-resistant (DTXR) prostate cancer cell lines. In DU145 DTXR and PC-3 DTXR cells as well as age-matched parental controls, the expression of selected ABC transporters was analyzed by quantitative PCR, Western blot, flow cytometry and immunofluorescence. ABCB1 effluxing activity was studied using the fluorescent ABCB1 substrate rhodamine 123. The influence of ABCB1 inhibitors (elacridar, tariquidar), ABCB1-specific siRNA and inhibition of post-translational glycosylation on DTX tolerance was assessed by cell viability and colony formation assays. In DTXR cells, only ABCB1 was highly upregulated, which was accompanied by a strong effluxing activity and additional post-translational glycosylation of ABCB1. Pharmacological inhibition and siRNA-mediated knockdown of ABCB1 completely resensitized DTXR cells to DTX. Inhibition of glycosylation with tunicamycin affected DTX resistance partially in DU145 DTXR cells, which was accompanied by a slight intracellular accumulation and decreased effluxing activity of ABCB1. In conclusion, DTX resistance can be reversed by various strategies with small molecule inhibitors representing the most promising and feasible approach.
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Huličiak, Martin, Ivan Vokřál, Ondřej Holas, Ondřej Martinec, František Štaud, and Lukáš Červený. "Evaluation of the Potency of Anti-HIV and Anti-HCV Drugs to Inhibit P-Glycoprotein Mediated Efflux of Digoxin in Caco-2 Cell Line and Human Precision-Cut Intestinal Slices." Pharmaceuticals 15, no. 2 (February 18, 2022): 242. http://dx.doi.org/10.3390/ph15020242.
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The inhibition of P-glycoprotein (ABCB1) could lead to increased drug plasma concentrations and hence increase drug toxicity. The evaluation of a drug’s ability to inhibit ABCB1 is complicated by the presence of several transport-competent sites within the ABCB1 binding pocket, making it difficult to select appropriate substrates. Here, we investigate the capacity of antiretrovirals and direct-acting antivirals to inhibit the ABCB1-mediated intestinal efflux of [3H]-digoxin and compare it with our previous rhodamine123 study. At concentrations of up to 100 µM, asunaprevir, atazanavir, daclatasvir, darunavir, elbasvir, etravirine, grazoprevir, ledipasvir, lopinavir, rilpivirine, ritonavir, saquinavir, and velpatasvir inhibited [3H]-digoxin transport in Caco-2 cells and/or in precision-cut intestinal slices prepared from the human jejunum (hPCIS). However, abacavir, dolutegravir, maraviroc, sofosbuvir, tenofovir disoproxil fumarate, and zidovudine had no inhibitory effect. We thus found that most of the tested antivirals have a high potential to cause drug–drug interactions on intestinal ABCB1. Comparing the Caco-2 and hPCIS experimental models, we conclude that the Caco-2 transport assay is more sensitive, but the results obtained using hPCIS agree better with reported in vivo observations. More inhibitors were identified when using digoxin as the ABCB1 probe substrate than when using rhodamine123. However, both approaches had limitations, indicating that inhibitory potency should be tested with at least these two ABCB1 probes.
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Makrecka-Kuka, Marina, Jelena Vasiljeva, Pavels Dimitrijevs, and Pavel Arsenyan. "Olaparib Conjugates with Selenopheno[3,2-c]quinolinone Inhibit PARP1 and Reverse ABCB1-Related Multidrug Resistance." Pharmaceutics 14, no. 12 (November 23, 2022): 2571. http://dx.doi.org/10.3390/pharmaceutics14122571.
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The restoration of the efficacy of antitumor medicines is a cornerstone in the combat with multidrug resistant (MDR) cancers. The overexpression of the ABCB1 transporter is a major obstacle to conventional doxorubicin therapy. The synergy of ABCB1 suppression and PARP1 activity inhibition that hampers malignant cell DNA repair could be a powerful tool in anticancer therapy. Herein, we report the design and synthesis of three novel olaparib conjugates with selenophenoquinolinones, their ability to reverse doxorubicin resistance in uterus sarcoma cells as well as their mechanism of action. It was found that the most potent chemosensitizer among studied compounds preserves PARP1 inhibitory activity and attenuates cells’ resistance to doxorubicin by inhibiting ABCB1 transporter activity. These results demonstrate that the conjugation of PARP inhibitors with selenophenoquinolinones is a prospective direction for the development of agents for the treatment of MDR cancers.
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McCorkle, J. Robert, Justin W. Gorski, Jinpeng Liu, McKayla B. Riggs, Anthony B. McDowell, Nan Lin, Chi Wang, Frederick R. Ueland, and Jill M. Kolesar. "Lapatinib and poziotinib overcome ABCB1-mediated paclitaxel resistance in ovarian cancer." PLOS ONE 16, no. 8 (August 4, 2021): e0254205. http://dx.doi.org/10.1371/journal.pone.0254205.
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Conventional frontline treatment for ovarian cancer consists of successive chemotherapy cycles of paclitaxel and platinum. Despite the initial favorable responses for most patients, chemotherapy resistance frequently leads to recurrent or refractory disease. New treatment strategies that circumvent or prevent mechanisms of resistance are needed to improve ovarian cancer therapy. We established in vitro paclitaxel-resistant ovarian cancer cell line and organoid models. Gene expression differences in resistant and sensitive lines were analyzed by RNA sequencing. We manipulated candidate genes associated with paclitaxel resistance using siRNA or small molecule inhibitors, and then screened the cells for paclitaxel sensitivity using cell viability assays. We used the Bliss independence model to evaluate the anti-proliferative synergy for drug combinations. ABCB1 expression was upregulated in paclitaxel-resistant TOV-21G (q < 1x10-300), OVCAR3 (q = 7.4x10-156) and novel ovarian tumor organoid (p = 2.4x10-4) models. Previous reports have shown some tyrosine kinase inhibitors can inhibit ABCB1 function. We tested a panel of tyrosine kinase inhibitors for the ability to sensitize resistant ABCB1-overexpressing ovarian cancer cell lines to paclitaxel. We observed synergy when we combined poziotinib or lapatinib with paclitaxel in resistant TOV-21G and OVCAR3 cells. Silencing ABCB1 expression in paclitaxel-resistant TOV-21G and OVCAR3 cells reduced paclitaxel IC50 by 20.7 and 6.2-fold, respectively. Furthermore, we demonstrated direct inhibition of paclitaxel-induced ABCB1 transporter activity by both lapatinib and poziotinib. In conclusion, lapatinib and poziotinib combined with paclitaxel synergizes to inhibit the proliferation of ABCB1-overexpressing ovarian cancer cells in vitro. The addition of FDA-approved lapatinib to second-line paclitaxel therapy is a promising strategy for patients with recurrent ovarian cancer.
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Ivnitski-Steele, Irena, Richard S. Larson, Debbie M. Lovato, Hadya M. Khawaja, Stuart S. Winter, Tudor I. Oprea, Larry A. Sklar, and Bruce S. Edwards. "High-Throughput Flow Cytometry to Detect Selective Inhibitors of ABCB1, ABCC1, and ABCG2 Transporters." ASSAY and Drug Development Technologies 6, no. 2 (April 2008): 263–76. http://dx.doi.org/10.1089/adt.2007.107.
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Pajeva, Ilza K, Christoph Globisch, and Michael Wiese. "Combined Pharmacophore Modeling, Docking, and 3D QSAR Studies of ABCB1 and ABCC1 Transporter Inhibitors." ChemMedChem 4, no. 11 (November 2, 2009): 1883–96. http://dx.doi.org/10.1002/cmdc.200900282.
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Vasiljeva, Jelena, Marina Makrecka-Kuka, Ilona Domracheva, Karlis Vilks, Pavels Dimitrijevs, and Pavel Arsenyan. "Development of prospective non-toxic inhibitors of ABCB1 activity and expression in a series of selenophenoquinolinones." New Journal of Chemistry 46, no. 16 (2022): 7424–32. http://dx.doi.org/10.1039/d2nj00340f.
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Giannoudis, Athina, Andrea Davies, Claire M. Lucas, Robert J. Harris, Munir Pirmohamed, and Richard E. Clark. "Effective dasatinib uptake may occur without human organic cation transporter 1 (hOCT1): implications for the treatment of imatinib-resistant chronic myeloid leukemia." Blood 112, no. 8 (October 15, 2008): 3348–54. http://dx.doi.org/10.1182/blood-2007-10-116236.
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Abstract We have previously shown that imatinib uptake into chronic myeloid leukemia (CML) cells is dependent on human organic cation transporter 1 (hOCT1; SLC22A1), and that low hOCT1 expression is an important determinant of clinical outcome to imatinib treatment. We hypothesized that dasatinib might be transported differently than imatinib, possibly accounting for its favorable effects in imatinib-resistant patients. 14C-dasatinib uptake was greater in KCL22-transfected cells with pcDNA3-hOCT1 plasmid (high hOCT1-expressing cells) than in control cells (P = .02). However, hOCT inhibitors did not decrease dasatinib uptake into either control or primary cells, in contrast to their block on imatinib uptake. Dasa-tinib decreased the level of phosphorylated CrkL to 49.9% in control and 40.3% in high hOCT1-expressing cells. Dasa-tinib efflux was investigated in confluent ABCB1-transfected MDCKII cell monolayers. Both dasatinib and imatinib were transported from the basal to the apical layer, indicating that they were transported by ABCB1, which was confirmed using the ABCB1 inhibitor PSC833 (P = .001 and P < .001, respectively). Compared with imatinib, dasatinib achieved superior intracellular levels and BCR-ABL suppression even in cells with low or blocked hOCT1. Efflux of dasatinib and imatinib appear similar via ABCB1. Dasatinib may therefore offer an advantage over imatinib in patients with low hOCT1 expression.
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Moss, Darren M., Wai San Kwan, Neill J. Liptrott, Darren L. Smith, Marco Siccardi, Saye H. Khoo, David J. Back, and Andrew Owen. "Raltegravir Is a Substrate for SLC22A6: a Putative Mechanism for the Interaction between Raltegravir and Tenofovir." Antimicrobial Agents and Chemotherapy 55, no. 2 (November 15, 2010): 879–87. http://dx.doi.org/10.1128/aac.00623-10.
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ABSTRACTThe identification of transporters of the HIV integrase inhibitor raltegravir could be a factor in an understanding of the pharmacokinetic-pharmacodynamic relationship and reported drug interactions of raltegravir. Here we determined whether raltegravir was a substrate for ABCB1 or the influx transporters SLCO1A2, SLCO1B1, SLCO1B3, SLC22A1, SLC22A6, SLC10A1, SLC15A1, and SLC15A2. Raltegravir transport by ABCB1 was studied with CEM, CEMVBL100, and Caco-2 cells. Transport by uptake transporters was assessed by using aXenopus laevisoocyte expression system, peripheral blood mononuclear cells, and primary renal cells. The kinetics of raltegravir transport and competition between raltegravir and tenofovir were also investigated using SLC22A6-expressing oocytes. Raltegravir was confirmed to be an ABCB1 substrate in CEM, CEMVBL100, and Caco-2 cells. Raltegravir was also transported by SLC22A6 and SLC15A1 in oocyte expression systems but not by other transporters studied. TheKmandVmaxfor SLC22A6 transport were 150 μM and 36 pmol/oocyte/h, respectively. Tenofovir and raltegravir competed for SLC22A6 transport in a concentration-dependent manner. Raltegravir inhibited 1 μM tenofovir with a 50% inhibitory concentration (IC50) of 14.0 μM, and tenofovir inhibited 1 μM raltegravir with an IC50of 27.3 μM. Raltegravir concentrations were not altered by transporter inhibitors in peripheral blood mononuclear cells or primary renal cells. Raltegravir is a substrate for SLC22A6 and SLC15A1 in the oocyte expression system. However, transport was limited compared to endogenous controls, and these transporters are unlikely to have a great impact on raltegravir pharmacokinetics.
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Sava, Georgina P., Hailing Fan, Rosemary A. Fisher, Sabrina Lusvarghi, Sunil Pancholi, Suresh V. Ambudkar, Lesley-Ann Martin, R. Charles Coombes, Lakjaya Buluwela, and Simak Ali. "ABC-transporter upregulation mediates resistance to the CDK7 inhibitors THZ1 and ICEC0942." Oncogene 39, no. 3 (September 17, 2019): 651–63. http://dx.doi.org/10.1038/s41388-019-1008-y.
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Abstract The CDK7 inhibitors (CDK7i) ICEC0942 and THZ1, are promising new cancer therapeutics. Resistance to targeted drugs frequently compromises cancer treatment. We sought to identify mechanisms by which cancer cells may become resistant to CDK7i. Resistant lines were established through continuous drug selection. ABC-transporter copy number, expression and activity were examined using real-time PCR, immunoblotting and flow cytometry. Drug responses were measured using growth assays. ABCB1 was upregulated in ICEC0942-resistant cells and there was cross-resistance to THZ1. THZ1-resistant cells upregulated ABCG2 but remained sensitive to ICEC0942. Drug resistance in both cell lines was reversible upon inhibition of ABC-transporters. CDK7i response was altered in adriamycin- and mitoxantrone-resistant cell lines demonstrating ABC-transporter upregulation. ABCB1 expression correlated with ICEC0942 and THZ1 response, and ABCG2 expression with THZ2 response, in a panel of cancer cell lines. We have identified ABCB1 upregulation as a common mechanism of resistance to ICEC0942 and THZ1, and confirmed that ABCG2 upregulation is a mechanism of resistance to THZ1. The identification of potential mechanisms of CDK7i resistance and differences in susceptibility of ICEC0942 and THZ1 to ABC-transporters, may help guide their future clinical use.
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Silbermann, Katja, Jiyang Li, Vigneshwaran Namasivayam, Fabian Baltes, Gerd Bendas, Sven Marcel Stefan, and Michael Wiese. "Superior Pyrimidine Derivatives as Selective ABCG2 Inhibitors and Broad-Spectrum ABCB1, ABCC1, and ABCG2 Antagonists." Journal of Medicinal Chemistry 63, no. 18 (July 31, 2020): 10412–32. http://dx.doi.org/10.1021/acs.jmedchem.0c00961.
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Tamaki, Akina, Caterina Ierano, Gergely Szakacs, Robert W. Robey, and Susan E. Bates. "The controversial role of ABC transporters in clinical oncology." Essays in Biochemistry 50 (September 7, 2011): 209–32. http://dx.doi.org/10.1042/bse0500209.
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The phenomenon of multidrug resistance in cancer is often associated with the overexpression of the ABC (ATP-binding cassette) transporters Pgp (P-glycoprotein) (ABCB1), MRP1 (multidrug resistance-associated protein 1) (ABCC1) and ABCG2 [BCRP (breast cancer resistance protein)]. Since the discovery of Pgp over 35 years ago, studies have convincingly linked ABC transporter expression to poor outcome in several cancer types, leading to the development of transporter inhibitors. Three generations of inhibitors later, we are still no closer to validating the ‘Pgp hypothesis’, the idea that increased chemotherapy efficacy can be achieved by inhibition of transporter-mediated efflux. In this chapter, we highlight the difficulties and past failures encountered in the development of clinical inhibitors of ABC transporters. We discuss the challenges that remain in our effort to exploit decades of work on ABC transporters in oncology. In learning from past mistakes, it is hoped that ABC transporters can be developed as targets for clinical intervention.
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Onafuye, Hannah, Sebastian Pieper, Dennis Mulac, Jindrich Cinatl Jr., Mark N. Wass, Klaus Langer, and Martin Michaelis. "Doxorubicin-loaded human serum albumin nanoparticles overcome transporter-mediated drug resistance in drug-adapted cancer cells." Beilstein Journal of Nanotechnology 10 (August 14, 2019): 1707–15. http://dx.doi.org/10.3762/bjnano.10.166.
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Resistance to systemic drug therapy is a major reason for the failure of anticancer therapies. Here, we tested doxorubicin-loaded human serum albumin (HSA) nanoparticles in the neuroblastoma cell line UKF-NB-3 and its ABCB1-expressing sublines adapted to vincristine (UKF-NB-3rVCR1) and doxorubicin (UKF-NB-3rDOX20). Doxorubicin-loaded nanoparticles displayed increased anticancer activity in UKF-NB-3rVCR1 and UKF-NB-3rDOX20 cells relative to doxorubicin solution, but not in UKF-NB-3 cells. UKF-NB-3rVCR1 cells were re-sensitised by nanoparticle-encapsulated doxorubicin to the level of UKF-NB-3 cells. UKF-NB-3rDOX20 cells displayed a more pronounced resistance phenotype than UKF-NB-3rVCR1 cells and were not re-sensitised by doxorubicin-loaded nanoparticles to the level of parental cells. ABCB1 inhibition using zosuquidar resulted in similar effects like nanoparticle incorporation, indicating that doxorubicin-loaded nanoparticles successfully circumvent ABCB1-mediated drug efflux. The limited re-sensitisation of UKF-NB-3rDOX20 cells to doxorubicin by circumvention of ABCB1-mediated efflux is probably due to the presence of multiple doxorubicin resistance mechanisms. So far, ABCB1 inhibitors have failed in clinical trials probably because systemic ABCB1 inhibition results in a modified body distribution of its many substrates including drugs, xenobiotics, and other molecules. HSA nanoparticles may provide an alternative, more specific way to overcome transporter-mediated resistance.
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Özenver, Nadire, Joelle C. Boulos, and Thomas Efferth. "Activity of Cordycepin From Cordyceps sinensis Against Drug-Resistant Tumor Cells as Determined by Gene Expression and Drug Sensitivity Profiling." Natural Product Communications 16, no. 2 (February 2021): 1934578X2199335. http://dx.doi.org/10.1177/1934578x21993350.
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Cordycepin is one of the substantial components of the parasitic fungus Cordyceps sinensis as well as other Cordyceps species. It exerts various effects such as antimetastatic, antiinflammatory, antioxidant, and neuroprotective activities. Assorted studies revealed in vitro and in vivo anticancer influence of cordycepin and put forward its potential for cancer therapy. However, the role of multidrug resistance-associated mechanisms for the antitumor effect of cordycepin has not been investigated in great detail thus far. Therefore, we searched cordycepin’s cytotoxicity with regard to well-known anticancer drug resistance mechanisms, including ABCB1, ABCB5, ABCC1, ABCG2, EGFR, and TP53, and identified putative molecular determinants related to the cellular responsiveness of cordycepin. Bioinformatic analyses of NCI microarray data and gene promoter transcription factor binding motif analyses were performed to specify the mechanisms of cordycepin towards cancer cells. COMPARE and hierarchical analyses led to the detection of the genes involved in cordycepin’s cytotoxicity and sensitivity and resistance of cell lines towards cordycepin. Tumor-type dependent response and cross-resistance profiles were further unravelled. We found transcription factors potentially involved in the common transcriptional regulation of the genes identified by COMPARE analyses. Cordycepin bypassed resistance mediated by the expression of ATP-binding cassete (ABC) transporters (P-gp, ABCB5, ABCC1 and BCRP) and mutant epidermal growth factor receptor (EGFR). The drug sensitivity profiles of several DNA Topo I and II inhibitors were significantly correlated with those of cordycepin’s activity. Among eight different tumor types, prostate cancer was the most sensitive, whereas renal carcinoma was the most resistant to cordycepin. NF-κB was discovered as a common transcription factor. The potential of cordycepin is set forth as a potential new drug lead by bioinformatic evaluations. Further experimental studies are warranted for better understanding of cordycepin’s activity against cancer.
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Kochanek, Stanton J., David A. Close, Allen Xinwei Wang, Tongying Shun, Philip E. Empey, Julie L. Eiseman, and Paul A. Johnston. "Confirmation of Selected Synergistic Cancer Drug Combinations Identified in an HTS Campaign and Exploration of Drug Efflux Transporter Contributions to the Mode of Synergy." SLAS DISCOVERY: Advancing the Science of Drug Discovery 24, no. 6 (April 30, 2019): 653–68. http://dx.doi.org/10.1177/2472555219844566.
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Systematic unbiased high-throughput screening (HTS) of drug combinations (DCs) in well-characterized tumor cell lines is a data-driven strategy to identify novel DCs with potential to be developed into effective therapies. Four DCs from a DC HTS campaign were selected for confirmation; only one appears in clinicaltrials.gov and limited preclinical in vitro data indicates that the drug pairs interact synergistically. Nineteen DC-tumor cell line sets were confirmed to interact synergistically in three pharmacological interaction models. We developed an imaging assay to quantify accumulation of the ABCG2 efflux transporter substrate Hoechst. Gefitinib and raloxifene enhanced Hoechst accumulation in ABCG2 (BCRP)-expressing cells, consistent with inhibition of ABCG2 efflux. Both drugs also inhibit ABCB1 efflux. Mitoxantrone, daunorubicin, and vinorelbine are substrates of one or more of the ABCG2, ABCB1, or ABCC1 efflux transporters expressed to varying extents in the selected cell lines. Interactions between ABC drug efflux transporter inhibitors and substrates may have contributed to the observed synergy; however, other mechanisms may be involved. Novel synergistic DCs identified by HTS were confirmed in vitro, and plausible mechanisms of action studied. Similar approaches may justify the testing of novel HTS-derived DCs in mouse xenograft human cancer models and support the clinical evaluation of effective in vivo DCs in patients.
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Fedina, L. V., I. N. Sychev, T. D. Rastvorova, E. V. Strigunkova, A. A. Kachanova, Z. A. Sozaeva, P. O. Bochkov, A. V. Vardanyan, K. B. Mirzayev, and D. A. Sychev. "Effect of ABCB1 Gene Carriage and Drug-Drug Interactions on Apixaban and Rivaroxaban Pharmacokinetics and Clinical Outcomes in Patients with Atrial Fibrillation and Deep Vein Thrombosis." Rational Pharmacotherapy in Cardiology 18, no. 6 (January 6, 2023): 624–29. http://dx.doi.org/10.20996/1819-6446-2022-12-02.
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Aim. To investigate the effect of ABCB1 gene carriage and interdrug interactions on apixaban pharmacokinetics and clinical outcomes in patients with atrial fibrillation and deep vein thrombosis.Material and methods. Patients hospitalized at Yudin State Clinical Hospital participated in the study. A total of 92 patients (50 patients received apixaban and 42 – rivaroxaban) with non-valvular atrial fibrillation and deep vein thrombosis were included. Genotyping was performed by real-time polymerase chain reaction. Direct oral anticoagulants concentrations were measured using an electrospray ionization mass spectrometer in positive ionization mode.Results. In our study we found that in patients carrying the CT+TT ABCB1 (rs4148738) C>T genotype encoding the carrier protein (P-gp), the plasma concentration of rivaroxaban was statistically significantly higher p= 0.026. In addition, we found that patients taking apixaban together with a CYP3A4/P-gp inhibitor were 3.5 times more likely to have hemorrhagic complications than those without inhibitors p = 0.004.Conclusion. Our study revealed that the plasma concentration of rivaroxaban was higher in patients carrying the ABCB1 (rs4148738) C>T polymorphism T allele. And patients taking apixaban together with CYP3A4/P-gp inhibitor had higher risk of hemorrhagic complications in comparison with patients not taking such drugs. Further studies are needed on the influence of pharmacogenetics and pharmacokinetics on the safety and efficacy profile of apixaban and rivaroxaban, taking into account the trend of systemic approach to optimization of anticoagulant therapy of direct oral anticoagulants based on pharmacokinetic, pharmacogenetic biomarkers.
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Eadie, Laura N., Verity A. Saunders, Tamara M. Leclercq, Susan Branford, Deborah L. White, and Timothy P. Hughes. "The Allosteric Inhibitor ABL001 Is Susceptible to Resistance in Vitro Mediated By Overexpression of the Drug Efflux Transporters ABCB1 and ABCG2." Blood 126, no. 23 (December 3, 2015): 4841. http://dx.doi.org/10.1182/blood.v126.23.4841.4841.
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Abstract Tyrosine Kinase Inhibitors (TKIs) result in excellent responses in Chronic Myeloid Leukaemia (CML) patients. However, secondary resistance is observed in ~35% of patients, mostly due to Bcr-Abl kinase domain (KD) mutations. Overexpression of the efflux transporter ABCB1 is a known mediator of primary resistance to imatinib (IM) and nilotinib (NIL). ABL001, a potent allosteric inhibitor, binds to the myristate pocket of the Bcr-Abl KD. In this study, we modelled ABL001 resistance in vitro with particular focus on ABCB1 and ABCG2. ABL001 sensitivity was evaluated in BCR -ABL1 + cell lines K562, K562-Dox (ABCB1 overexpressing) and K562-ABCG2 (transfected with ABCG2 overexpression vector). Also, K562, K562-Dox and KU812 cells were made resistant to ABL001 by culturing long term in increasing concentrations of ABL001 (increased once >80% survival in culture for >10 days). Parental ABL001 naive cells were maintained in parallel culture. Initial onset of resistance was characterised by significantly increased IC50ABL001 based on AnnexinV/7-AAD cytotoxicity assays performed at each ABL001 dose escalation. Resistance mechanisms were interrogated during escalation and once 10 μM was reached (based on clinically achievable doses). Protein expression levels of efflux transporters ABCB1/ABCG2 were examined by flow cytometry; KD mutation sequencing and Bcr-Abl protein quantitation were performed. K562-Dox and K562-ABCG2 cells demonstrated significantly increased IC50ABL001 compared with parental K562 control cells: 256 and 299 nM respectively vs 23 nM, p<0.001 suggesting both ABCB1 and ABCG2 are important in ABL001 transport. Furthermore, resistance was reversible through use of specific inhibitors cyclosporin (ABCB1) and Ko143 (ABCG2). IC50ABL001 +inhibitors was comparable to that of control K562 cells: K562-Dox +cyclosporin=11 nM; K562-ABCG2 +Ko143=15 nM. A prior study identified ABCB1 overexpression as an initiator of resistance to IM and NIL (expression increased up to 7- and 5-fold in IM and NIL resistant cells compared with respective control cells p<0.002). In this study, expression levels of ABCB1 and ABCG2 were interrogated in 3 ABL001 resistant cell lines; results indicated overexpression of both transporters was integral in development of resistance. Up to 17- and 60-fold greater levels of ABCB1 and ABCG2 respectively was observed in resistant vs control cells (Table 1). No KD mutations were detected in early resistance intermediates; however, a myristate pocket mutation was detected in later stage KU812 ABL001 resistant cells (percentage mutation correlated with IC50ABL001). Table 1. ABL001 resistance characteristics in selected resistant intermediates IC50ABL001 (nM) Fold change (MFI) Bcr-Abl over expression KD mutation ABCB1 ABCG2 K562 Control 23 N N K562 500 nM ABL001 >2500 p <0.001 -1.4 p =0.003 48.3 p =0.007 Y N K562 10 μM ABL001 27, 800p <0.001 -2.3 p =0.535 60.3 p <0.001 Y N K562 10 μM ABL001 +Ko143 89 p <0.001 K562-Dox Control 256 N N K562-Dox 500 nM ABL001 2393 p <0.001 2.3 p <0.001 -2.9 p <0.001 N N K562-Dox 500 nM ABL001 +cyclosporin 17 p <0.001 K562-Dox 10 μM ABL001 >50, 000 p <0.001 -1.6 p <0.001 -0.1 p <0.001 N N KU812 Control 2.7 N N KU812 5 nM ABL001 6.4 p <0.001 2.2 p =0.008 4.0 p =0.002 Y N KU812 10 μM ABL001 33, 300 p <0.001 8.1 p =0.003 11.0 p =0.010 Y Y (90%) p-value: resistant vs respective control; n>3; N=No; Y=Yes While further in vitro and in vivo studies will determine the clinical relevance of efflux-mediated resistance to ABL001 mono- and combination therapy, our preclinical data provide evidence that kinase domain mutations may not be the predominant cause of ABL001 resistance; drug transporters likely play an important role as well. Susceptibility to ABCB1 overexpression is well recognised for IM and NIL resistance and we now show it is also relevant for ABL001. ABCG2-mediated resistance has not been observed with NIL or IM but is clearly important for ABL001. ABL001 treatment induced ABCG2 overexpression in two cell lines; both have negligible basal expression. ABCG2 overexpression preceded Bcr-Abl overexpression and mutation emergence. Importantly, ABL001 resistance was completely reversible in the presence of the ABCG2 inhibitor Ko143 (K562 10 μM ABL001 cells). Given the lack of strong evidence for ABCG2-mediated transport of NIL or IM at clinically relevant concentrations, our data provide a strong rationale for the use of ABL001 in combination with either of these TKIs. Disclosures Branford: Ariad: Research Funding; Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; BMS: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Qiagen: Membership on an entity's Board of Directors or advisory committees. White:Novartis: Honoraria, Research Funding; BMS: Honoraria, Research Funding. Hughes:ARIAD: Honoraria, Research Funding; Bristol-Myers Squibb: Honoraria, Research Funding; Novartis: Honoraria, Research Funding.
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Liu, Hongming, Zhiguo Ma, and Baojian Wu. "Structure-activity relationships andin silicomodels of P-glycoprotein (ABCB1) inhibitors." Xenobiotica 43, no. 11 (April 25, 2013): 1018–26. http://dx.doi.org/10.3109/00498254.2013.791003.
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Hilgeroth, Andreas, Marc Hemmer, Sebastian Neuber, Josef Molnar, and Hermann Lage. "Discovery of 9,10-Dihydroacridines as Novel Class of ABCB1 Inhibitors." Medicinal Chemistry 11, no. 4 (April 29, 2015): 329–35. http://dx.doi.org/10.2174/1573406410666141111100720.
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von Richter, Oliver, Hristos Glavinas, Peter Krajcsi, Stephanie Liehner, Beate Siewert, and Karl Zech. "A novel screening strategy to identify ABCB1 substrates and inhibitors." Naunyn-Schmiedeberg's Archives of Pharmacology 379, no. 1 (August 29, 2008): 11–26. http://dx.doi.org/10.1007/s00210-008-0345-0.
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Huang, Ying, Paul E. Blower, Ruqing Liu, Zunyan Dai, Anh-Nhan Pham, Hojin Moon, Jialong Fang, and Wolfgang Sadée. "Chemogenomic Analysis Identifies Geldanamycins as Substrates and Inhibitors of ABCB1." Pharmaceutical Research 24, no. 9 (April 25, 2007): 1702–12. http://dx.doi.org/10.1007/s11095-007-9300-x.
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Hiwase, Devendra K., Jane Engler, Verity Saunders, Deborah L. White, and Timothy Hughes. "In Contrast to Imatinib, Dasatinib Intracellular Concentration In CML-CD34+ Progenitors Is Not Significantly Different Than That Observed In CD34- Mature Cells." Blood 116, no. 21 (November 19, 2010): 1205. http://dx.doi.org/10.1182/blood.v116.21.1205.1205.
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Abstract Abstract 1205 Long term follow up of imatinib (IM) clinical studies and in vitro studies suggest that tyrosine kinase inhibitors (TKI) do not eradicate leukemic stem cells. Refractoriness of leukemic stem cells is postulated to be due to inadequate Bcr-Abl kinase inhibition which in turn could be due to low intracellular uptake and retention (IUR) of TKI. We have previously demonstrated that IM cellular uptake is predominantly mediated by the organic cation transporter protein (OCT-1) and patients with low OCT-1 activity have suboptimal response as compared to patients with high OCT-1 activity. More recently Engler et al (Leukemia 2010) demonstrated that IM IUR is significantly lower in CML-CD34+ cells compared to CD34- cells. This could be due to low expression and activity of the OCT-1 protein and/or high expression of ABCB1 and/or ABCG2 (Jiang et al Leukemia 2007). Although dasatinib is clinically available there are no published data assessing dasatinib IUR in CML-CD34+ progenitors. We and others have previously demonstrated that dasatinib cellular uptake is predominantly OCT-1 independent and dasatinib is a substrate of ABCB1 and ABCG2. We hypothesized that dasatinib IUR would be lower in CML-CD34+ cells compared to CD34- cells. In this study we compare dasatinib and IM IUR; and OCT-1 and ABCB1 mRNA expression in CML-CD34+ and CD34- cells of newly diagnosed CML-CP patients. CD34+ and CD34- cells were incubated with 14C-dasatinib (100 nM and 1 μM) or 14C-IM (2 μM) for 2h and IUR was assessed as described previously (Hiwase et al Clin Cancer Res. 2008). As shown previously, the OCT-1 expression and activity was lower in CML-CD34+ cells, and resulted in lower IM IUR in CML-CD34+ cells compared to CML-CD34- cells (15±5 vs. 27±5; p=0.04; Fig 1A and C). However at a therapeutically achievable concentration (100 nM dasatinib) and at higher concentration (1 μM dasatinib), there was no significant difference in dasatinib IUR in CML-CD34+ and CD34- cells (Fig 1B). Low OCT-1 expression and activity in CML-CD34+ cells did not influence the dasatinib IUR; further confirming that dasatinib cellular influx is predominantly OCT-1 independent. Despite higher ABCB1 mRNA expression in CML-CD34+ cells, the dasatinib IUR was not lower in CML-CD34+ cells compared to CD34- cells. High ABCB1 mRNA expression may not necessarily translate into high ABCB1 activity. To this end, we assessed the effect of PSC-833, an ABCB1 inhibitor, on dasatinib IUR and dasatinib mediated Bcr-Abl kinase inhibition in CML-CD34+ cells. The baseline p-Crkl, a surrogate marker of Bcr-Abl kinase activity, was significantly higher in CML-CD34+ compared to CML-CD34- cells (67±5% vs. 55±8%; p=0.002; n=9). PSC-833 neither increased dasatinib IUR, nor enhanced dasatinib mediated Bcr-Abl kinase inhibition in CML-CD34+ cells (% p-Crkl at 10 nM dasatinib: 20±6 vs. 27±10). Similarly, Ko143, an ABCG2 inhibitor, did not significantly change dasatinib IUR or Bcr-Abl kinase inhibition (% p-Crkl at 10 nM dasatinib: 21±3 vs. 27±10). In summary, although dasatinib is an ABCB1 and ABCG2 substrate, ABCB1 and ABCG2 inhibitors neither increase dasatinib IUR, nor enhance dasatinib mediated Bcr-Abl kinase inhibition in CML-CD34+ cells. This data suggest that dasatinib IUR in CML-CD34+ cells is not influenced by ABCB1 and ABCG2. Hatziieremia et al (Exp. Hematology, 2009) reported that ABCB1 activity is low in CML-CD34+ cells and suggested that it did not influence IM level in CML-CD34+ cells. We further demonstrated that 100 nM dasatinib inhibited ≥95% Bcr-Abl kinase activity in CML-CD34+ and CD34- cells. In summary our data demonstrates that in contrast to IM, the intracellular concentration of dasatinib is equivalent in mature and immature CML cell compartments which may contribute to better targeting of early CML progenitors with dasatinib. Fig. 1: In contrast to IM, dasatinib intracellular uptake and retention (IUR) is not significantly different in CML-CD34+and mature CD34-cells: (A) OCT-1 activity and IM IUR is significantly lower in CML-CD34+ than CD34- cells (p=0.04). (B) However, dasatinib IUR is not significantly different in CD34+ and CD34- cells (p=0.8) (C) OCT-1 mRNA expression is lower in CML-CD34+ cells than CD34- cells. While ABCB1 expression is significantly higher in CML-CD34+ compared to CD34- cells (p=0.007). Fig. 1:. In contrast to IM, dasatinib intracellular uptake and retention (IUR) is not significantly different in CML-CD34+ and mature CD34- cells: (A) OCT-1 activity and IM IUR is significantly lower in CML-CD34+ than CD34- cells (p=0.04). (B) However, dasatinib IUR is not significantly different in CD34+ and CD34- cells (p=0.8) (C) OCT-1 mRNA expression is lower in CML-CD34+ cells than CD34- cells. While ABCB1 expression is significantly higher in CML-CD34+ compared to CD34- cells (p=0.007). Disclosures: White: Novartis: Honoraria, Research Funding; BMS: Research Funding. Hughes:Novartis: Honoraria, Research Funding; Bristol-Myers Squibb: Honoraria, Research Funding.
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White, Deborah L., Liu Lu, Timothy P. Clackson, Verity A. Saunders, and Timothy P. Hughes. "ATP Dependent Efflux Transporters ABCB1 and ABCG2 Are Unlikely to Impact the Efficacy, or Mediate Resistance to the Tyrosine Kinase Inhibitor, Ponatinib." Blood 118, no. 21 (November 18, 2011): 2745. http://dx.doi.org/10.1182/blood.v118.21.2745.2745.
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Abstract Abstract 2745 Ponatinib is a potent pan-BCR-ABL tyrosine kinase inhibitor (TKI) currently in a pivotal phase 2 clinical trial. Ponatinib (PON) was specifically designed to target both native and all mutant forms of BCR-ABL, including T315I. The phase I study of oral ponatinib in patients with refractory CML/ALL or other hematologic malignancies recently reported that 66% and 53% of patients with CP-CML achieved MCyR and CCyR respectively (Cortes et al., ASH 2011 abstract #210). While extensive modelling experiments in BaF3 cells have been performed characterising in vitro response to ponatinib, little is known about the interactions of this drug and drug transporters that impact the response of other tyrosine kinase inhibitors (TKIs). To explore this we have examined both the degree of in vitro kinase inhibition mediated by ponatinib in BCR-ABL+ cell lines, and the intracellular uptake and retention (IUR) of ponatinib achieved. The IC50 was determined by assessing the reduction in %p-Crkl in response to increasing concentrations of ponatinib in vitro. The IUR assay was performed as previously using [14-C]-ponatinib. To determine the role of ABCB1 and ABCG2, both previously implicated in the transport of other TKIs, IC50 analysis was performed on K562 cells, and variants; ABCB1 overexpressing K562-DOX and ABCG2 overexpressing K562-ABCG2. As shown in Table 1, in contrast to the results previously observed with imatinib (IM), nilotinib (NIL) and dasatinib (DAS) there was no significant difference in the IC50ponatinib between these three cell lines, suggesting neither ABCB1 nor ABCG2 play a major role in ponatinib transport. Furthermore, the addition of either the ABCB1 and ABCG2 inhibitor pantoprazole, or the multidrug resistance (MDR) inhibitor cyclosporin did not result in a significant change in the IC50ponatinib in any of the cell lines tested. In contrast the addition of either pantoprazole or cyclosporin resulted in a significant reduction in IC50IM, IC50NIL. and IC50DAS of K562-DOX cells, supporting the notion that these TKIs interact with ABCB1.Table 1:The IC50 of ponatinib (compared to IM, NIL and DAS) in K562 cells and the over-expressing variants DOX and ABCG2 in the presence of the ABC inhibitors pantoprazole and cyclosporin. n=5. *p<0.05IC50% reduction in IC50+ pantoprazole+ cyclosporinPON (nM)IM (μM)NIL (nM)DAS (nM)PONIMNILDASPONIMNILDASK5627.793751111544*NA−107NA2DOX7.919*598*100*1018*63*1655*88*ABCG26.4730025*6NA To further examine the effect of ABC transporters on ponatinib efflux we have determined the IUR of [14-C]-ponatinib in K562, DOX and ABCG2 cell lines. We demonstrate no significant difference in the IUR between these cell lines at 37°C (n=6) (K562 vs DOX p=0.6; K562 vs ABCG2 p=0.37 and DOX vs ABCG2 p=0.667 at 2uM respectively). Temperature dependent IUR experiments reveal a significant reduction in the ponatinib IUR at 4°C compared to 37°C in K562 cells (n=6) (p=0.008), DOX cells (p=0.004) and ABCG2 cells (p=0.002) supporting the likely involvement of an ATP/temperature dependent, and yet to be determined, component of ponatinib influx. There was no significant difference in the IUR between these cell lines at 4°C (p=0.824, p=0.7 and p=0.803 respectively). Importantly, these data are consistent with the IC50ponatinib findings. If ATP dependent efflux pumps (ABCB1 and ABCG2) were actively transporting ponatinib, a significant decrease in IUR in DOX and ABCG2 at 37°C compared to K562 cells would be expected, but is not observed here. Analysis of ponatinib IUR in the prototypic ABCB1 over-expressing CEM-VBL100 cells, and their parental, ABCB1 null counterparts (CCRF-CEM) further confirmed these findings. The IUR in VBL100 cells was significantly higher than that observed in CEM's (p<0.001; n=5), providing further evidence that ponatinib was not being exported from the cell actively via ABCB1. These data suggest that the transport of ponatinib is, at least in part, temperature-dependent indicating a yet to be determined ATP transporter may be involved in the transport of ponatinib into leukaemic cells. Importantly, this data suggests that ponatinib is unlikely to be susceptible to resistance via the major ATP efflux transporters (ABCB1 or ABCG2) that have been previously demonstrated to significantly impact the transport of, and mediate resistance to other clinically available TKIs. Disclosures: White: BMS: Honoraria, Research Funding; Novartis Pharmaceuticals: Honoraria, Research Funding. Clackson:ARIAD: Employment. Hughes:Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; BMS: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; ARIAD: Honoraria, Membership on an entity's Board of Directors or advisory committees.
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Eadie, Laura, Timothy P. Hughes, and Deborah L. White. "Increasing Expression Of The Efflux Transporter ABCB1 May Predispose CML Cells To Overt TKI Resistance." Blood 122, no. 21 (November 15, 2013): 5157. http://dx.doi.org/10.1182/blood.v122.21.5157.5157.
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Abstract Tyrosine kinase inhibitors (TKIs) result in excellent responses in most Chronic Myeloid Leukemia (CML) patients. However, up to 35% of patients treated with imatinib (IM) exhibit resistance and more recently nilotinib (NIL) and dasatinib (DAS) resistance have also been observed. Mutations in the BCR-ABL kinase domain (KD) are the main cause of secondary TKI resistance. Other mechanisms include overexpression of BCR-ABL, LYN and ABCB1. Predicting patients with susceptibility to mutation development and disease progression is crucial, thus we investigated the kinetics of TKI resistance emergence in vitro and in vivo. ABCB1 is implicated in TKI efflux hence we postulated that overexpression of ABCB1 leads to reduced intracellular TKI concentrations, resulting in inferior inhibition of Bcr-Abl predisposing cells to resistance development. Accordingly, 3 CML blast crisis (BC) cell lines (K562, K562-Dox, KU812) were cultured in increasing concentrations of IM to 2 μM, NIL to 2 μM and DAS to 200 nM until we observed overt resistance defined as a significant increase in survival in cytotoxicity assays and p-Crkl dependent IC50. Mechanisms of resistance were investigated in cell line intermediates: BCR-ABL, ABCB1 and LYN mRNA expression levels were determined by RT-PCR and KD mutation sequencing was performed. In our TKI resistant cell lines (Table 1), an increase in ABCB1 mRNA was the initial change observed prior to the development of additional resistance mechanisms (KD mutations, ABCB1 BCR-ABL and LYN overexpression). Interestingly, in 4/6 cells lines ABCB1 mRNA reduced to basal levels or below following establishment of these additional resistance mechanisms. ABCB1 levels were assessed in 37 de novo CML patients treated with IM who achieved major molecular response (MMR) compared with patients who progressed to BC, lost MMR or developed KD mutations. ABCB1 levels were determined in blood at diagnosis and following therapy (selected patients summarized in Table 2). A sustained >2 fold rise in ABCB1 was observed prior to disease progression in 3/3 patients and in 13/16 patients who did not achieve MMR. Importantly, the same was not observed in patients who achieved MMR (1/6 patients). The fold change of ABCB1 mRNA at day 22 vs diagnosis in patients achieving MMR was significantly different to that in patients not achieving MMR (p=0.004). ABCB1 increased by >2 fold post therapy and decreased following mutation development in 3/12 patients, confirming observations made in vitro, while 6/12 patients demonstrated sustained increase in ABCB1 post mutation similar to results observed in progression patients. ABCB1 mRNA did not change during therapy in 3/12 patients with mutations. While we recognize the majority of cells present in patients who achieve MMR are normal rather than leukemic, it is important to note that in patients who do not achieve MMR, ABCB1 expression increases in the remaining leukemic cells. We conclude ABCB1 overexpression acts as an initial mediator of resistance, providing a favorable environment for development of further resistance. Sustained increased levels of ABCB1 may contribute to disease progression and lack of response to IM. Additionally, ABCB1 may serve as a prognostic indicator (eg: level at day 22) and potentially assist in development of treatment strategies using TKIs in combination with other medications to enhance intracellular TKI concentration. Disclosures: Hughes: Ariad: Honoraria, Membership on an entity’s Board of Directors or advisory committees, Research Funding; BMS: Honoraria, Membership on an entity’s Board of Directors or advisory committees, Research Funding; Novartis: Honoraria, Membership on an entity’s Board of Directors or advisory committees, Research Funding; CSL: Research Funding. White:Novartis: Research Funding; BMS: Research Funding, Speakers Bureau; Ariad: Research Funding; CSL: Research Funding.
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Adorni, Maria Pia, Maricla Galetti, Silvia La Monica, Matteo Incerti, Alessandro Ruffoni, Lisa Elviri, Ilaria Zanotti, et al. "A New ABCB1 Inhibitor Enhances the Anticancer Effect of Doxorubicin in Both In Vitro and In Vivo Models of NSCLC." International Journal of Molecular Sciences 24, no. 2 (January 4, 2023): 989. http://dx.doi.org/10.3390/ijms24020989.
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In tumors, the multi drug resistance phenomenon may occur through the efflux of chemotherapeutic drugs out of cancer cells, impeding their accumulation, and eventually reducing their toxicity. This process is mediated by transporters overexpressed in the plasma membranes of tumor cells, among which is the P-glycoprotein/multidrug resistance 1/ATP-binding cassette B1 (P-gp/MDR1/ABCB1). The aim of this study was to explore the effect of a new molecule, called AIF-1, on ABCB1 activity. In a cellular model of non-small cell lung cancer (NSCLC), AIF-1 significantly inhibited ABCB1 activity, which was evaluated by the fluorimetric measurement of the intracellular accumulation of calcein. AIF-1 also significantly increased the intracellular content of doxorubicin, which was evaluated by confocal microscopy and LC-MS/MS analysis. This effect translated to higher cytotoxicity of doxorubicin and reduced cellular proliferation. Finally, in a murine xenograft model, the tumor volume increased by 267% and 148% on average in mice treated with vehicle and doxorubicin alone, respectively. After the co-administration of doxorubicin with AIF-1, tumor volume increased by only 13.4%. In conclusion, these results suggest enhancement of the efficacy of the chemotherapeutic drug doxorubicin by AIF-1, laying the basis for the future development of new ABCB1 inhibitors for tumor treatment.
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Hiwase, Devendra K., Deborah L. White, Verity A. Saunders, Phuong Dang, Amity Venables, Laura Eadie, Steph Zrim, L. Bik To, Junia V. Melo, and Timothy P. Hughes. "In Contrast to Imatinib, OCT-1 Mediated Influx Has Minimal Impact on Cellular Uptake of Dasatinib in CML Patients at Diagnosis." Blood 110, no. 11 (November 16, 2007): 1937. http://dx.doi.org/10.1182/blood.v110.11.1937.1937.
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Abstract Molecular response to imatinib in newly diagnosed CML patients correlates with intrinsic sensitivity to imatinib (IC50imatinib) which is related to imatinib intracellular uptake and retention (IUR). Imatinib cellular uptake is OCT-1 dependent and low OCT-1 activity in newly diagnosed CML patients is a major contributor to suboptimal response to imatinib. The cellular transport pathways for dasatinib, a more potent BCR-ABL kinase inhibitor, are yet to be defined. We have assessed dasatinib cellular transport pathways using mononuclear cells (MNCs) from newly diagnosed CML patients and cell lines. Dasatinib IUR was determined with 14C-labelled dasatinib (Bristol-Myers Squibb) at 4°C and 37°C, with/without influx/efflux inhibitors. There was no significant difference in dasatinib IUR at 37°C and 4°C in KU812 cell line (27 ng vs. 26 ng) and MNCs of CML patients (17 ng vs. 17 ng, n=10), indicating cellular uptake was not temperature dependent and most likely passive. Prazosin and progesterone (OCT-1 and OCT-3 inhibitors) did not significantly reduce IUR and did not reduce inter-patient variation in dasatinib IUR in contrast to imatinib IUR (P=0.45 and P=0.93 respectively, Fig. 1). Procainamide (OCT-1 and OCT-2 inhibitor), nicotinamide (OCT-2) and corticosterone (OCT-3 inhibitor) reduced dasatinib IUR but this did not reach significance (P=0.42, 0.74, 0.79 respectively). These data suggest that dasatinib uptake is not OCT dependent. Using ABCB1 overexpressing cell lines, we found that dasatinib IUR is lower in K562-Dox (P<0.001) and VBL-100 (P=0.05) compared to parental cell lines (K562 and CCRF-CEM respectively). PSC-833 (ABCB1 inhibitor, Novartis) significantly increased dasatinib IUR (P<0.0001), reduced IC50dasatinib in K562-Dox cell line (100 nM vs. 16 nM) and increased IUR in VBL-100 (P=0.01) suggesting ABCB1 actively efflux dasatinib. Median IC50dasatinib in 18 newly diagnosed CML patients was 2.20 nM (± 0.8), which is significantly below median plasma concentration achieved (100–200 nM). Interpatient variability in IC50dasatinib and IUR is therefore unlikely to be clinically significant for newly diagnosed CML patients receiving dasatinib. In conclusion dasatinib cellular uptake is predominantly passive and may be OCT independent. dasatinib is effluxed actively by ABCB1 in cell lines over-expressing these proteins. Inter-patient variation in dasatinib intracellular uptake and intrinsic sensitivity may not be clinically significant in newly diagnosed CML patients receiving dasatinib, in contrast to recipients of imatinib. Figure 1: Prazosin significantly reduced imatinib (IM) IUR and eliminated interpatient variability, however it did not reduce dasatinib IUR and did not eliminate interpatient variability. Figure 1:. Prazosin significantly reduced imatinib (IM) IUR and eliminated interpatient variability, however it did not reduce dasatinib IUR and did not eliminate interpatient variability.
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Sambyalova, A. Yu, T. A. Bairova, T. L. Manaenkova, A. V. Belskikh, E. V. Belyaeva, O. A. Ershova, D. V. Kumratov, et al. "Some pharmacogenetic aspects of the <i>ABCB1</i> gene in lopinavir / ritonavir concentration variability in children with HIV infection: A pilot study." Acta Biomedica Scientifica 7, no. 5-1 (December 6, 2022): 53–61. http://dx.doi.org/10.29413/abs.2022-7.5-1.7.
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Polymorphic variants of the multidrug resistance gene (ABCB1 or MDR1) are associated with changes in the absorption and transport of drugs in the body. One of the substrates of the ABCB1 transporter is an antiretroviral drug from the class of protease inhibitors, lopinavir. The aim. To research the effect of polymorphic variants C1236T and C3435T in the ABCB1 gene on the plasma concentration of lopinavir / ritonavir in children and adolescents living with HIV infection. Methods. The genotypes of polymorphic variants of the ABCB1 gene were identified in 136 HIV infected children and adolescents; median age – 10 [7–12] years. The plasma concentration of lopinavir / ritonavir was measured from blood taken during the next scheduled appointment as part of dispensary observation at the Irkutsk Regional AIDS Centre using high performance liquid chromatography. Results. The average duration of lopinavir/ritonavir use as part of an antiretroviral therapy was 55 months. Median viral load in patients was 1 [1–2.03] log 10 copies/ mL; the number of CD4 + T cells – 38.36 %. The frequency of occurrence of the 3435T and 1236T alleles of the ABCB1 gene was ~50 %. In carriers of the 3435TT genotype, the median lopinavir concentrations 2 and 12 hours after drug intake were 5050.8 [3615.8–5847.7] and 2665.5 [216–4896.3] ng/mL, respectively. In carriers of the 1236TT genotype, median lopinavir concentrations 2 and 12 hours after drug intake were 4913.5 [3355.1–5733.7] and 3290.6 [159.1–4972.5] ng/mL, respectively. Conclusions. The study did not reveal a significant relationship between the carriage of the C3435T and C1236T genotypes of the ABCB1 gene and the concentrations of lopinavir and ritonavir 2 and 12 hours after drug intake.
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Giannoudis, Athina, Andrea Davies, Claire M. Lucas, Robert J. Harris, Munir Pirmohamed, and Richard E. Clark. "Unlike Imatinib, Dasatinib Uptake into Chronic Myeloid Leukaemia Cells Is Independent of hOCT1 Expression." Blood 110, no. 11 (November 16, 2007): 3458. http://dx.doi.org/10.1182/blood.v110.11.3458.3458.
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Abstract In chronic myeloid leukaemia (CML), resistance to imatinib is an important clinical issue. We have previously shown that imatinib uptake into CML cells and cell lines is dependent on the uptake transporter human Organic Cation Transporter 1 (hOCT1; SLCA22). Furthermore, in clinical samples, low hOCT1 expression is an important mechanism of imatinib resistance (Wang L et al, Clinical Pharmacology and Therapeutics; epub June 13th 2007). Dasatinib is a second generation novel tyrosine kinase inhibitor that is effective in many imatinib-resistant patients. Its mechanism of transport into and out of CML cells has not been previously investigated. We hypothesised that dasatinib might be transported differently to imatinib, which might account for its favourable effects in imatinib-resistant patients. The CML cell line KCL22 was selected for transfection work as it has a particularly low basal level of hOCT1 expression. KCL22 cells were transfected with pcDNA3-hOCT1 plasmid (kind gift of D Gründemann, Germany) and stable lines were selected with high hOCT1 expression. The uptake of radiolabelled dasatinib (kind gift from Bristol Myers Squibb) was greater in high-hOCT1 expressing cells than in mock transfected cells (p=0.0149, n=3). However, prazosin and amantadine, both inhibitors of hOCT transport, did not decrease dasatinib uptake into mock transfected KCL22 cells, in sharp contrast to the block on imatinib uptake seen with both these inhibitors. Dasatinib decreased the level of phosphorylated CRKL (surrogate marker for BCR-ABL) by 49.9% in mock transfected KCL22 cells and 40.3% in high-hOCT1 expressing cells. The distribution coefficient (logD) of dasatinib between 1-octanol and HEPES medium was 2.05, compared with 0.81 for imatinib, demonstrating that dasatinib is more lipophilic. The efflux of dasatinib was investigated in confluent monolayers of Madin-Darby canine kidney (MDCKII) cells on a semipermeable membrane. These cells stably express ABCB1 (MDR1) on their apical but not their basal aspect. Both dasatinib and imatinib were transported from the basal to the apical layer, indicating ABCB1 transporter-mediated efflux of both drugs (p=0.001, p<0.0001, respectively). Addition of the ABCB1 inhibitor PSC833 blocked transport of both drugs (p=0.0013, p<0.0001 respectively). Taken together, the data are consistent with the view that dasatinib, unlike imatinib, may achieve adequate intracellular levels and BCR-ABL suppression even in cells with low or blocked hOCT1 function. However, in cells with high hOCT1 expression, dasatinib uptake may be augmented. Efflux of dasatinib and imatinib appear similar and via ABCB1. Dasatinib may be useful therapeutically in patients with imatinib resistance related to low hOCT1 expression.
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Deng, Minjuan, Kang Qin, Yang Li, Yu Jiang, Wei Jin, and Zhirong Shen. "Abstract 5338: Pamiparib as a non-P-glycoprotein substrate PARP inhibitor can overcome ABCB1-mediated multidrug resistance in ovarian cancer cells." Cancer Research 82, no. 12_Supplement (June 15, 2022): 5338. http://dx.doi.org/10.1158/1538-7445.am2022-5338.
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Abstract Ovarian cancer is commonly treated with debulking surgery followed by platinum-taxane based chemotherapy and targeted therapy (PARP inhibitors). However, a key challenge facing these therapies is drug resistance caused by various mechanisms, one of which is the P-glycoprotein (P-gp, encoded by the ABCB1 gene) mediated multidrug resistance. Tumor cells with high P-gp protein expression extrude P-gp substrate drugs from intracellular space and reduce drug efficacy. Since Paclitaxel and some PARP inhibitors such as Olaparib are P-gp substrates, their clinical benefit may be compromised in primary or acquired P-gp positive patients. Pamiparib, on the other hand, is PARP inhibitor that is not P-gp substrate, therefore, it is reasonable to hypothesize that Pamiparib will have better efficacy in P-gp high ovarian tumors. To test this hypothesis, Paclitaxel or Olaparib acquired resistant cell lines were developed through prolonged in vitro culture of sensitive A2780 cells (P-gp negative) with stepwise increased concentrations of individual drug. Both Paclitaxel and Olaparib resistant cells (A2780pacR and A2780olaR, respectively) highly express P-gp protein and are cross-resistant to each other, indicating this acquired resistance is mediated by P-gp protein upregulation. Indeed, P-gp protein inhibitor Verapamil can largely restore sensitivity to Paclitaxel and Olaparib in A2780pacR and A2780olaR cells, while over-expression of P-gp protein into parental A2780 cell line recapitulated the resistant phenotype. In contrast to Paclitaxel and Olaparib, Pamiparib as non P-gp substrate, is equally sensitive to parental A2780, A2780pacR and A2780olaR cell lines and not affected by P-gp expression. Consistently, intracellular drug concentration of Pamiparib is equivalent in A2780 and A2780olaR cells, however, decreased cellular Olaparib was detected in resistant cell line. These findings have been further confirmed in Olaparib resistant CDX model, in which Pamiparib showed tumor growth inhibition while Olaparib did not. The results highly suggest that Pamiparib as a non-P-gp substrate PARP inhibitor can overcome ABCB1-mediated drug resistance in tumors and may provide additional clinical benefits to ovarian cancer patients. Citation Format: Minjuan Deng, Kang Qin, Yang Li, Yu Jiang, Wei Jin, Zhirong Shen. Pamiparib as a non-P-glycoprotein substrate PARP inhibitor can overcome ABCB1-mediated multidrug resistance in ovarian cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5338.
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Zhang, Sheng-lie, Yin-xiang Wei, Qian Li, Hao-peng Sun, Hui Peng, and Qi-dong You. "Pharmacophore-Based Drug Design and Biological Evaluation of Novel ABCB1 Inhibitors." Chemical Biology & Drug Design 81, no. 3 (December 26, 2012): 349–58. http://dx.doi.org/10.1111/cbdd.12081.
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Dastvan, Reza, Smriti Mishra, Yelena B. Peskova, Robert K. Nakamoto, and Hassane S. Mchaourab. "Mechanism of allosteric modulation of P-glycoprotein by transport substrates and inhibitors." Science 364, no. 6441 (May 16, 2019): 689–92. http://dx.doi.org/10.1126/science.aav9406.
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The ATP-binding cassette subfamily B member 1 (ABCB1) multidrug transporter P-glycoprotein plays a central role in clearance of xenobiotics in humans and is implicated in cancer resistance to chemotherapy. We used double electron electron resonance spectroscopy to uncover the basis of stimulation of P-glycoprotein adenosine 5′-triphosphate (ATP) hydrolysis by multiple substrates and illuminate how substrates and inhibitors differentially affect its transport function. Our results reveal that substrate-induced acceleration of ATP hydrolysis correlates with stabilization of a high-energy, post-ATP hydrolysis state characterized by structurally asymmetric nucleotide-binding sites. By contrast, this state is destabilized in the substrate-free cycle and by high-affinity inhibitors in favor of structurally symmetric nucleotide binding sites. Together with previous data, our findings lead to a general model of substrate and inhibitor coupling to P-glycoprotein.
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Yakusheva, Elena N., Aleksey V. Shchulkin, Ivan V. Chernykh, Natalia M. Popova, Anna A. Kotlyarova, and Alexandr A. Slepnev. "Assessment of drugs belonging to inhibitors and inductors of p-glycoprotein in vitro." Reviews on Clinical Pharmacology and Drug Therapy 17, no. 1 (May 30, 2019): 71–78. http://dx.doi.org/10.17816/rcf17171-78.
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The article describes modern approaches for testing of drugs belonging to substrates and inhibitors of P-glycoprotein (Pgp, ABCB1-protein, MDR1-protein) according to the recommendations of Food and Drug Administration (United States) and European Medicines Agency. In vitro methods on cell lines with hyperexpression of the transporter are presented. The same analysis was done on human colon adenocarcinoma cell line (Caco-2).
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Chernykh, Ivan V., Alexey V. Shchulkin, Natalya M. Popova, Ekaterina E. Kirichenko, and Elena N. Yakusheva. "Vegetative polysaccharides: new target and direction of clinical use." Reviews on Clinical Pharmacology and Drug Therapy 16, no. 4 (December 15, 2018): 5–9. http://dx.doi.org/10.17816/rcf1645-9.
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P-glycoprotein (Pgp, ABCB1-protein) is a membrane efflux protein transporter located in hepatocytes, enterocytes, epithelial cells of the proximal renal tubules, histogematic barriers, and also in tumor cells, which releases the substrates from cells and plays an important role in pharmacokinetics of drugs. Inhibition of Pgp functional activity is promising for increasing the effectiveness of drug treatment of oncological diseases and pharmacoresistant epilepsy, but today no synthetic inhibitor of the transporter can be used in clinical practice because of side effects development. The aim of this review is to prove the relevance to use polysaccharides of plant origin, their derivatives and chemical modifications as effective, safe and cost-effective inhibitors of the transporter protein. The article describes the chemical structure of Pgp substrates and inhibitors, the ability to predict the drugs belonging to the transporter protein substrates or inhibitors. Some results of investigations of the belonging of oligo- and polysaccharides to Pgp substrates and inhibitors are presented. They are premises for carrying out appropriate experiments for polysaccharides of plant origin both in vitro and in vivo.
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Lentz, Fabian, Norbert Reiling, Gabriella Spengler, Annamária Kincses, Andrea Csonka, Joseph Molnár, and Andreas Hilgeroth. "Dually Acting Nonclassical 1,4-Dihydropyridines Promote the Anti-Tuberculosis (Tb) Activities of Clofazimine." Molecules 24, no. 16 (August 8, 2019): 2873. http://dx.doi.org/10.3390/molecules24162873.
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The number of effective antituberculotic drugs is strongly limited to four first-line drugs in standard therapy. In case of resistances second-line antibiotics are used with a poor efficacy and tolerability. Therefore, novel antituberculotic drugs are urgently needed. We synthesized novel nonclassical 1,4-dihydropyridines and evaluated their antituberculotic properties depending on substituent effects. Preferred substituents could be identified. As related classical 1,4-dihydropyridines are known as inhibitors of the transmembrane efflux pump ABCB1 in cancer cells, we wondered whether a use of our compounds may be of favour to enhance the antituberculotic drug efficacy of the second-line antituberculotic drug clofazimine, which is a known substrate of ABCB1 by a suggested inhibition of a corresponding efflux pump in Mycobacterium tuberculosis (Mtb). For this, we determined the ABCB1 inhibiting properties of our compounds in a mouse T-lymphoma cell line model and then evaluated the drug-enhancing properties of selected compounds in a co-application with clofazimine in our Mtb strain. We identified novel enhancers of clofazimine toxicity which could prevent clofazimine resistance development mediated by an efflux pump activity.
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Micucci, Matteo, Maurizio Viale, Alberto Chiarini, Domenico Spinelli, Maria Frosini, Cinzia Tavani, Massimo Maccagno, Lara Bianchi, Rosaria Gangemi, and Roberta Budriesi. "3-Aryl-4-nitrobenzothiochromans S,S-dioxide: From Calcium-Channel Modulators Properties to Multidrug-Resistance Reverting Activity." Molecules 25, no. 5 (February 27, 2020): 1056. http://dx.doi.org/10.3390/molecules25051056.
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Our research groups have been involved for many years in studies aimed at identifying new active organic compounds endowed with pharmacological properties. In this work, we focused our attention on the evaluation of cardiovascular and molecular drug resistance (MDR) reverting activities of some nitrosubstituted sulphur-containing heterocycles. Firstly, we have examined the effects of 4-nitro-3-(4-methylphenyl)-3,6-dihydro-2H-thiopyran S,S-dioxide 5, and have observed no activity. Then we have extended our investigation to the 3-aryl-4-nitrobenzothiochromans S,S-dioxide 6 and 7, and have observed an interesting biological profile. Cardiovascular activities were assessed for all compounds using ex vivo studies, while the MDR reverting effect was evaluated only for selected compounds using tumor cell lines. All compounds were shown to affect cardiovascular parameters. Compound 7i exerted the most effect on negative inotropic activity, while 6d and 6f could be interesting molecules for the development of more active ABCB1 inhibitors. Both 6 and 7 represent structures of large possible biological interest, providing a scaffold for the identification of new ABCB1 inhibitors.
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Tsai, Yo-Ting, Gerard Lozanski, Amy M. Lehman, Ellen Sass, Erin K. Hertlein, Santosh Salunke, Ching-Shih Chen, Michael R. Grever, John C. Byrd, and David M. Lucas. "BRAFV600E Induces ABCB1/P-Glycoprotein Expression and Drug Resistance in B-Cells Via AP-1 Activation." Blood 126, no. 23 (December 3, 2015): 2477. http://dx.doi.org/10.1182/blood.v126.23.2477.2477.
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Abstract A subset of patients with chronic lymphocytic leukemia (CLL) and nearly all patients with classic hairy cell leukemia (HCL) harbor somatic BRAF activating mutations. However, the pathological role of activated BRAF in B-cell leukemia development and progression remains unclear. In addition, although HCL patients respond well to the BRAFV600E inhibitor vemurafenib, relapses are being observed, suggesting the development of drug resistance in patients with this mutation. Cell line models to study the mechanism of BRAFV600E in B cell leukemia do not exist. Therefore, we utilized the CLL-like B-cell line OSUCLL (PLoS One 2013, 8(10):e76607) to generate cells with doxycycline (dox)-inducible BRAFV600E expression to examine transcriptional and biochemical features induced by this mutation in B-cells. We observed that BRAFV600E induction enhanced proliferation and activation of MAPK signaling in these cells. Microarray using Affymetrix U133 Plus 2.0 GeneChips demonstrated that 235 genes were up-regulated at least 2-fold, and 227 genes were down-regulated at least 2-fold. Several were confirmed by real-time RT-PCR analysis, including ABCB1 (p<0.001, vehicle versus dox-treated). Increased expression of the ABCB1 gene product, P-glycoprotein (P-gp), was also confirmed by immunoblot. This increase resulted in enhanced P-gp function as determined by rhodamine exclusion assays (p<0.005), an effect that was significantly reversed by the P-gp inhibitor verapamil (p<0.005). As an additional demonstration of P-gp function, BRAFV600E induction via dox treatment resulted in a significant increase in resistance to the P-gp substrate vincristine, and the addition of verapamil significantly reduced vincristine resistance. Importantly, pharmacological inhibition of BRAFV600E and MEK, by vemurafenib and CI-1040 respectively, diminished BRAFV600E-induced MAPK pathway activation and enhancement of ABCB1/P-gp expression. To further understand the transcriptional mechanism of BRAFV600E-induced P-gp expression, we performed luciferase assays using a vector containing 1 kb of ABCB1 promoter driving a luciferase reporter, co-transfected into HEK293 cells with either an empty vector or a vector containing BRAFV600E. These assays showed that BRAFV600E expression enhanced luciferase activity (p<0.001), and this effect was down-regulated by vemurafenib and CI-1040 (p<0.001). The role of AP-1 has been reported in ABCB1 regulation. Thus, we conducted electrophoretic mobility shift assays in the BRAFV600E-transfected OSUCLL B-cells, with or without dox treatment, to identify AP-1 factor(s) involved in ABCB1 regulation by BRAFV600E. In these experiments, a supershift in the AP-1 complex was produced by an antibody to JunD in the presence of dox treatment, demonstrating that at least JunD activity is important in this mechanism. Based on these observations, we conclude that BRAFV600E activates AP-1 proteins including JunD to induce ABCB1/P-gp expression and drug resistance in B-cells. This study uncovers a new pathological role for BRAFV600E in B-cell leukemia, and provides further evidence that combination strategies with inhibitors of BRAFV600E and MEK may be beneficial in delaying disease progression and occurrence of resistance to drugs that are substrates of P-gp. Disclosures Byrd: Acerta Pharma BV: Research Funding.