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PloS One 2018This paper describes the preparation of giant unilamellar vesicles with reconstituted hamster P-glycoprotein (Pgp, ABCB1) for studying the transport activity of this...
This paper describes the preparation of giant unilamellar vesicles with reconstituted hamster P-glycoprotein (Pgp, ABCB1) for studying the transport activity of this efflux pump in individual liposomes using optical microscopy. Pgp, a member of ABC (ATP-binding cassette) transporter family, is known to contribute to the cellular multidrug resistance (MDR) against variety of drugs. The efficacy of many therapeutics is, thus, hampered by this efflux pump, leading to a high demand for simple and effective strategies to monitor the interactions of candidate drugs with this protein. Here, we applied small Pgp proteoliposomes to prepare giant Pgp-bearing liposomes via modified electroformation techniques. The presence of Pgp in the membrane of giant proteoliposomes was confirmed using immunohistochemistry. Assessment of Pgp ATPase activity suggested that this transporter retained its activity upon reconstitution into giant liposomes, with an ATPase specific activity of 439 ± 103 nmol/mg protein/min. For further confirmation, we assessed the transport activity of Pgp in these proteoliposomes by monitoring the translocation of rhodamine 123 (Rho123) across the membrane using confocal microscopy at various ATP concentrations (0-2 mM) and in the presence of Pgp inhibitors. Rate of change in Rho123 concentration inside the liposomal lumen was used to estimate the Rho123 transport rates (1/s) for various ATP concentrations, which were then applied to retrieve the Michaelis-Menten constant (Km) of ATP in Rho123 transport (0.42 ± 0.75 mM). Similarly, inhibitory effects of verapamil, colchicine, and cyclosporin A on Pgp were studied in this system and the IC50 values for these Pgp inhibitors were found 26.6 ± 6.1 μM, 94.6 ± 47.6 μM, and 0.21 ± 0.07 μM, respectively. We further analyzed the transport data using a kinetic model that enabled dissecting the passive diffusion of Rho123 from its Pgp-mediated transport across the membrane. Based on this model, the permeability coefficient of Rho123 across the liposomal membrane was approximately 1.25×10-7 cm/s. Comparing the membrane permeability in liposomes with and without Pgp revealed that the presence of this protein did not have a significant impact on membrane integrity and permeability. Furthermore, we used this model to obtain transport rate constants for the Pgp-mediated transport of Rho123 (m3/mol/s) at various ATP and inhibitor concentrations, which were then applied to estimate values of 0.53 ± 0.66 mM for Km of ATP and 25.2 ± 5.0 μM for verapamil IC50, 61.8 ± 34.8 μM for colchicine IC50, and 0.23 ± 0.09 μM for cyclosporin A IC50. The kinetic parameters obtained from the two analyses were comparable, suggesting a minimal contribution from the passive Rho123 diffusion across the membrane. This approach may, therefore, be applied for screening the transport activity of Pgp against potential drug candidates.
Topics: ATP Binding Cassette Transporter, Subfamily B, Member 1; Adenosine Triphosphatases; Adenosine Triphosphate; Animals; Biological Transport; Cricetinae; Drug Resistance, Multiple; Proteolipids; Rhodamine 123
PubMed: 29912971
DOI: 10.1371/journal.pone.0199279 -
Fundamental & Clinical Pharmacology Dec 2016Ticagrelor is the unique reversible oral antiplatelet drug commercialized today. During this study, the intestinal permeability of ticagrelor and its potential...
Ticagrelor is the unique reversible oral antiplatelet drug commercialized today. During this study, the intestinal permeability of ticagrelor and its potential P-glycoprotein (P-gp)-mediated active transport were assessed. To this end, bidirectional transport of ticagrelor was performed across Caco-2 (human epithelial colorectal adenocarcinoma) monolayer model in the presence and absence of potent P-gp inhibitor valspodar. Ticagrelor presented an apical-basolateral apparent permeability coefficient (P ) of 6.0 × 10 cm/s. On the other hand, mean efflux ratio (ER) of 2.71 was observed for ticagrelor describing a higher efflux permeability compared to the influx component. Valspodar showed a significant inhibitory effect on the efflux of ticagrelor suggesting involvement of P-gp in its oral disposition. Co-incubation of the P-gp inhibitor decreased the efflux P of ticagrelor from 1.60 × 10 to 1.13 × 10 cm/s and decreased its ER by 70%. Results suggest a modest active transport of ticagrelor by P-gp across the Caco-2 cell monolayer. The co-administration of ticagrelor with a P-gp inhibitor seems altogether unlikely to have an extended impact on pharmacokinetics of ticagrelor and cause bleeding events in patients.
Topics: ATP Binding Cassette Transporter, Subfamily B, Member 1; Adenosine; Biological Transport, Active; Caco-2 Cells; Cell Line, Tumor; Cyclosporins; Humans; Intestinal Mucosa; Permeability; Protein Transport; Ticagrelor
PubMed: 27416295
DOI: 10.1111/fcp.12219 -
Molecules (Basel, Switzerland) Apr 2017Adenosine triphosphate (ATP)-binding cassette (ABC) transporters are highly expressed in tumor cells, as well as in organs involved in absorption and secretion... (Review)
Review
Adenosine triphosphate (ATP)-binding cassette (ABC) transporters are highly expressed in tumor cells, as well as in organs involved in absorption and secretion processes, mediating the ATP-dependent efflux of compounds, both endogenous substances and xenobiotics, including drugs. Their expression and activity levels are modulated by the presence of inhibitors, inducers and/or activators. In vitro, ex vivo and in vivo studies with both known and newly synthesized P-glycoprotein (P-gp) inducers and/or activators have shown the usefulness of these transport mechanisms in reducing the systemic exposure and specific tissue access of potentially harmful compounds. This article focuses on the main ABC transporters involved in multidrug resistance [P-gp, multidrug resistance-associated protein 1 (MRP1) and breast cancer resistance protein (BCRP)] expressed in tissues of toxicological relevance, such as the blood-brain barrier, cardiovascular system, liver, kidney and intestine. Moreover, it provides a review of the available cellular models, in vitro and ex vivo assays for the screening and selection of safe and specific inducers and activators of these membrane transporters. The available cellular models and in vitro assays have been proposed as high throughput and low-cost alternatives to excessive animal testing, allowing the evaluation of a large number of compounds.
Topics: ATP Binding Cassette Transporter, Subfamily B, Member 1; ATP Binding Cassette Transporter, Subfamily G, Member 2; Animals; Drug Discovery; Drug Evaluation, Preclinical; Drug Resistance; Humans; Models, Biological; Multidrug Resistance-Associated Proteins; Organ Specificity; Structure-Activity Relationship
PubMed: 28397762
DOI: 10.3390/molecules22040600 -
Molecules (Basel, Switzerland) Nov 2023P-glycoprotein (P-gp) is a crucial membrane transporter situated on the cell's apical surface, being responsible for eliminating xenobiotics and endobiotics. P-gp... (Review)
Review
P-glycoprotein (P-gp) is a crucial membrane transporter situated on the cell's apical surface, being responsible for eliminating xenobiotics and endobiotics. P-gp modulators are compounds that can directly or indirectly affect this protein, leading to changes in its expression and function. These modulators can act as inhibitors, inducers, or activators, potentially causing drug-drug interactions (DDIs). This comprehensive review explores diverse models and techniques used to assess drug-induced P-gp modulation. We cover several approaches, including , , , and methods, with their respective strengths and limitations. Additionally, we explore the therapeutic implications of DDIs involving P-gp, with a special focus on the renal and intestinal elimination of P-gp substrates. This involves enhancing the removal of toxic substances from proximal tubular epithelial cells into the urine or increasing the transport of compounds from enterocytes into the intestinal lumen, thereby facilitating their excretion in the feces. A better understanding of these interactions, and of the distinct techniques applied for their study, will be of utmost importance for optimizing drug therapy, consequently minimizing drug-induced adverse and toxic effects.
Topics: ATP Binding Cassette Transporter, Subfamily B, Member 1; Membrane Transport Proteins; ATP Binding Cassette Transporter, Subfamily B; Kidney; Drug Interactions
PubMed: 38005253
DOI: 10.3390/molecules28227532 -
Journal of Chemical Information and... Oct 2020The efflux transporter P-glycoprotein (P-gp) is responsible for the extrusion of a wide variety of molecules, including drug molecules, from the cell. Therefore,...
The efflux transporter P-glycoprotein (P-gp) is responsible for the extrusion of a wide variety of molecules, including drug molecules, from the cell. Therefore, P-gp-mediated efflux transport limits the bioavailability of drugs. To identify potential P-gp substrates early in the drug discovery process, models have been developed based on structural and physicochemical descriptors. In this study, we investigate the use of molecular dynamics fingerprints (MDFPs) as an orthogonal descriptor for the training of machine learning (ML) models to classify small molecules into substrates and nonsubstrates of P-gp. MDFPs encode the information from short MD simulations of the molecules in different environments (water, membrane, or protein pocket). The performance of the MDFPs, evaluated on both an in-house dataset (3930 compounds) and a public dataset from ChEMBL (1114 compounds), is compared to that of commonly used 2D molecular descriptors, including structure-based and property-based descriptors. We find that all tested classifiers interpolate well, achieving high accuracy on chemically diverse subsets. However, by challenging the models with external validation and prospective analysis, we show that only tree-based ML models trained on MDFPs or property-based descriptors generalize well to regions of the chemical space not covered by the training set.
Topics: ATP Binding Cassette Transporter, Subfamily B; ATP Binding Cassette Transporter, Subfamily B, Member 1; Machine Learning; Molecular Dynamics Simulation; Prospective Studies
PubMed: 32786699
DOI: 10.1021/acs.jcim.0c00525 -
Journal of Veterinary Pharmacology and... Jul 2023The P-glycoprotein (P-gp) substrate status of antineoplastic drugs intended for veterinary patients is an important characteristic to define for two reasons. First,...
The P-glycoprotein (P-gp) substrate status of antineoplastic drugs intended for veterinary patients is an important characteristic to define for two reasons. First, neoplastic cells expressing P-gp can actively efflux drugs that are P-gp substrates curtailing their efficacy. Second, antineoplastic drugs tend to have a narrow therapeutic index. Antineoplastic drugs that are P-gp substrates can cause severe adverse reactions in animals with P-gp dysfunction such as dogs with ABCB1-1Δ and cats with ABCB11930_1931del TC. Animals with P-gp dysfunction experience greater overall exposure to P-gp substrate drugs due to mechanisms such as increased intestinal absorption, decreased biliary clearance and greater central nervous system penetration compared with animals with normal P-gp function. Accordingly, knowing the P-gp substrate status of antineoplastic drugs is an important safety consideration prior to use in canine or feline cancer patients. This study used a cell line overexpressing canine P-gp to assess the P-gp substrate status of verdinexor. Based on both a cytotoxicity assay and a competitive flow cytometry assay verdinexor is not a substrate for canine P-gp.
Topics: Animals; Dogs; Cats; ATP Binding Cassette Transporter, Subfamily B, Member 1; ATP Binding Cassette Transporter, Subfamily B; Acrylamides; Hydrazines
PubMed: 36924353
DOI: 10.1111/jvp.13123 -
Leukemia & Lymphoma Jan 2015Multiple myeloma (MM) is a malignant neoplastic cancer of the plasma cells that involves the bone marrow. The majority of patients with MM initially respond to... (Review)
Review
Multiple myeloma (MM) is a malignant neoplastic cancer of the plasma cells that involves the bone marrow. The majority of patients with MM initially respond to chemotherapy, but they eventually become resistant to later drug therapy. One of the reasons for drug resistance in patients with MM is efflux transporters. P-glycoprotein (P-gp) is the most studied of the multidrug resistance proteins, and is up-regulated in response to many chemotherapeutic drugs. This up-regulation of P-gp causes a decrease in the intracellular accumulation of these drugs, limiting their therapeutic efficacy. In this review, we focus on the role of P-gp in drugs used for patients with MM. P-gp has been found to be an important factor with regard to drug resistance in many of the drug classes used in the treatment of MM (proteasome inhibitors, anthracyclines, alkylating agents and immunomodulators are examples). Thus, our further understanding of its mechanism and inhibitory effects will help us decrease drug resistance in patients with MM.
Topics: ATP Binding Cassette Transporter, Subfamily B, Member 1; Animals; Antineoplastic Agents; Drug Resistance, Multiple; Drug Resistance, Neoplasm; Humans; Multiple Myeloma; Neoplasms
PubMed: 24678978
DOI: 10.3109/10428194.2014.907890 -
Clinical Pharmacokinetics Feb 2016As the effect of P-glycoprotein (P-gp) transport on antidepressant delivery has been extensively evaluated using in vitro cellular and in vivo rodent models, an... (Review)
Review
As the effect of P-glycoprotein (P-gp) transport on antidepressant delivery has been extensively evaluated using in vitro cellular and in vivo rodent models, an increasing number of publications have addressed the effect of P-gp in limiting brain penetration of antidepressants and causing treatment-resistant depression in current clinical therapies. However, contradictory results have been observed in different systems. It is of vital importance to understand the potential for drug interactions related to P-gp at the blood-brain barrier (BBB), and whether coadministration of a P-gp inhibitor together with an antidepressant is a good clinical strategy for dosing of patients with treatment-resistant depression. In this review, the complicated construction of the BBB, the transport mechanisms for compounds that cross the BBB, and the basic characteristics of antidepressants are illustrated. Further, the reliability of different systems related to antidepressant brain delivery, including in vitro bidirectional transport cell lines, in vivo Mdr1 knockout mice, and chemical inhibition studies in rodents are analyzed, supporting a low possibility that P-gp affects currently marketed antidepressants when these results are extrapolated to the human BBB. These findings can also be applied to other central nervous system drugs.
Topics: ATP Binding Cassette Transporter, Subfamily B, Member 1; Animals; Antidepressive Agents; Brain; Humans
PubMed: 26293617
DOI: 10.1007/s40262-015-0310-2 -
The Veterinary Clinics of North... Nov 2018Overdoses of macrocyclic lactones in dogs and cats can result in such signs as tremors, ataxia, seizures, coma, and blindness. Dogs with the ABCB1-1Δ gene defect are... (Review)
Review
Overdoses of macrocyclic lactones in dogs and cats can result in such signs as tremors, ataxia, seizures, coma, and blindness. Dogs with the ABCB1-1Δ gene defect are predisposed to macrocyclic lactone toxicosis at lower dosages than dogs without the defect. Intravenous lipid emulsion therapy has been suggested for treatment of macrocyclic lactone toxicosis but evidence of efficacy is limited. Initial decontamination and supportive care remain the mainstays of therapy for macrocyclic lactone toxicosis.
Topics: ATP Binding Cassette Transporter, Subfamily B, Member 1; Animals; Anthelmintics; Cat Diseases; Cats; Dog Diseases; Dogs; Fat Emulsions, Intravenous; Genotype; Ivermectin; Lactones; Macrolides; Seizures
PubMed: 30139545
DOI: 10.1016/j.cvsm.2018.07.002 -
Journal of Pharmaceutical Sciences Jan 2024This report focuses on pharmacokinetic drug-endogenous substrate interactions (DEIs). We hypothesized that P-glycoprotein (P-gp)-mediated DEI might affect androgen...
This report focuses on pharmacokinetic drug-endogenous substrate interactions (DEIs). We hypothesized that P-glycoprotein (P-gp)-mediated DEI might affect androgen kinetics, especially its blood-brain barrier (BBB) permeability. The intracellular accumulation of the endogenous substrates of P-gp, testosterone (TES) and androstenedione (ADO) was increased by several tested drugs in uptake studies using P-gp overexpressing cells, indicating that these drugs inhibit P-gp-mediated efflux of TES of ADO from the cells. In a transport study using rat BBB kit, we found that the BBB limited the penetration of TES and ADO into the central nervous system. In addition, tested drugs that cause DEI were found to increase BBB permeability of TES and ADO via P-gp inhibition. In short, this study provides new findings regarding the possibility that DEI may affect the kinetics of endogenous substrates of P-gp.
Topics: Rats; Animals; Blood-Brain Barrier; Androgens; Biological Transport; ATP Binding Cassette Transporter, Subfamily B; ATP Binding Cassette Transporter, Subfamily B, Member 1; Permeability; Testosterone
PubMed: 37898165
DOI: 10.1016/j.xphs.2023.10.034