ATP-binding Cassette Efflux Transporters Research Articles

Low Doses of Deoxynivalenol and Zearalenone Alone or in Combination with a Mycotoxin Binder Affect ABCB1 mRNA and ABCC2 mRNA Expression in the Intestines of Pigs.

Mycotoxin binders, in combination with enzymes degrading some mycotoxins, contribute to feed detoxification. Their use reduces economic losses and the negative impacts of mycotoxins on animal health and productivity in farm animals. The aim of this study was to evaluate the efficacy of a mycotoxin detoxifier on the expression of the ATP-binding cassette efflux transporters ABCB1 mRNA and ABCC2 mRNA, which transport xenobiotics and thus have a barrier function, in the tissues of pigs exposed to low doses of deoxynivalenol (DON, 1 mg/kg feed) and zearalenone (ZEN, 0.4 mg/kg feed) for 37 days. The levels of expression were determined by an RT-PCR, and the effect of the mycotoxin detoxifier (Mycofix Plus3.E) was evaluated by a comparison of results between healthy pigs (n = 6), animals treated with DON and ZEN (n = 6), and a group that received both mycotoxins and the detoxifier (n = 6). A significant downregulation of ABCB1 mRNA and ABCC2 mRNA was observed in the jejunum (p < 0.05). A tendencies toward the downregulation of ABCB1 mRNA and ABCC2 mRNA were found in the ileum and duodenum, respectively. The mycotoxin detoxifier restored the expression of ABCB1 mRNA to the level found in healthy animals but did not restore that of ABCC2 mRNA to the level of healthy animals in the jejunum.

Chronic exposure to polystyrene microplastics increased the chemosensitivity of normal human liver cells via ABC transporter inhibition

Microplastics (MPs) are ubiquitous in environmental compartments and consumer products. Although liver is frequently reported to be a target organ of MP accumulation in mammals, few studies have focused on MP hepatoxicity in humans. In this study, we used normal human liver cells, THLE-2, to assess the acute and chronic toxicity of polystyrene (PS) MPs with sizes of 0.1 and 1 μm. The results showed that after 48 h of exposure, both kinds of PS MPs could enter THLE-2 cells and cause no obviously acute cytotoxicity at <20 μg/mL. In contrast, metabolomic analysis revealed that 90 days of PS MPs exposure at environmentally relevant dose (0.2 μg/mL) could significantly alter the metabolic profiles of the cells, especially the nanosized MPs. KEGG pathway analysis showed that the ATP-binding cassette (ABC) transporter pathway was the most significantly changed pathway. Cell functional tests confirmed that chronic PS MP treatment could inhibit the activity of the ABC efflux transporter and further increase the cytotoxicity of arsenic, indicating that the PS MPs had a chemosensitizing effect. These findings underline the chronic risk of MPs to human liver.

Dissimilar Effect of P-Glycoprotein and Breast Cancer Resistance Protein Inhibition on the Distribution of Erlotinib to the Retina and Brain in Humans and Mice.

P-glycoprotein (P-gp) and breast cancer resistance protein (BCRP) are two ATP-binding cassette efflux transporters that are coexpressed at the human blood-brain barrier (BBB) and blood-retina barrier (BRB). While pharmacological inhibition of P-gp and/or BCRP results in increased brain distribution of dual P-gp/BCRP substrate drugs, such as the tyrosine kinase inhibitor erlotinib, the effect of P-gp and/or BCRP inhibition on the retinal distribution of such drugs has hardly been investigated. In this study, we used positron emission tomography (PET) imaging to assess the effect of transporter inhibition on the distribution of [11C]erlotinib to the human retina and brain. Twenty two healthy volunteers underwent two PET scans after intravenous (i.v.) injection of a microdose (<5 μg) of [11C]erlotinib, a baseline scan, and a second scan either with concurrent i.v. infusion of tariquidar to inhibit P-gp (n = 5) or after oral intake of single ascending doses of erlotinib (300 mg, 650 mg, or 1000 mg, n = 17) to saturate erlotinib transport. In addition, transport of [3H]erlotinib to the retina and brain was assessed in mice by in situ carotid perfusion under various drug transporter inhibition settings. In comparison to the baseline PET scan, coadministration of tariquidar or erlotinib led to a significant decrease of [11C]erlotinib total volume of distribution (VT) in the human retina by -25 ± 8% (p ≤ 0.05) and -41 ± 16% (p ≤ 0.001), respectively. In contrast, erlotinib intake led to a significant increase in [11C]erlotinib VT in the human brain (+20 ± 16%, p ≤ 0.001), while administration of tariquidar did not result in any significant changes. In situ carotid perfusion experiments showed that both P-gp and BCRP significantly limit the distribution of erlotinib to the mouse retina and brain but revealed a similar discordant effect at the mouse BRB and BBB following co-perfusion with tariquidar and erlotinib as in humans. Co-perfusion with prototypical inhibitors of solute carrier transporters did not reveal a significant contribution of organic cation transporters (e.g., OCTs and OCTNs) and organic anion-transporting polypeptides (e.g., OATP2B1) to the retinal and cerebral distribution of erlotinib. In conclusion, we observed a dissimilar effect after P-gp and/or BCRP inhibition on the retinal and cerebral distribution of [11C]erlotinib. The exact mechanism for this discrepancy remains unclear but may be related to the function of an unidentified erlotinib uptake carrier sensitive to tariquidar inhibition at the BRB. Our study highlights the great potential of PET to study drug distribution to the human retina and to assess the functional impact of membrane transporters on ocular drug distribution.

Synthesis and Anticancer Activity of A-Ring-Modified Derivatives of Dihydrobetulin.

Multidrug resistance (MDR) is a common phenomenon in clinical oncology, whereby cancer cells become resistant to chemotherapeutic drugs. A common MDR mechanism is the overexpression of ATP-binding cassette efflux transporters in cancer cells, with P-glycoprotein (P-gp) being one of them. New 3,4-seco-lupane triterpenoids, and the products of their intramolecular cyclization with the removed 4,4-gem-dimethyl group, were synthesized by the selective transformations of the A-ring of dihydrobetulin. Among the semi-synthetic derivatives, the MT-assay-enabled methyl ketone 31 (MK), exhibiting the highest cytotoxicity (0.7-16.6 µM) against nine human cancer cell lines, including P-gp overexpressing subclone HBL-100/Dox, is identified. In silico, MK has been classified as a potential P-gp-inhibitor; however, the Rhodamine 123 efflux test, and the combined use of P-gp-inhibitor verapamil with MK in vitro, showed the latter to be neither an inhibitor nor a substrate of P-gp. As the studies have shown, the cytotoxic effect of MK against HBL-100/Dox cells is, arguably, induced through the activation of the ROS-mediated mitochondrial pathway, as evidenced by the positive Annexin V-FITC staining of apoptotic cells, the cell cycle arrest in the G0/G1 phase, mitochondrial dysfunction, cytochrome c release, and the activation of caspase-9 and -3.

Overcoming Multidrug Resistance by Base-Editing-Induced Codon Mutation.

Multidrug resistance (MDR) is the main obstacle in cancer chemotherapy. ATP binding cassette (ABC) transporters on the MDR cell membrane can transport a wide range of antitumor drugs out of cells, which is one of the main causes of MDR. Therefore, disturbing ABC transporters becomes the key to reversing MDR. In this study, we implement a cytosine base editor (CBE) system to knock out the gene encoding ABC transporters by base editing. When the CBE system works in MDR cells, the MDR cells are manipulated, and the genes encoding ABC transporters can be inactivated by precisely changing single in-frame nucleotides to induce stop (iSTOP) codons. In this way, the expression of ABC efflux transporters is reduced and intracellular drug retention is significantly increased in MDR cells. Ultimately, the drug shows considerable cytotoxicity to the MDR cancer cells. Moreover, the substantial downregulation of P-glycoprotein (P-gp) and breast cancer resistance protein (BCRP) implies the successful application of the CBE system in the knockout of different ABC efflux transporters. The recovery of chemosensitivity of MDR cancer cells to the chemotherapeutic drugs revealed that the system has a satisfactory universality and applicability. We believe that the CBE system will provide valuable clues for the use of CRISPR technology to defeat the MDR of cancer cells.

In vitro interaction of the pesticides flupyradifurone, bupirimate and its metabolite ethirimol with the ATP-binding cassette transporter G2 (ABCG2)

ABCG2 is an ATP-binding cassette efflux transporter that is expressed in absorptive and excretory organs such as liver, intestine, kidney, brain and testis where it plays a crucial physiological and toxicological role in protecting cells against xenobiotics, affecting pharmacokinetics of its substrates. In addition, the induction of ABCG2 expression in mammary gland during lactation is related to active secretion of many toxicants into milk. In this study, the in vitro interactions between ABCG2 and three pesticides flupyradifurone, bupirimate and its metabolite ethirimol were investigated to check whether these compounds are substrates and/or inhibitors of this transporter. Using in vitro transepithelial assays with cells transduced with murine, ovine and human ABCG2, we showed that ethirimol and flupyradifurone were transported efficiently by murine Abcg2 and ovine ABCG2 but not by human ABCG2. Bupirimate was not found to be an in vitro substrate of ABCG2 transporter. Accumulation assays using mitoxantrone in transduced MDCK-II cells suggest that none of the tested pesticides were efficient ABCG2 inhibitors, at least in our experimental conditions. Our studies disclose that ethirimol and flupyradifurone are in vitro substrates of murine and ovine ABCG2, opening the possibility of a potential relevance of ABCG2 in the toxicokinetics of these pesticides.

Reduction in ABCG2 mRNA Expression in Human Immortalised Brain Microvascular Endothelial Cells by Ferric Ammonium Citrate is Mediated by Reactive Oxygen Species and Activation of ERK1/2 Signalling.

The ATP-binding cassette (ABC) transport protein ABCG2 (also known as breast cancer resistance protein (BCRP)) is expressed at the luminal face of the blood-brain barrier (BBB), where it limits the brain uptake of a number of therapeutic drugs. We recently reported that the ABC efflux transporter P-glycoprotein (P-gp) was downregulated in human immortalised brain endothelial (hCMEC/D3) cells treated with ferric ammonium citrate (FAC). The aim of the present study, therefore, was to assess whether BCRP expression is also affected by FAC and identify any signalling mechanisms involved. ABCG2 mRNA was assessed by RT-qPCR. Protein levels of BCRP, phosphorylated extracellular-regulated kinases 1 and 2 (p-ERK1/2) and total ERK 1/2 were assessed by Western blot. Reactive oxygen species (ROS) levels were determined using 2',7'-dichlorofluorescin diacetate. Treatment of hCMEC/D3 cells with FAC (250 µM, 72 h) significantly reduced ABCG2 mRNA levels (32.2 ± 3.7%) without a concomitant reduction in BCRP protein expression. ABCG2 mRNA levels were restored to control levels when co-treated with the antioxidant N-acetylcysteine (NAC), suggesting the effect of FAC was mediated by a ROS-sensitive pathway. We also found that FAC-treatment was associated with increased levels of p-ERK1/2, suggesting involvement of the ERK1/2 signalling pathway in the observed ABCG2 mRNA downregulation. The ERK1/2 signalling pathway inhibitor U0126 restored p-ERK1/2 levels and partially attenuated the FAC-induced reduction in ABCG2 mRNA. This study suggests that FAC-induced downregulation of ABCG2 mRNA isdriven byROS and ERK1/2 signalling, mechanisms which may be exploited to modulate BCRP expression at the BBB.

Synthesis, characterization, and radiosynthesis of fluorine-18-AVT-011 as a Pgp chemoresistance imaging marker

P-glycoprotein (Pgp) is the most studied ATP-binding cassette (ABC) efflux transporter and contributes to chemoresistance. A few tracers have been developed to detect the in-vivo status of chemoresistance using positron emission tomography (PET) imaging. In our study, we have synthesized labeled AVT-011 with fluorine-18 (18F) followed by in-vitro and in-vivo analysis. Tosylate AVT-011 precursor was synthesized and characterized by 1H-NMR and 13C-NMR. AVT-011 was labeled with 18F using the nucleophilic substitution method, and a standard set of quality control was performed. The specificity for Pgp was tested in U87MG cells with and without an inhibitor (tariquidar). The biodistribution and in-vivo stability were tested in the small animals (mice). The biodistribution data of [18F]-AVT-011 was extracted from the PET-CT imaging of breast cancer patients (n = 6). The precursor was synthesized with 36 ± 4% yield and 97 ± 2% purity. The labeling was more than 95% with a 42 ± 2% yield, as evaluated by Radio-HPLC. The cell-binding assay showed a specificity of the tracer for Pgp as the uptake increased by twice after blocking the Pgp receptors. The radiotracer showed a hepatorenal excretion pathway for clearance in an animal study. The uptake was higher in the liver, lungs, spleen, and heart at 15 min and decreased at 60 min. The patients' distribution showed similar uptake patterns as observed in the small animals. [18F]AVT-011 was characterized successfully with high radiochemical purity and yield. The in-vitro and in-vivo studies proved its specificity for Pgp and safe for patient use.

In silico drug repurposing and lipid bilayer molecular dynamics puzzled out potential breast cancer resistance protein (BCRP/ABCG2) inhibitors

Multidrug resistance (MDR) is a fundamental reason for the fiasco of carcinoma chemotherapy. A wide variety of anticarcinoma drugs are expelled from neoplasm cells through the ATP-binding cassette (ABC) transporter superfamily, rendering the neoplasm cells resistant to treatment. The ATP-binding cassette transporter G2 (ABCG2, gene symbol BCRP) is an ABC efflux transporter that plays a key function in MDR to antineoplastic therapies. For these reasons, the identification of medicaments as BCRP inhibitors could assist in discovering better curative approaches for breast cancer therapy. Because of the deficiency of prospective BCRP inhibitors, the SuperDRUG2 database was virtually screened for inhibitor activity towards the BCRP transporter using molecular docking computations. The most potent drug candidates were then characterized utilizing molecular dynamics (MD) simulations. Furthermore, molecular mechanics-generalized Born surface area (MM-GBSA) binding affinities of the most potent drug candidates were estimated. Based on the MM-GBSA binding affinities throughout 150 ns MD simulations, three drugs—namely zotarolimus (SD002595), temsirolimus (SD003393), and glecaprevir (SD006009)—revealed greater binding affinities towards BCRP transporter compared to the co-crystallized BWQ ligand with ΔG binding values of –86.6 ± 5.6, –79.5 ± 8.0, –75.8 ± 4.6 and –59.5 ± 4.1 kcal/mol, respectively. The steadiness of these promising drugs bound with BCRP transporter was examined utilizing their structural and energetical analyses throughout a 150 ns MD simulation. To imitate the physiological environment, 150 ns MD simulations for the identified drugs bound with BCRP transporter were conducted in the 1-palmitoyl-2-oleoyl-phosphatidylcholine lipid bilayer. These findings identify zotarolimus, temsirolimus and glecaprevir as auspicious anti-MDR drug leads that warrant further experimental assays. Communicated by Ramaswamy H. Sarma

P‐gp and Bcrp Exert Differential Impacts on Maternal and Fetal Exposure to Tetrahydrocannabinol and Its Major Metabolites in Pregnant Mice

In the United States, cannabis consumption is increasing. (‐)‐Δ9‐tetrahydrocannabinol (THC) is the primary pharmacological active constituent of cannabis, while 11‐hydroxy‐THC (11‐OH‐THC) and 11‐nor‐9‐carboxy‐THC (THC‐COOH) are active and nonactive circulating metabolites of THC in humans, respectively. P‐glycoprotein (P‐gp) and breast cancer resistance protein (BCRP) are ATP‐binding cassette efflux transporters important for drug disposition, including brain penetration and fetal exposure. Our previous in vitro transport study showed that only THC‐COOH is a weak substrate of human BCRP, while previous animal studies reported that THC and its metabolites could be substrates of the P‐gp and BCRP orthologs in mice and macaques. To further investigate the roles of P‐gp and BCRP in the maternal‐fetal disposition of THC and its metabolites, we administrated 3 mg/kg THC (retro‐orbitally) to FVB wild‐type (WT), P‐gp‐/‐, and Bcrp‐/‐ pregnant mice on gestational day 18, and determined maternal, placental, and fetal concentrations of THC and its metabolites by LC‐MS/MS. The AUCs of THC and its major metabolites were estimated using a pooled data bootstrap approach. Differences in tissue/maternal plasma AUC ratios between WT and P‐gp‐/‐ or Bcrp‐/‐ were compared via unpaired parametric t‐test, and differences with p values of &lt; 0.05 were considered statistically significant. In Bcrp‐/‐ pregnant mice, the placenta/maternal plasma AUC ratio for THC was 188% (p &lt; 0.05) of respective AUC ratio in WT pregnant mice. In P‐gp‐/‐ pregnant mice, the maternal brain/maternal plasma AUC ratio for THC, maternal brain/maternal plasma and fetus/maternal plasma AUC ratios for 11‐OH‐THC, and fetus/maternal plasma AUC ratio for THC‐COOH were 29%, 65%, 49%, and 54% (p &lt; 0.05) of respective AUC ratio in WT pregnant mice. There were no significant differences in fetal exposure of THC in either P‐gp‐/‐ or Bcrp‐/‐ mice, and in fetal exposure of 11‐OH‐THC and THC‐COOH in Bcrp‐/‐ mice, compared to WT mice. In conclusion, this study revealed that P‐gp and Bcrp exerted differential impacts on maternal brain, placental, and fetal exposure to cannabinoids in pregnant mice. P‐gp and Bcrp do not seem to play a role in limiting fetal exposure to THC and its major metabolites; but could differentially alter maternal brain exposure to the cannabinoids.

Dabrafenib inhibits ABCG2 and cytochrome P450 isoenzymes; potential implications for combination anticancer therapy

Dabrafenib is a BRAF inhibitor used in combination treatment of malignant melanoma and non-small cell lung carcinoma. In this study, we aimed to characterize its interactions with cytochrome P450 (CYP) isoenzymes and ATP-binding cassette (ABC) efflux transporters that have critical impact on the pharmacokinetics of drugs and play a role in drug resistance development. Using accumulation assays, we showed that dabrafenib inhibited ABCG2 and, less potently, ABCB1 transporter. We also confirmed dabrafenib as a CYP2C8, CYP2C9, CYP3A4, and CYP3A5 inhibitor. Importantly, inhibition of ABCG2 and CYP3A4 by dabrafenib led to the potentiation of cytotoxic effects of mitoxantrone and docetaxel toward respective resistant cell lines in drug combination studies. On the contrary, the synergistic effect was not consistently observed in ABCB1-expressing models. We further demonstrated that mRNA levels of ABCB1, ABCG2, ABCC1, and CYP3A4 were increased after 24 h and 48 h exposure to dabrafenib. Overall, our data confirm dabrafenib as a drug frequently and potently interacting with ABC transporters and CYP isoenzymes. This feature should be addressed with caution when administering dabrafenib to patients with polypharmacy but also could be utilized advantageously when designing new dabrafenib-containing drug combinations to improve the therapeutic outcome in drug-resistant cancer.

Tissue Type Differences in ABCB1 Expression and Paclitaxel Tissue Pharmacokinetics in Patients With Esophageal Cancer.

Background: Data from previous work suggests that there is no correlation between systemic (plasma) paclitaxel exposure and efficacy in patients treated for esophageal cancer. In this trial, we investigated ATP-binding cassette efflux transporter expression and intratumoral pharmacokinetics of paclitaxel to identify changes which could be a first sign of chemoresistance. Methods: Patients with esophageal cancer treated with paclitaxel and carboplatin (± concomitant radiotherapy) were included. During the first and last cycle of weekly paclitaxel, blood samples and biopsies of esophageal mucosa and tumor tissue were taken. Changes in paclitaxel exposure and expression of ABCB1 (P-glycoprotein) over time were studied in both tumor tissue and normal appearing esophageal mucosa. Results: ABCB1 was significantly higher expressed in tumor tissue compared to esophageal tissue, during both the first and last cycle of paclitaxel (cycle 1: p < 0.01; cycle 5/6: p = 0.01). Interestingly, ABCB1 expression was significantly higher in adenocarcinoma than in squamous cell carcinoma (p < 0.01). During the first cycle, a trend towards a higher intratumoral paclitaxel concentration was observed compared to the esophageal mucosa concentration (RD:43%; 95%CI: −3% to 111% p = 0.07). Intratumoral and plasma paclitaxel concentrations were significantly correlated during the first cycle (AUC0–48 h: r = 0.72; p < 0.01). Conclusion: Higher ABCB1 expression in tumor tissue, and differences between histological tumor types might partly explain why tumors respond differently to systemic treatment. Resistance by altered intratumoral paclitaxel concentrations could not be demonstrated because the majority of the biopsies taken at the last cycle of paclitaxel did contain a low amount of tumor cells or no tumor.

The role of dietary flavonoids for modulation of ATP binding cassette transporter mediated multidrug resistance

Flavonoids are widely existing compounds with enormous pharmacological effects from food and medicine. However, the low bioavailability in intestinal absorption and metabolism limits their clinical application. Intestinal efflux ABC (ATP binding cassette) transporters, including P‐glycoprotein (P‐gp), breast cancer resistance protein (BCRP) and multidrug resistance‐ associated proteins (MRPs), act as ’’pumping doors” to regulate the efflux of flavonoids from intestinal epithelial cells into the intestinal cavity or the systemic circulation. The present review describes the critical effect of ABC transporters involved in the efflux of flavonoids which depend on its efflux direction. And the role of flavonoids for modulation of intestinal ABC transporters was emphasized and several examples were given. We summarized that the resistance effect of flavonoid‐mediated multidrug on ABC transporters may influence the bioavailability of drugs, bioactive ingredients and/or toxic compounds upon dietary uptake. Meanwhile, flavonoids functionalized as reversing agents of the ABC transporter maybe an important mechanism for unexpected food‐drug, food‐toxin or food‐food interactions. The overview also indicates that elucidation of the action and mechanism of the intestinal metabolic enzymes‐efflux transporters coupling will lay a foundation for improving the bioavailability of flavonoids in vivo and increasing their clinical efficacy.

Novel nanomedicines to overcome cancer multidrug resistance.

Chemotherapy remains a powerful tool to eliminate malignant cells. However, the efficacy of chemotherapy is compromised by the frequent emergence of intrinsic and acquired multidrug resistance (MDR). These chemoresistance modalities are based on a multiplicity of molecular mechanisms of drug resistance, including : 1) Impaired drug uptake into cancer cells; 2) Increased expression of ATP-binding cassette efflux transporters; 3) Loss of function of pro-apoptotic factors; 4) Enhanced DNA repair capacity; 5) Qualitative or quantitative alterations of specific cellular targets; 6) Alterations that allow cancer cells to tolerate adverse or stressful conditions; 7) Increased biotransformation or metabolism of anticancer drugs to less active or completely inactive metabolites; and 8) Intracellular and intercellular drug sequestration in well-defined organelles away from the cellular target. Hence, one of the major aims of cancer research is to develop novel strategies to overcome cancer drug resistance. Over the last decades, nanomedicine, which focuses on targeted delivery of therapeutic drugs into tumor tissues using nano-sized formulations, has emerged as a promising tool for cancer treatment. Therefore, nanomedicine has been introduced as a reliable approach to improve treatment efficacy and minimize detrimental adverse effects as well as overcome cancer drug resistance. With rationally designed strategies including passively targeted delivery, actively targeted delivery, delivery of multidrug combinations, as well as multimodal combination therapy, nanomedicine paves the way towards efficacious cancer treatment and hold great promise in overcoming cancer drug resistance. Herein, we review the recent progress of nanomaterials used in medicine, including liposomal nanoparticles, polymeric nanoparticles, inorganic nanoparticles and hybrid nanoparticles, to surmount cancer multidrug resistance. Finally, the future perspectives of the application of nanomedicine to reverse cancer drug resistance will be addressed.

Review of Transporter Substrate, Inhibitor, and Inducer Characteristics of Cladribine.

Cladribine is a nucleoside analog that is phosphorylated in its target cells (B- and T-lymphocytes) to its active adenosine triphosphate form (2-chlorodeoxyadenosine triphosphate). Cladribine tablets 10 mg (Mavenclad®) administered for up to 10 days per year in 2 consecutive years (3.5-mg/kg cumulative dose over 2 years) are used to treat patients with relapsing multiple sclerosis. The ATP-binding cassette, solute carrier, and nucleoside transporter substrate, inhibitor, and inducer characteristics of cladribine are reviewed in this article. Available evidence suggests that the distribution of cladribine across biological membranes is facilitated by a number of uptake and efflux transporters. Among the key ATP-binding cassette efflux transporters, only breast cancer resistance protein has been shown to be an efficient transporter of cladribine, while P-glycoprotein does not transport cladribine well. Intestinal absorption, distribution throughout the body, and intracellular uptake of cladribine appear to be exclusively mediated by equilibrative and concentrative nucleoside transporters, specifically by ENT1, ENT2, ENT4, CNT2 (low affinity), and CNT3. Renal excretion of cladribine appears to be most likely driven by breast cancer resistance protein, ENT1, and P-glycoprotein. The latter may play a role despite its poor cladribine transport efficiency in view of the renal abundance of P-glycoprotein. There is no evidence that solute carrier uptake transporters such as organic anion transporting polypeptides, organic anion transporters, and organic cation transporters are involved in the transport of cladribine. Available in vitro studies examining the inhibitor characteristics of cladribine for a total of 13 major ATP-binding cassette, solute carrier, and CNT transporters indicate that in vivo inhibition of any of these transporters by cladribine is unlikely.

Rifampicin Induces Gene, Protein, and Activity of P-Glycoprotein (ABCB1) in Human Precision-Cut Intestinal Slices.

P-glycoprotein (ABCB1), an ATP-binding cassette efflux transporter, limits intestinal absorption of its substrates and is a common site of drug–drug interactions. Drug-mediated induction of intestinal ABCB1 is a clinically relevant phenomenon associated with significantly decreased drug bioavailability. Currently, there are no well-established human models for evaluating its induction, so drug regulatory authorities provide no recommendations for in vitro/ex vivo testing drugs’ ABCB1-inducing activity. Human precision-cut intestinal slices (hPCISs) contain cells in their natural environment and express physiological levels of nuclear factors required for ABCB1 induction. We found that hPCISs incubated in William’s Medium E for 48 h maintained intact morphology, ATP content, and ABCB1 efflux activity. Here, we asked whether rifampicin (a model ligand of pregnane X receptor, PXR), at 30 μM, induces functional expression of ABCB1 in hPCISs over 24- and 48-h incubation (the time to allow complete induction to occur). Rifampicin significantly increased gene expression, protein levels, and efflux activity of ABCB1. Moreover, we described dynamic changes in ABCB1 transcript levels in hPCISs over 48 h incubation. We also observed that peaks of induction are achieved among donors at different times, and the extent of ABCB1 gene induction is proportional to PXR mRNA levels in the intestine. In conclusion, we showed that hPCISs incubated in conditions comparable to those used for inhibition studies can be used to evaluate drugs’ ABCB1-inducing potency in the human intestine. Thus, hPCISs may be valuable experimental tools that can be prospectively used in complex experimental evaluation of drug–drug interactions.

Role of the Abcg2 transporter in plasma levels and tissue accumulation of the anti-inflammatory tolfenamic acid in mice

The Breast Cancer Resistance Protein (BCRP/ABCG2) is an ATP-binding cassette efflux transporter that is expressed in the apical membrane of cells from relevant tissues involved in drug pharmacokinetics such as liver, intestine, kidney, testis, brain and mammary gland, among others. Tolfenamic acid is an anti-inflammatory drug used as an analgesic and antipyretic in humans and animals. Recently, tolfenamic acid has been repurposed as an antitumoral drug and for use in chronic human diseases such as Alzheimer. The aim of this work was to study whether tolfenamic acid is an in vitro Abcg2 substrate, and to investigate the potential role of Abcg2 in plasma exposure, secretion into milk and tissue accumulation of this drug. Using in vitro transepithelial assays with cells transduced with Abcg2, we showed that tolfenamic acid is an in vitro substrate of Abcg2. The in vivo effect of this transporter was tested using wild-type and Abcg2−/− mice, showing that after oral and intravenous administration of tolfenamic acid, its area under the plasma concentration-time curve in Abcg2−/− mice was between 1.7 and 1.8-fold higher compared to wild-type mice. Abcg2−/− mice also showed higher liver and testis accumulation of tolfenamic acid after intravenous administration. In this study, we demonstrate that tolfenamic acid is transported in vitro by Abcg2 and that its plasma levels as well as its tissue distribution are affected by Abcg2, with potential pharmacological and toxicological consequences.

Tebuconazole induces liver injury coupled with ROS-mediated hepatic metabolism disorder

Tebuconazole, the most widely used fungicide, is reported to cause various environmental problems and have serious health risks in humans. Despite numerous advances in toxicity studies, its internal metabolic process and the underlying mechanisms have not been systemically studied. The present study administered low doses (0.02 g/kg bw and 0.06 g/kg bw) of tebuconazole to C57BL/6 mice in vivo. The high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) was developed and validated to analyze the tebuconazole in different organs, and our data revealed that tebuconazole mainly accumulated in the liver and that histopathological damage were exhibited in this organ. Tebuconazole significantly dysregulated phase Ⅰ- and phase II-metabolizing enzymes, ATP-binding cassette (ABC) efflux transporters (Abcc2 and Abcc3) and fatty acid metabolism-related genes (Cdkn1a and Fasn), thereby directly causing liver hypertrophy and steatosis. Importantly, the excessive induction of reactive oxygen species (ROS) and oxidative stress partially accounted for the metabolic abnormalities mediated by tebuconazole. Moreover, these alterations were related to the abnormal transcriptional levels of peroxisome proliferator-activated receptor α (PPAR-α) and liver x receptor α (LXR-α), which were predicted to bind to tebuconazole via hydrogen bonding interactions. The current findings provide new insight into the molecular mechanisms of metabolic abnormalities induced by tebuconazole at low concentration, and are conducive to a better understanding of the environmental risk posed by this fungicide.

Uptake, intracellular dissolution, and cytotoxicity of silver nanowires in cell models

The uptake, intracellular dissolution, and cytotoxicity of silver nanowires (AgNWs) in two cell models (human keratinocytes - HaCaT cells and murine macrophages) were systemically investigated for the first time. Cellular uptake of AgNWs occurred mainly via pathways of clathrin-dependent endocytosis, caveolae-dependent endocytosis, and phagocytosis. AgNWs could be internalized by two types of cells with numerous lysosomal vesicles detected in close vicinity to AgNWs. Meanwhile, AgNWs exposure caused lysosomal permeabilization and release of cathepsisn B into cytoplasm. Furthermore, for the first time, this study found that AgNWs exposure inhibited the transmembrane ATP binding cassette (ABC) efflux transporter activity, which could make AgNWs as chemosensitizers to increase the toxicity of other xenobiotic pollutants. Toxicity assays evaluating reactive oxygen species production and mitochondrial activity indicated that cytotoxicity differed for different cell types and particles. The intracellular presence of AgNWs with different diameters induced similar toxic events but to different extents. AgNWs were absorbed by macrophages more efficiently than HaCaT cells, while AgNWs exhibited only marginal cytotoxicity towards macrophages compared to HaCaT cells. Using an Ag+ fluorescence probe, it was found that a fraction of AgNWs was dissolved inside the lysosomes. A higher amount of released Ag+ was detected in HaCaT cells than in macrophages, which might partially contribute to their higher cytotoxicity in HaCaT cells. The toxicity of AgNWs in HaCaT cells and macrophages is due to the high-aspect nature of the nanowires rather than the extracellular release of Ag+. This study may be useful for risk assessments of AgNWs in their practical applications in the biomedical field.

Tetrahydrocannabinol and Its Major Metabolites are Not (or are poor) Substrates and Inhibitors of Human P‐glycoprotein (P‐gp/MDR1/ABCB1) and Breast Cancer Resistance Protein (BCRP/ABCG2)

Cannabis consumption in the United States is rising. (-)-Δ9-tetrahydrocannabinol (THC) is the primary psychoactive constituent of cannabis. In humans, 11-hydroxy-THC (11-OH-THC) and 11-nor-9-carboxy-THC (THC-COOH) are psychoactive and non-psychoactive circulating metabolites of THC, respectively. P-glycoprotein (P-gp) and breast cancer resistance protein (BCRP) are ATP-binding cassette (ABC) efflux transporters important in drug disposition. Previous animal studies suggest that THC and its metabolites could be substrates of P-gp and BCRP orthologs in mice and macaques; however, whether these cannabinoids are substrates or inhibitors of human P-gp and BCRP is unknown. Therefore, we performed transwell transport, cellular accumulation, and vesicular transport assays using Madin-Darby canine kidney II (MDCKII) cells or plasma membrane vesicles overexpressing human P-gp or BCRP to determine whether THC, 11-OH-THC or THC-COOH, at pharmacologically relevant concentrations, are substrates or inhibitors of these human ABC transporters. Neither THC nor 11-OH-THC was found to be a substrate or inhibitor of P-gp or BCRP. The efflux ratio of THC-COOH in MDCKII-BCRP cells was 1.6, which was significantly decreased to 1.0 by the BCRP inhibitor Ko143. Likewise, cellular accumulation of THC-COOH was significantly increased 1.6-fold in the presence vs. absence of Ko143. THC-COOH also significantly inhibited BCRP-mediated transport of lucifer yellow, a BCRP substrate; however, THC-COOH was neither a substrate nor an inhibitor of P-gp. Collectively, these results indicate that THC and 11-OH-THC are not substrates or inhibitors (at pharmacologically relevant concentrations) of either P-gp or BCRP. THC-COOH is a weak substrate and inhibitor of BCRP, but not of P-gp. Based on these data we predict that P-gp or BCRP will not modulate the brain/fetal distribution or intestinal absorption of these cannabinoids in humans. In addition, significant in vivo drug interactions between these cannabinoids and P-gp or BCRP inhibitors/inducers are unlikely.