Cryo-EM structure of P-glycoprotein bound to triple elacridar inhibitor molecules.

P-glycoprotein (P-gp) is an ATP-binding cassette transporter known for its roles in expelling xenobiotic compounds from cells and contributing to cellular drug resistance through multidrug efflux. This mechanism is particularly problematic in cancer cells, where it diminishes the therapeutic efficacy of anticancer drugs. P-gp inhibitors, such as elacridar, have been developed to circumvent the decrease in drug efficacy due to P-gp efflux. An earlier study reported the cryo-EM structure of human P-gp-Fab (MRK-16) complex bound by two elacridar molecules, at a resolution of 3.6 Å. In this study, we have obtained a higher resolution (2.5 Å) structure of the P-gp- Fab (UIC2) complex bound by three elacridar molecules. This finding, which exposes a larger space for compound-binding sites than previously acknowledged, has significant implications for the development of more selective inhibitors and enhances our understanding of the compound recognition mechanism of P-gp.

Role of ABCB1 and ABCB4 in renal and biliary excretion of perfluorooctanoic acid in mice.

BACKGROUND: Perfluorooctanoic acid (PFOA) is one of the major per- and polyfluoroalkyl substances. The role of ATP-binding cassette (ABC) transporters in PFOA toxicokinetics is unknown. METHODS: In this study, two ABC transporters, ABCB1 and ABCB4, were examined in mice with single intravenous PFOA administration (3.13 µmol/kg). To identify candidate renal PFOA transporters, we used a microarray approach to evaluate changes in gene expression of various kidney transporters in Abcb4 null mice. RESULTS: Biliary PFOA concentrations were lower in Abcb4 null mice (mean ± standard deviation: 0.25 ± 0.12 µg/mL) than in wild-type mice (0.87 ± 0.02 µg/mL). Immunohistochemically, ABCB4 expression was confirmed at the apical region of hepatocytes. However, renal clearance of PFOA was higher in Abcb4 null mice than in wild-type mice. Among 642 solute carrier and ABC transporters, 5 transporters showed significant differences in expression between wild-type and Abcb4 null mice. These candidates included two major xenobiotic transporters, multidrug resistance 1 (Abcb1) and organic anion transporter 3 (Slc22a8). Abcb1 mRNA levels were higher in Abcb4 null mice than in wild-type mice in kidney. In Abcb4 null mice, Abcb1b expression was enhanced in proximal tubules immunohistochemically, while that of Slc22a8 was not. Finally, in Abcb1a/b null mice, there was a significant decrease in the renal clearance of PFOA (0.69 ± 0.21 vs 1.1 mL ± 0.37/72 h in wild-type mice). A homology search of ABCB1 showed that several amino acids are mutated in humans compared with those in rodents and monkeys. CONCLUSIONS: These findings suggest that, in the mouse, Abcb4 and Abcb1 are excretory transporters of PFOA into bile and urine, respectively.

Electroacupuncture stimulation enhances the permeability of the blood-brain barrier: A systematic review and meta-analysis of preclinical evidence and possible mechanisms.

An important cellular barrier to maintain the stability of the brain's internal and external environment is the blood-brain barrier (BBB). It also prevents harmful substances from entering brain tissue through blood circulation while providing protection for the central nervous system. It should be noted, however, that the intact BBB can be a barrier to the transport of most drugs into the brain via the conventional route of administration, which can prevent them from reaching effective concentrations for the treatment of disorders affecting the central nervous system. Electroacupuncture stimulation has been shown to be effective at opening the BBB in a series of experimental studies. This study systematically analyzes the possibility and mechanism by which electroacupuncture opens the BBB. In PubMed, Web of Science, VIP Database, Wanfang Database, and the Chinese National Knowledge Infrastructure, papers have been published for nearly 22 years aimed at opening the BBB and its associated structures. A comparison of EB content between electroacupuncture and control was selected as the primary outcome. There were also results on vascular endothelial growth factor (VEGF), nerve growth factor (NGF), P-Glycoprotein (P-gp), Matrix Metalloproteinase 9 (MMP-9), and glial fibrillary acidic protein (GFAP). We utilized Review Manager software analysis to analyze correlations between studies with a view to exploring the mechanisms of similarity. Evans Blue infiltration forest plot: pooled effect size of 2.04, 95% CI: 1.21 to 2.87, P < 0.01. These results indicate that electroacupuncture significantly increases EB penetration across the BBB. Most studies have reported that GFAP, MMP-9, and VEGF were upregulated after treatment. P-gp expression decreased as well. Electroacupuncture can open the BBB, and the sparse-dense wave is currently the most effective electroacupuncture frequency for opening the BBB. VEGF plays an important role in opening the BBB. It is also important to regulate the expression of MMP-9 and GFAP and inhibit the expression of P-gp.

Co-delivery of Paclitaxel/Atovaquone/Quercetin to regulate energy metabolism to reverse multidrug resistance in ovarian cancer by PLGA-PEG nanoparticles.

Ovarian cancer is a malignant tumor that seriously endangers the lives of women, with chemotherapy being the primary clinical treatment. However, chemotherapy encounters the problem of generating multidrug resistance (MDR), mainly due to drug efflux induced by P-glycoprotein (P-gp), which decreases intracellular accumulation of chemotherapeutic drugs. The drugs efflux mediated by P-gp requires adenosine triphosphate (ATP) hydrolysis to provide energy. Therefore, modulating energy metabolism pathways and inhibiting ATP production may be a potential strategy to reverse MDR. Herein, we developed a PTX-ATO-QUE nanoparticle (PAQNPs) based on a PLGA-PEG nanoplatform capable of loading the mitochondrial oxidative phosphorylation (OXPHOS) inhibitor atovaquone (ATO), the glycolysis inhibitor quercetin (QUE), and the chemotherapeutic drug paclitaxel (PTX) to reverse MDR by inhibiting energy metabolism through multiple pathways. Mechanistically, PAQNPs could effectively inhibit the OXPHOS and glycolytic pathways of A2780/Taxol cells by suppressing the activities of mitochondrial complex III and hexokinase II (HK II), respectively, ultimately decreasing intracellular ATP levels in tumor cells. Energy depletion can effectively inhibit cell proliferation and reduce P-gp activity, increasing the chemotherapeutic drug PTX accumulation in the cells. Moreover, intracellular reactive oxygen species (ROS) is increased with PTX accumulation and leads to chemotherapy-resistant cell apoptosis. Furthermore, PAQNPs significantly inhibited tumor growth in the A2780/Taxol tumor-bearing NCG mice model. Immunohistochemical (IHC) analysis of tumor tissues revealed that P-gp expression was suppressed, demonstrating that PAQNPs are effective in reversing MDR in tumors by inducing energy depletion. In addition, the safety study results, including blood biochemical indices, major organ weights, and H&E staining images, showed that PAQNPs have a favorable in vivo safety profile. In summary, the results suggest that the combined inhibition of the two energy pathways, OXPHOS and glycolysis, can enhance chemotherapy efficacy and reverse MDR in ovarian cancer.

Model of P-Glycoprotein Ligand Binding and Validation with Efflux Substrate Matched Pairs.

The blood-brain barrier (BBB) poses a significant obstacle in developing therapeutics for neurodegenerative diseases and central nervous system (CNS) disorders. P-glycoprotein (P-gp), a multidrug resistance protein, is a critical gatekeeper in the BBB and plays a role in cancer chemoresistance. This paper uses cryo-EM P-gp structures as starting points with an induced fit docking (IFD) model to evaluate 19 pairs of compounds with known P-gp efflux data. The study reveals significant differences in binding energy and sheds light on structural modifications' impact on efflux properties. In the cases examined, fluorine incorporation influences the efflux by altering the molecular conformation rather than proximal heteroatom basicity. Although there are limitations in addressing covalent interactions or when binding extends into the more flexible vestibule region of the protein, the results provide valuable insights and potential strategies to overcome P-gp efflux, contributing to the advancement of drug development for both CNS disorders and cancer therapies.

Design, synthesis and bioactivity study on oxygen-heterocyclic-based pyran analogues as effective P-glycoprotein-mediated multidrug resistance in MCF-7/ADR cell.

P-glycoprotein (P-gp) imparts multi-drug resistance (MDR) on the cancers cell and malignant tumor clinical therapeutics. We report a class of newly designed and synthesized oxygen-heterocyclic-based pyran analogues (4a-l) bearing different aryl/hetaryl-substituted at the 1-postion were synthesized, aiming to impede the P-gp function. These compounds (4a-l) have been tested against cancerous PC-3, SKOV-3, HeLa, and MCF-7/ADR cell lines as well as non-cancerous HFL-1 and WI-38 cell lines to determine their anti-proliferative potency.The findings demonstrated the superior potency of 4a-c with 4-F, 2-Cl, and 3-Cl derivatives and 4h,g with 4-NO2, 4-MeO derivatives against PC-3, SKOV-3, HeLa, and MCF-7/ADR cell lines.Compounds 4a-c were tested for P-gp inhibition and demonstrated significant vigour against MCF-7/ADR cells with IC50 = 5.0-10.7 µM. The Rho123 accumulation assay showed that compounds 4a-c adequately inhibited P-gp function, as predicted. Furthermore, 4a or 4b administration resulted in MCF-7/ADR cell accumulation in the S phase, while compound 4c induced apoptosis by causing cell cycle arrest at G2/M. The molecular docking was applied to understand the likely modes of action and guide us in the rational design of more potent analogs. The investigate derivatives showed their good binding potential for p-gp active site with excellent docking scores and interactions. Finally, the majority of investigated derivatives 4a-c derivatives showed high oral bioavailability, but they did not cross the blood-brain barrier. These results suggest that they have favorable pharmacokinetic properties. Therefore, these compounds could serve as leads for designing more potent and stable drugs in the future.

Exploring the potential of P-glycoprotein inhibitors in the targeted delivery of anti-cancer drugs: A comprehensive review.

Due to the high prevalence of cancer, progress in the management of cancer is the need of the hour. Most cancer patients develop chemotherapeutic drug resistance, and many remain insidious due to overexpression of Multidrug Resistance Protein 1 (MDR1), also known as Permeability-glycoprotein (P-gp) or ABCB1 transporter (ATP-binding cassette subfamily B member 1). P-gp, a transmembrane protein that protects vital organs from outside chemicals, expels medications from malignant cells. The blood-brain barrier (BBB), gastrointestinal tract (GIT), kidneys, liver, pancreas, and cancer cells overexpress P-gp on their apical surfaces, making treatment inefficient and resistant. Compounds that compete with anticancer medicines for transportation or directly inhibit P-gp may overcome biological barriers. Developing nanotechnology-based formulations may help overcome P-gp-mediated efflux and improve bioavailability and cell chemotherapeutic agent accumulation. Nanocarriers transport pharmaceuticals via receptor-mediated endocytosis, unlike passive diffusion, which bypasses ABCB1. Anticancer drugs and P-gp inhibitors in nanocarriers may synergistically increase drug accumulation and chemotherapeutic agent toxicity. The projection of desirable binding and effect may be procured initially by molecular docking of the inhibitor with P-gp, enabling the reduction of preliminary trials in formulation development. Here, P-gp-mediated efflux and several possible outcomes to overcome the problems associated with currently prevalent cancer treatments are highlighted.

Design, synthesis, and bioactivity evaluation of novel indole-selenide derivatives as P-glycoprotein inhibitors against multi-drug resistance in MCF-7/ADR cell.

The inhibition of P-glycoprotein (P-gp) has emerged as an intriguing strategy for circumventing multidrug resistance (MDR) in anticancer chemotherapy. In this study, we have designed and synthesized 30 indole-selenides as a new class of P-gp inhibitors based on the scaffold hopping strategy. Among them, the preferred compound H27 showed slightly stronger reversal activity (reversal fold: 271.7 vs 261.6) but weaker cytotoxicity (inhibition ratio: 33.7% vs 45.1%) than the third-generation P-gp inhibitor tariquidar on the tested MCF-7/ADR cells. Rh123 accumulation experiments and Western blot analysis demonstrated that H27 displayed excellent MDR reversal activity by dose-dependently inhibiting the efflux function of P-gp rather than its expression. Besides, UIC-2 reactivity shift assay revealed that H27 could bind to P-gp directly and induced a conformation change of P-gp. Moreover, docking study revealed that H27 matched well in the active pockets of P-gp by forming some key H-bonding interactions, arene-H interactions and hydrophobic contacts. These results suggested that H27 is worth to be a starting point for the development of novel Se-containing P-gp inhibitors for clinic use.

Lycorine and homolycorine derivatives for chemo-sensitizing resistant human ovarian adenocarcinoma cells.

BACKGROUND: Multidrug resistance is the major obstacle to cancer chemotherapy. Modulation of P-glycoprotein and drug combination approaches have been considered important strategies to overcome drug resistance. PURPOSE: Aiming at generating a small library of Amaryllidaceae-type alkaloids to overcome drug resistance, two major alkaloids, isolated from Pancratium maritimum, lycorine (1), and 2α-10bα-dihydroxy-9-O-demethylhomolycorine (2), were derivatized, giving rise to nineteen derivatives (3 - 21). METHODS: The main chemical transformation of lycorine resulted from the cleavage of ring E of the diacetylated lycorine derivative (3) to obtain compounds that have carbamate and amine functions (5 - 16), while acylation of compound 2 provided derivatives 17 - 21. Compounds 1 - 21 were evaluated for their effects on cytotoxicity, and drug resistance reversal, using resistant human ovarian carcinoma cells (HOC/ADR), overexpressing P-glycoprotein (P-gp/ABCB1), as model. RESULTS: Excluding lycorine (1) (IC50 values of 1.2- 2.5 µM), the compounds were not cytotoxic or showed moderate/weak cytotoxicity. Chemo-sensitization assays were performed by studying the in vitro interaction between the compounds and the anticancer drug doxorubicin. Most of the compounds have shown synergistic interactions with doxorubicin. Compounds 5, 6, 9 - 14, bearing both carbamate and aromatic amine moieties, were found to have the highest sensitization rate, reducing the dose of doxorubicin 5-35 times, highlighting their potential to reverse drug resistance in combination chemotherapy. Selected compounds (4 - 6, 9 - 14, and 21), able of re-sensitizing resistant cancer cells, were further evaluated as P-gp inhibitors. Compound 11, which has a para­methoxy-N-methylbenzylamine moiety, was the strongest inhibitor. In the ATPase assay, compounds 9-11 and 13 behaved as verapamil, suggesting competitive inhibition of P-gp. At the same time, none of these compounds affected P-gp expression at the mRNA or protein level. CONCLUSIONS: This study provided evidence of the potential of Amaryllidaceae alkaloids as lead candidates for the development of MDR reversal agents.

Novel betulin derivatives as multidrug reversal agents targeting P-glycoprotein.

Chemotherapy is a powerful means of cancer treatment but its efficacy is compromised by the emergence of multidrug resistance (MDR), mainly linked to the efflux transporter ABCB1/P-glycoprotein (P-gp). Based on the chemical structure of betulin, identified in our previous work as an effective modulator of the P-gp function, a series of analogs were designed, synthesized and evaluated as a source of novel inhibitors. Compounds 6g and 6i inhibited rhodamine 123 efflux in the P-gp overexpressed leukemia cells, K562/Dox, at concentrations of 0.19 µM and 0.39 µM, respectively, and increased the intracellular accumulation of doxorubicin at the submicromolar concentration of 0.098 µM. Compounds 6g and 6i were able to restore the sensitivity of K562/Dox to Dox at 0.024 µM and 0.19 µM, respectively. Structure-activity relationship analysis and molecular modeling revealed important information about the structural features conferring activity. All the active compounds fitted in a specific region involving mainly transmembrane helices (TMH) 4-6 from one homologous half and TMH 7 and 12 from the other, also showing close contacts with TMH 6 and 12. Compounds that bound preferentially to another region were inactive, regardless of their free energy of binding. It should be noted that compounds 6g and 6i were devoid of toxic effects against peripheral blood mononuclear normal cells and erythrocytes. The data obtained indicates that both compounds might be proposed as scaffolds for obtaining promising P-gp inhibitors for overcoming MDR.

Impact of P-Glycoprotein-Mediated Drug-Endogenous Substrate Interactions on Androgen and Blood-Brain Barrier Permeability.

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.

A physiologically based toxicokinetic model of P-glycoprotein transporter-mediated placenta perfusion of dexamethasone in the pregnant rat.

The present dosage of Dexamethasone (DEX) administered to pregnant women may pose a risk of toxicity to their unborn offspring. We aimed to develop a maternal-fetal physiologically based toxicokinetic (PBTK) model for DEX in pregnant rats, with a specific focus on the role of the P-glycoprotein (P-gp) transporter in placenta perfusion, and finally facilitate the optimization of clinical DEX dosage. We conducted animal experiments to determine DEX concentrations in various rat tissues, and constructed the PBTK model using MATLAB software. Sensitivity analysis was performed to assess input parameters and the model stability, with fold error (FE) values serving as evaluation indices. Our results indicate the successful construction of the PBTK model, with the fitting key parameters such as the absorption rate constant (Ka), intrinsic hepatic clearance (CLh,int) and intrinsic P-gp clearance (CLint,P-gp). The median concentration of DEX in maternal plasma, fetal plasma, fetal lung, and fetal brain were determined, which allowed us to fit the tissue-to-plasma partition coefficients for the fetal lung (Kp,lung,f) and fetal brain (Kp,brain,f). After making adjustments, all calculated FE values were found to be less than 2, demonstrating the acceptability and accuracy of our model's predictions. Our model integrated external literature data and internal animal experimentation to comprehensively evaluate the maternal-fetal PK characteristics of DEX. These findings provide valuable support for the optimization of clinical DEX dosing.

Mosloflavone from Fissistigma petelotii ameliorates oncogenic multidrug resistance by STAT3 signaling modulation and P-glycoprotein blockade.

BACKGROUND: Oncogenic multidrug resistance (MDR) is a tough question in cancer therapy. However, no effective medications targeting oncogenic MDR are currently available. Studies have demonstrated that mosloflavone exerts anti-inflammatory effects, yet, its potential to ameliorate MDR remains unclear. PURPOSE: This study aimed to access the capability and elucidate molecular mechanisms of mosloflavone as a MDR resensitizing candidate. METHODS: We investigated the ability of mosloflavone to reverse oncogenic MDR and investigated its underlying mechanisms through cytotoxicity assay, cell cycle assay, apoptosis assay, and zebrafish xenograft model. The modulatory interplay between mosloflavone and P-gp was investigated through analysis of calcein-AM uptake, substrate efflux, ATPase assays, and molecular docking simulation. RESULTS: Mosloflavone inhibited P-gp efflux function in an uncompetitive manner without altering ABCB1 gene expression. In addition, it stimulated P-gp ATPase activity by binding to an active site distinct from that of verapamil. Regarding MDR reversal potential, mosloflavone resensitized MDR cancer cells to chemotherapies by arresting cell cycle and triggering apoptosis, possibly by enhancing reactive oxygen species accumulation and reducing phospho-STAT3. Moreover, in the zebrafish xenograft model, mosloflavone significantly potentiated the antitumor effect of paclitaxel. CONCLUSION: Our findings underscore the potential of mosloflavone as a novel dual modulator of STAT3 and P-gp, indicating it is a promising candidate for overcoming MDR in cancer treatment.

Association of ABCB1 gene polymorphisms rs1128503, rs2032582, rs4148738 with anemia in patients receiving dabigatran after total knee arthroplasty.

PURPOSE: Dabigatran is usually prescribed in recommended doses without monitoring of the blood coagulation for the prevention of venous thromboembolism after joint arthroplasty. ABCB1 is a key gene in the metabolism of dabigatran etexilate. Its allele variants are likely to play a pivotal role in the occurrence of hemorrhagic complications. METHODS: The prospective study included 127 patients with primary knee osteoarthritis undergoing total knee arthroplasty. Patients with anemia and coagulation disorders, elevated transaminase and creatinine levels as well as already receiving anticoagulant and antiplatelet therapy were excluded from the study. The association of ABCB1 gene polymorphisms rs1128503, rs2032582, rs4148738 with anemia as the outcome of dabigatran therapy was evaluated by single-nucleotide polymorphism analysis with a real-time polymerase chain reaction assay and laboratory blood tests. The beta regression model was used to predict the effect of polymorphisms on the studied laboratory markers. The probability of the type 1 error (p) was less than 0.05 was considered statistically significant. BenjaminiHochberg was used to correct for significance levels in multiple hypothesis tests. All calculations were performed using Rprogramming language v3.6.3. RESULTS: For all polymorphisms there was no association with the level of platelets, protein, creatinine, alanine transaminase, prothrombin, international normalized ratio, activated partial thromboplastin time and fibrinogen. Carriers of rs1128503 (TT) had a significant decrease of hematocrit (p = 0.001), red blood count and hemoglobin (p = 0.015) while receiving dabigatran therapy during the postoperative period compared to the CC, CT. Carriers of rs2032582 (TT) had a significant decrease of hematocrit (p = 0.001), red blood count and hemoglobin (p = 0.006) while receiving dabigatran therapy during the postoperative period compared to the GG, GT phenotypes. These differences were not observed in carriers of rs4148738. CONCLUSION: It might be necessary to reconsider thromboprophylaxis with dabigatran in carriers of rs1128503 (TT) or rs2032582 (TT) polymorphisms in favor of other new oral anticoagulants. The long-term implication of these findings would be the reduction of bleeding complications after total joint arthroplasty.

Enhanced Brain Targeting Delivery of Salvianic Acid Using Borneol as a Promoter of Blood/Brain Transport and Regulator of P-gp.

BACKGROUND: Borneol can enhance the blood-brain barrier (BBB) permeability of some drugs and suppress the efflux transport of P-glycoprotein (P-gp), which will contribute to the brain delivery of salvianic acid A (SAA). OBJECTIVE: The study aimed to develop an approach to improve the brain targeting delivery of SAA with the aid of borneol. MATERIALS AND METHODS: "Borneol" was involved in SAA via esterified prodrug SAA borneol ester (SBE) and combined administration (SAA-borneol, SAA-B). Subsequently, the blood-brain transport of SAA through brain/blood distribution and P-gp regulation via expression and function assay were investigated in rats. RESULTS: The SBE and SAA-B-treated group received a three-fold brain concentration and longer t1/2 and retention period of active SAA than that of SAA alone (20.18/13.82 min vs. 6.48 min; 18.30/17.42 min vs. 11.46 min). In addition, blood to brain transport of active SAA in SBE was altered in comparison to that of SAA-B, ultimately resulting in a better drug targeting index (9.93 vs. 3.63). Further studies revealed that SBE-induced downregulation of P-gp expression occurred at the later stage of administration (60 min, P < 0.01), but SBE always showed a more powerful drug transport activity across BBB represented by Kp value of rhodamine 123 than SAA-B (30, 60 min, P < 0.05). CONCLUSION: The comparative results indicate that SBE exhibits prominent efficiency on SAA's targeting delivery through improved blood/brain metabolic properties and sustained inhibitory effect of "borneol" on P-gp efflux. Therefore, prodrug modification can be applied as a more effective approach for brain delivery of SAA.

Utilizing a Dual Human Transporter MDCKII-MDR1-BCRP Cell Line to Assess Efflux at the Blood Brain Barrier.

To facilitate the design of drugs readily able to cross the blood brain barrier (BBB), a Madin-Darby canine kidney (MDCK) cell line was established that over expresses both P-glycoprotein (Pgp) and breast cancer resistance protein (BCRP), the main human efflux transporters of the BBB. Proteomics analyses indicate BCRP is expressed at a higher level than Pgp in this cell line. This cell line shows good activity for both transporters [BCRP substrate dantrolene efflux ratio (ER) 16.3 ± 0.9, Pgp substrate quinidine ER 27.5 ± 1.2], and use of selective transporter inhibitors enables an assessment of the relative contributions to overall ERs. The MDCKII-MDR1-BCRP ER negatively correlates with rat unbound brain/unbound plasma ratio, Kpuu Highly brain penetrant compounds with rat Kpuu ≥ 0.3 show ERs ≤ 2 in the MDCKII-MDR1-BCRP assay while compounds predominantly excluded from the brain, Kpuu ≤ 0.05, demonstrate ERs ≥ 20. A subset of compounds with MDCKII-MDR1-BCRP ER < 2 and rat Kpuu < 0.3 were shown to be substrates of rat Pgp using a rat transfected cell line, MDCKII-rMdr1a. These compounds also showed ERs > 2 in the human National Institutes of Health (NIH) MDCKI-MDR1 (high Pgp expression) cell line, which suggests that they are weak human Pgp substrates. Characterization of 37 drugs targeting the central nervous system in the MDCKII-MDR1-BCRP efflux assay show 36 have ERs < 2. In drug discovery, use of the MDCKII-MDR1-BCRP in parallel with the NIH MDCKI-MDR1 cell line is useful for identification of compounds with high brain penetration. SIGNIFICANCE STATEMENT: A single cell line that includes both the major human efflux transporters of the blood brain barrier (MDCKII-MDR1-BCRP) has been established facilitating the rapid identification of efflux substrates and enabling the design of brain penetrant molecules. Efflux ratios using this cell line demonstrate a clear relationship with brain penetration as defined by rat brain Kpuu.

[11C]metoclopramide is a sensitive radiotracer to measure moderate decreases in P-glycoprotein function at the blood-brain barrier.

The efflux transporter P-glycoprotein (P-gp) at the blood-brain barrier limits the cerebral uptake of various xenobiotics. To assess the sensitivity of [11C]metoclopramide to measure decreased cerebral P-gp function, we performed [11C]metoclopramide PET scans without (baseline) and with partial P-gp inhibition by tariquidar in wild-type, heterozygous Abcb1a/b(+/-) and homozygous Abcb1a/b(-/-) mice as models with controlled levels of cerebral P-gp expression. Brains were collected to quantify P-gp expression with immunohistochemistry. Brain uptake of [11C]metoclopramide was expressed as the area under the brain time-activity curve (AUCbrain) and compared with data previously obtained with (R)-[11C]verapamil and [11C]N-desmethyl-loperamide. Abcb1a/b(+/-) mice had intermediate P-gp expression compared to wild-type and Abcb1a/b(-/-) mice. In baseline scans, all three radiotracers were able to discriminate Abcb1a/b(-/-) from wild-type mice (2.5- to 4.6-fold increased AUCbrain, p ≤ 0.0001). However, only [11C]metoclopramide could discriminate Abcb1a/b(+/-) from wild-type mice (1.46-fold increased AUCbrain, p ≤ 0.001). After partial P-gp inhibition, differences in [11C]metoclopramide AUCbrain between Abcb1a/b(+/-) and wild-type mice (1.39-fold, p ≤ 0.001) remained comparable to baseline. There was a negative correlation between baseline [11C]metoclopramide AUCbrain and ex-vivo-measured P-gp immunofluorescence (r = -0.9875, p ≤ 0.0001). Our data suggest that [11C]metoclopramide is a sensitive radiotracer to measure moderate, but (patho-)physiologically relevant decreases in cerebral P-gp function without the need to co-administer a P-gp inhibitor.

Identification of two novel heterodimeric ABC transporters in melanoma: ABCB5ß/B6 and ABCB5ß/B9.

ABCB5 is a member of the ABC transporter superfamily composed of 48 transporters, which have been extensively studied for their role in cancer multidrug resistance and, more recently, in tumorigenesis. ABCB5 has been identified as a marker of skin progenitor cells, melanoma, and limbal stem cells. It has also been associated with multidrug resistance in several cancers. The unique feature of ABCB5 is that it exists as both a full transporter (ABCB5FL) and a half transporter (ABCB5ß). Several studies have shown that the ABCB5ß homodimer does not confer multidrug resistance, in contrast to ABCB5FL. In this study, using three complementary techniques, (1) nanoluciferase-based bioluminescence resonance energy transfer, (2) coimmunoprecipitation, and (3) proximity ligation assay, we identified two novel heterodimers in melanoma: ABCB5ß/B6 and ABCB5ß/B9. Both heterodimers could be expressed in High-Five insect cells and ATPase assays revealed that both functional nucleotide-binding domains of homodimers and heterodimers are required for their basal ATPase activity. These results are an important step toward elucidating the functional role of ABCB5ß in melanocytes and melanoma.

Overcoming multi drug resistance mediated by ABC transporters by a novel acetogenin- annonacin from Annona muricata L.

ETHNOPHARMACOLOGICAL RELEVANCE: Multi-Drug Resistance (MDR), mediated by P-glycoprotein (P-gp) is one of the barriers to successful chemotherapy in colon cancer patients. Annona muricata L. (A.muricata), commonly known as soursop/Graviola, is a medicinal plant that has been traditionally used in treating diverse diseases including cancer. Phytochemicals of A.muricata (Annonaceous Acetogenins-AGEs) have been well-reported for their anti-cancer effects on various cancers. AIM OF THE STUDY: The study aimed to examine the effect of AGEs in reversing MDR in colorectal cancer cells. METHODS: Based on molecular docking and molecular dynamic simulation, the stability of annonacin upon P-gp was investigated. Further in vitro studies were carried in oxaliplatin-resistant human colon cancer cells (SW480R) to study the biological effect of annonacin, in reversing drug resistance in these cells. RESULTS: Molecular docking and simulation studies have indicated that annonacin stably interacted at the drug binding site of P-gp. In vitro analysis showed that annonacin was able to significantly reduce the expression of P-gp by 2.56 folds. It also induced apoptosis in the drug-resistant colon cancer cells. Moreover, the intracellular accumulation of P-gp substrate (calcein-AM) was observed to increase in resistant cells upon treatment with annonacin. CONCLUSION: Our findings suggest that annonacin could inhibit the efflux of chemotherapeutic drugs mediated by P-gp and thereby help in reversing MDR in colon cancer cells. Further in vivo studies are required to decipher the underlying mechanism of annonacin in treating MDR cancers.

Coadministration of ABCB1/P-glycoprotein inhibitor elacridar improves tissue distribution of ritonavir-boosted oral cabazitaxel in mice.

Developing an oral formulation for the chemotherapeutic cabazitaxel might improve its patient-friendliness, costs, and potentially exposure profile. Cabazitaxel oral availability is restricted by CYP3A-mediated first-pass metabolism, but can be substantially boosted with the CYP3A inhibitor ritonavir. We here tested whether adding the ABCB1/P-glycoprotein inhibitor elacridar to ritonavir-boosted oral cabazitaxel could further improve its tissue exposure using wild-type, CYP3A4-humanized and Abcb1a/b-/- mice. The plasma AUC0-2h of cabazitaxel was increased 2.3- and 1.9-fold in the ritonavir- and ritonavir-plus-elacridar groups of wild-type, and 10.5- and 8.8-fold in CYP3A4-humanized mice. Elacridar coadministration did not influence cabazitaxel plasma exposure. The brain-to-plasma ratio of cabazitaxel was not increased in the ritonavir group, 7.3-fold in the elacridar group and 13.4-fold in the combined booster group in wild-type mice. This was 0.4-, 4.6- and 3.6-fold in CYP3A4-humanized mice, illustrating that Abcb1 limited cabazitaxel brain exposure also during ritonavir boosting. Ritonavir itself was also a potent substrate for the Abcb1 efflux transporter, limiting its oral availability (3.3-fold) and brain penetration (10.6-fold). Both processes were fully reversed by elacridar. The tissue disposition of ritonavir-boosted oral cabazitaxel could thus be markedly enhanced by elacridar coadministration without affecting the plasma exposure. This approach should be verified in selected patient populations.