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

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

Crossing the blood-brain barrier: A review on drug delivery strategies using colloidal carrier systems.

The blood-brain barrier represents the major challenge for delivering drugs to the central nervous system (CNS). It separates the blood circulation from the brain tissue, thereby protecting the CNS and maintaining its ion homeostasis. Unfortunately, most drugs are not able to cross this barrier in vivo despite promising in vitro results. One approach to solve this problem is the delivery of drugs via surface modified nanocarrier systems. This review will give an overview on currently tested systems, mainly liposomes and solid nanoparticles and inform about new developments.

Aryl hydrocarbon receptor ligands increase ABC transporter activity and protein expression in killifish (Fundulus heteroclitus) renal proximal tubules.

Many widespread and persistent organic pollutants, for example, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and some polychlorinated biphenyls, activate the aryl hydrocarbon receptor (AhR) causing it to translocate to the cell nucleus where it transactivates target genes, increasing expression of a number of xenobiotic metabolizing enzymes as well as some transporters. AhR's ability to target transporters within the kidney is essentially unexplored. We show here that exposing isolated killifish (Fundulus heteroclitus) renal proximal tubules to micromolar ß-naphthoflavone (BNF) or nanomolar TCDD roughly doubled the transport activity of Multidrug resistance-associated proteins Mrp2 and Mrp4, P-glycoprotein (P-gp) and Breast cancer resistance protein (Bcrp), all ATP-driven xenobiotic efflux pumps and critical determinants of renal xenobiotic excretion. These effects were abolished by actinomycin D and cycloheximide and by the AhR antagonist, α-naphthoflavone, indicating that increased transport activity was dependent on transcription and translation as well as ligand binding to AhR. Quantitative immunostaining of renal tubules exposed to BNF and TCDD showed increased luminal membrane expression of Mrp2, Mrp4, P-gp and Bcrp. Thus, in these renal tubules, the four ABC transporters are targets of AhR action.

Radioligands for Tropomyosin Receptor Kinase (Trk) Positron Emission Tomography Imaging.

The tropomyosin receptor kinases family (TrkA, TrkB, and TrkC) supports neuronal growth, survival, and differentiation during development, adult life, and aging. TrkA/B/C downregulation is a prominent hallmark of various neurological disorders including Alzheimer's disease (AD). Abnormally expressed or overexpressed full-length or oncogenic fusion TrkA/B/C proteins were shown to drive tumorigenesis in a variety of neurogenic and non-neurogenic human cancers and are currently the focus of intensive clinical research. Neurologic and oncologic studies of the spatiotemporal alterations in TrkA/B/C expression and density and the determination of target engagement of emerging antineoplastic clinical inhibitors in normal and diseased tissue are crucially needed but have remained largely unexplored due to the lack of suitable non-invasive probes. Here, we review the recent development of carbon-11- and fluorine-18-labeled positron emission tomography (PET) radioligands based on specifically designed small molecule kinase catalytic domain-binding inhibitors of TrkA/B/C. Basic developments in medicinal chemistry, radiolabeling and translational PET imaging in multiple species including humans are highlighted.

The concerted amyloid-beta clearance of LRP1 and ABCB1/P-gp across the blood-brain barrier is linked by PICALM.

The accumulation of neurotoxic amyloid-beta (Aß) in the brain is a characteristic hallmark of Alzheimer's disease (AD). The blood-brain barrier (BBB) provides a large surface area and has been shown to be an important mediator for removal of brain Aß. Both, the ABC transporter P-glycoprotein (ABCB1/P-gp) and the receptor low-density lipoprotein receptor-related protein 1 (LRP1) have been implicated to play crucial roles in Aß efflux from brain. Here, with immunoprecipitation experiments, co-immunostainings and dual inhibition of ABCB1/P-gp and LRP1, we show that both proteins are functionally linked, mediating a concerted transcytosis of Aß through endothelial cells. Late-onset AD risk factor Phosphatidylinositol binding clathrin assembly protein (PICALM) is associated with both ABCB1/P-gp and LRP1 representing a functional link and guiding both proteins through the brain endothelium. Together, our results give more mechanistic insight on Aß transport across the BBB and show that the functional interplay of different clearance proteins is needed for the rapid removal of Aß from the brain.

Reversible opening of the blood-brain barrier by claudin-5-binding variants of Clostridium perfringens enterotoxin's claudin-binding domain.

The blood-brain barrier (BBB) prevents entry of neurotoxic substances but also that of drugs into the brain. Here, the paracellular barrier is formed by tight junctions (TJs) with claudin-5 (Cldn5) being the main sealing constituent. Transient BBB opening by targeting Cldn5 could improve paracellular drug delivery. The non-toxic C-terminal domain of Clostridium perfringens enterotoxin (cCPE) binds to a subset of claudins, e.g., Cldn3, -4. Structure-based mutagenesis was used to generate Cldn5-binding variants (cCPE-Y306W/S313H and cCPE-N218Q/Y306W/S313H). These cCPE-variants were tested for transient TJ opening using multiple in vitro BBB models: Primary porcine brain endothelial cells, coculture of primary rat brain endothelial cells with astrocytes and mouse cerebEND cells. cCPE-Y306W/S313H and cCPE-N218Q/Y306W/S313H but neither cCPE-wt nor cCPE-Y306A/L315A (not binding to claudins) decreased transendothelial electrical resistance in a concentration-dependent and reversible manner. Furthermore, permeability of carboxyfluorescein (with size of CNS drugs) was increased. cCPE-Y306W/S313H but neither cCPE-wt nor cCPE-Y306A/L315A bound to Cldn5-expressing brain endothelial cells. However, freeze-fracture EM showed that cCPE-Y306W/S313H did not cause drastic TJ breakdown. In sum, Cldn5-binding cCPE-variants enabled mild and transient opening of brain endothelial TJs. Using reliable in vitro BBB models, the results demonstrate that cCPE-based biologics designed to bind Cldn5 improve paracellular drug delivery across the BBB.

Identification of [18F]TRACK, a Fluorine-18-Labeled Tropomyosin Receptor Kinase (Trk) Inhibitor for PET Imaging.

Changes in expression and dysfunctional signaling of TrkA/B/C receptors and oncogenic Trk fusion proteins are found in neurological diseases and cancers. Here, we describe the development of a first 18F-labeled optimized lead suitable for in vivo imaging of Trk, [18F]TRACK, which is radiosynthesized with ease from a nonactivated aryl precursor concurrently combining largely reduced P-gp liability and improved brain kinetics compared to previous leads while displaying high on-target affinity and human kinome selectivity.

A Kinome-Wide Selective Radiolabeled TrkB/C Inhibitor for in Vitro and in Vivo Neuroimaging: Synthesis, Preclinical Evaluation, and First-in-Human.

The proto-oncogenes NTRK1/2/3 encode the tropomyosin receptor kinases TrkA/B/C which play pivotal roles in neurobiology and cancer. We describe herein the discovery of [11C]-(R)-3 ([11C]-(R)-IPMICF16), a first-in-class positron emission tomography (PET) TrkB/C-targeting radiolabeled kinase inhibitor lead. Relying on extensive human kinome vetting, we show that (R)-3 is the most potent and most selective TrkB/C inhibitor characterized to date. It is demonstrated that [11C]-(R)-3 readily crosses the blood-brain barrier (BBB) in rodents and selectively binds to TrkB/C receptors in vivo, as evidenced by entrectinib blocking studies. Substantial TrkB/C-specific binding in human brain tissue is observed in vitro, with specific reduction in the hippocampus of Alzheimer's disease (AD) versus healthy brains. We additionally provide preliminary translational data regarding the brain disposition of [11C]-(R)-3 in primates including first-in-human assessment. These results illustrate for the first time the use of a kinome-wide selective radioactive chemical probe for endogenous kinase PET neuroimaging in human.

Genetic Mouse Models with Intestinal-Specific Tight Junction Deletion Resemble an Ulcerative Colitis Phenotype.

BACKGROUND AND AIMS: A key pathogenetic feature of ulcerative colitis [UC] is an intrinsic low mucus phosphatidylcholine[PC] content. Recently, a paracellular transport for PC across tight junctions[TJs] was described, suggesting TJ disturbance as a cause of diminished luminal PC transport. Therefore, we aimed to generate mutant mice with TJ deletion to evaluate whether a UC phenotype developed. METHODS: CL57BL/6 control wild-type mice were compared to mutant mice with tamoxifen-induced villin-Cre-dependent intestinal deletion of kindlin 1 and 2. RESULTS: Electron microscopy of mucosal biopsies obtained from both mutants before overt inflammation following only 2 days of tamoxifen exposure revealed a defective TJ morphology with extended paracellular space and, by light microscopy, expanded mucosal crypt lumina. PC secretion into mucus was reduced by >65% and the mucus PC content dropped by >50%, causing a >50 % decrease of mucus hydrophobicity in both mutants. Consequently, the microbiota was able to penetrate the submucosa. After 3 days of tamoxifen exposure, intestinal inflammation was present in both mutants, with loose bloody stools as well as macroscopic and histological features of colitis. Oral PC supplementation was able to suppress inflammation. By analogy, colonic biopsies obtained from patients with UC in remission also showed a defective epithelium with widened intercellular clefts, and enlarged crypt luminal diameters with functionally impaired luminal PC secretion. CONCLUSIONS: Genetic mouse models with intestinal deletion of kindlin 1 and 2 resulted in TJ deletion and revealed pathophysiological features of impaired PC secretion to the mucus leading to mucosal inflammation compatible with human UC.

Design and synthesis of a fluorinated quinazoline-based type-II Trk inhibitor as a scaffold for PET radiotracer development.

NTRK1/2/3 fusions have recently been characterized as low incidence oncogenic alterations across various tumor histologies. Tyrosine kinase inhibitors (TKIs) of the tropomyosin receptor kinase family TrkA/B/C (encoded by NTRK1/2/3) are showing promises in the clinic for the treatment of cancer patients whose diseases harbor NTRK tumor drivers. We describe herein the development of [18F]QMICF ([18F]-(R)-9), a quinazoline-based type-II pan-Trk radiotracer with nanomolar potencies for TrkA/B/C (IC50=85-650nM) and relevant TrkA fusions including TrkA-TPM3 (IC50=162nM). Starting from a racemic FLT3 (fms like tyrosine kinase 3) inhibitor lead with off-target TrkA activity ((±)-6), we developed and synthesized the fluorinated derivative (R)-9 in three steps and 40% overall chemical yield. Compound (R)-9 displays a favorable selectivity profile on a diverse set of kinases including FLT3 (>37-fold selectivity for TrkB/C). The mesylate precursor 16 required for the radiosynthesis of [18F]QMICF was obtained in six steps and 36% overall yield. The results presented herein support the further exploration of [18F]QMICF for imaging of Trk fusions in vivo.

ABC transporters at the blood-brain barrier.

INTRODUCTION: The blood-brain barrier (BBB) possesses an outstanding ability to protect the brain against xenobiotics and potentially poisonous metabolites. Owing to this, ATP binding cassette (ABC) export proteins have garnered significant interest in the research community. These transport proteins are predominantly localized to the luminal membrane of brain microvessels, where they recognize a wide range of different substrates and transport them back into the blood circulation. AREAS COVERED: This review summarizes recent findings on these transport proteins, including their expression in the endothelial cell membrane and their substrate recognition. Signaling cascades underlying the expression and function of these proteins will be discussed as well as their role in diseases such as Alzheimer's disease, epilepsy, amyotrophic lateral sclerosis and brain tumors. EXPERT OPINION: ABC transporters represent an integral part of the human transportome and are of particular interest at the blood-brain barrier they as they significantly contribute to brain homeostasis. In addition, they appear to be involved in myriad CNS diseases. Therefore studying their mechanisms of action as well as their signaling cascades and responses to internal and external stimuli will help us understand the pathogenesis of these diseases.

The pivotal role of astrocytes in an in vitro stroke model of the blood-brain barrier.

Stabilization of the blood-brain barrier during and after stroke can lead to less adverse outcome. For elucidation of underlying mechanisms and development of novel therapeutic strategies validated in vitro disease models of the blood-brain barrier could be very helpful. To mimic in vitro stroke conditions we have established a blood-brain barrier in vitro model based on mouse cell line cerebEND and applied oxygen/glucose deprivation (OGD). The role of astrocytes in this disease model was investigated by using cell line C6. Transwell studies pointed out that addition of astrocytes during OGD increased the barrier damage significantly in comparison to the endothelial monoculture shown by changes of transendothelial electrical resistance as well as fluorescein permeability data. Analysis on mRNA and protein levels by qPCR, western blotting and immunofluorescence microscopy of tight junction molecules claudin-3,-5,-12, occludin and ZO-1 revealed that their regulation and localisation is associated with the functional barrier breakdown. Furthermore, soluble factors of astrocytes, OGD and their combination were able to induce changes of functionality and expression of ABC-transporters Abcb1a (P-gp), Abcg2 (bcrp), and Abcc4 (mrp4). Moreover, the expression of proteases (matrixmetalloproteinases MMP-2, MMP-3, MMP-9, and t-PA) as well as of their endogenous inhibitors (TIMP-1, TIMP-3, PAI-1) was altered by astrocyte factors and OGD which resulted in significant changes of total MMP and t-PA activity. Morphological rearrangements induced by OGD and treatment with astrocyte factors were confirmed at a nanometer scale using atomic force microscopy. In conclusion, astrocytes play a major role in blood-brain barrier breakdown during OGD in vitro.

The xenoestrogens ethinylestradiol and bisphenol A regulate BCRP at the blood-brain barrier of rats.

1. Breast cancer resistance protein (BCRP) is an ABC-transporter at the blood-brain barrier (BBB) facilitating efflux of xenobiotics into blood. Expression and function are regulated via estrogen-receptors (ERs). 2. 17α-Ethinylestradiol (EE2) and bisphenol A (BPA) represent two prominent xenoestrogens. We studied whether EE2 and BPA regulate BCRP function and expression upon a 6 h treatment in an ER-dependent manner in a rat BBB-ex-vivo-model. 3. Isolated brain capillaries were incubated with EE2 or BPA. BCRP function and expression were analyzed by confocal microscopy and Western-Blot. ERα-antagonist MPP and ER-antagonist ICI182.780 were used to study involvement of ERs. 4. EE2 and BPA down-regulated BCRP transport function and expression. EE2 effects occurred at pharmacologically relevant doses, BPA exhibited only weak influences. Down-regulation by EE2 was reversed by ICI but not MPP. BPA effects were not reversed by either antagonist. 5. EE2 is a potent regulator of BCRP expression and function acting by ERß-stimulation. Oral contraception could alter uptake of pharmaceuticals to the brain and might thus be considered as an origin of central nervous system (CNS) side-effects. EE2 could also present a novel co-treatment to improve CNS-pharmacotherapy. BPA is a weak modulator of BCRP expression. Its effects appear not to be caused by ERs.

Cytotoxicity and inhibition of P-glycoprotein by selected medicinal plants from Thailand.

ETHNOPHARMACOLOGICAL RELEVANCE: Thai medicine has a long tradition of tonifying medicinal plants. In the present investigation, we studied the flower extracts of Jasminum sambac, Mammea siamensis, Mesua ferrea, Michelia alba, Mimusops elengi, and Nelumbo nucifera and speculated that these plants might influence metabolism and substance flow in the body. MATERIALS AND METHODS: Isolation of porcine brain capillary endothelial cells (PBCECs) as well as multidrug-resistance CEM/ADR5000 leukemia cells, MDA-M;B-231 breast cancer, U-251 brain tumor, and HCT-116 colon cancer cells were used. The calcein-acetoxymethylester (AM) assay was used to measure inhibition of P-glycoprotein transport. XTT and resazurin assays served for measuring cytotoxicity. RESULTS: The extracts revealed cytotoxicity towards CCRF-CEM leukemia cells to a different extent. The strongest growth inhibition was found for the n-hexane extracts of Mammea siamensis and Mesua ferrea, and the dichloromethane extracts of Mesua ferrea and Michelia alba. The flower extracts also inhibited P-glycoprotein function in porcine brain capillary endothelial cells and CEM/ADR5000 leukemia cells, indicating modulation of the blood-brain barrier and multidrug resistance of tumors. Bioactivity-guided isolation of coumarins from Mammea siamensis flowers revealed considerable cytotoxicity of mammea A/AA, deacetylmammea E/BA and deacetylmammea E/BB towards human MDA-MB-231 breast cancer, U-251 brain tumor, HCT-116 colon cancer, and CCRF-CEM leukemia cells. CONCLUSION: The plants analyzed may be valuable in developing novel treatment strategies to overcome the blood-brain barrier and multidrug-resistance in tumor cells mediated by P-glycoprotein.

Alkamides from Echinacea angustifolia Interact with P-glycoprotein of primary brain capillary endothelial cells isolated from porcine brain blood vessels.

The blood-brain barrier prevents the passage of toxic compounds from blood circulation into brain tissue. Unfortunately, drugs for the treatment of neurodegenerative diseases, brain tumors, and other diseases also do not cross the blood-brain barrier. In the present investigation, we used isolated porcine brain capillary endothelial cells and a flow cytometric calcein-AM assay to analyze inhibition of P-glycoprotein, a major constituent of the blood-brain barrier. We tested 8 alkamides isolated from Echinacea angustifolia and found that four of them inhibited P-glycoprotein-mediated calcein transport in porcine brain capillary endothelial cells.

The ABC of the blood-brain barrier - regulation of drug efflux pumps.

According to the World Health Organization Central nervous system disorders are the major medical challenge of the 21st Century, yet treatments for many CNS disorders are either inadequate or absent. One reason is the existence of the blood-brain barrier, which strictly limits the access of substances to the brain. A key element of the barrier function is the expression of ABC export proteins in the luminal membrane of brain microvessel endothelial cells. Understanding the signaling cascades and the response to endogenous and exogenous stimuli, which lead to altered expression or function of the transporters as well as subsequent modulation of the transporters, may offer novel strategies to overcome the barrier and to improve drug delivery to the brain. This review gives a short overview about structure of the key elements of the blood-brain barrier with emphasis on ABC transporters. An insight into regulation of function and expression of these transport proteins is given and the involvement of these transporters in CNS diseases is discussed.

Design of novel artemisinin-like derivatives with cytotoxic and anti-angiogenic properties.

Artemisinins are plant products with a wide range of medicinal applications. Most prominently, artesunate is a well tolerated and effective drug for treating malaria, but is also active against several protozoal and schistosomal infections, and additionally exhibits anti-angiogenic, anti-tumorigenic and anti-viral properties. The array of activities of the artemisinins, and the recent emergence of malaria resistance to artesunate, prompted us to synthesize and evaluate several novel artemisinin-like derivatives. Sixteen distinct derivatives were therefore synthesized and the in vitro cytotoxic effects of each were tested with different cell lines. The in vivo anti-angiogenic properties were evaluated using a zebrafish embryo model. We herein report the identification of several novel artemisinin-like compounds that are easily synthesized, stable at room temperature, may overcome drug-resistance pathways and are more active in vitro and in vivo than the commonly used artesunate. These promising findings raise the hopes of identifying safer and more effective strategies to treat a range of infections and cancer.

BCRP at the blood-brain barrier: genomic regulation by 17ß-estradiol.

At the blood-brain barrier (BBB), the ABC transporter breast cancer resistance protein (BCRP) actively extrudes a variety of therapeutic drugs, including cytostatics, and diminishes their pharmacological efficacy in the brain. Consequently, new strategies to circumvent BCRP-mediated multidrug resistance in the CNS are required. One major approach to increase brain drug levels is to manipulate signaling mechanisms that control transporter expression and function. In the present study, we investigated the long-term effect of 17ß-estradiol on BCRP in an ex vivo model of isolated rat brain capillaries. BCRP function and protein expression were decreased after 6 h of incubation with nanomolar concentrations of 17ß-estradiol in capillaries from male and female rats. Concomitantly, levels of BCRP mRNA were also reduced by 17ß-estradiol suggesting that the transporter is down-regulated via a genomic pathway. Additionally, we identified the presence of both estrogen receptor (ER) subtypes α and ß at the rat BBB. Experiments using selective ER agonists and antagonists revealed that ER subtype ß is responsible for the hormone-induced reduction of BCRP function and protein expression. These findings were confirmed by the use of ERKO mice. Blocking the proteasome-dependent degradation by lactacystin reversed the 17ß-estradiol-mediated decrease of BCRP supposing that transcriptional down-regulation of the efflux transporter is paralleled by protein degradation. This study demonstrates that 17ß-estradiol induces the down-regulation of BCRP on transcriptional and translational levels via the activation of ERß in rat brain capillaries after 6 h. These results could help to improve brain targeting of BCRP substrates in the treatment of CNS diseases such as brain tumors and also contribute to an enlarged understanding of BCRP-drug interactions at a chronic intake of phytoestrogens and oral contraceptives.

Inhibition of P-glycoprotein at the blood-brain barrier by phytochemicals derived from traditional Chinese medicine.

The blood-brain barrier (BBB) is a key determinant for drug transport through brain vessels. It restricts the pharmacological efficacy in numerous neurological diseases, including brain tumors. A major functional constituent of BBB is P-glycoprotein, which is also a major obstacle for effective chemotherapy of brain tumors. An appealing strategy is to selectively modulate BBB function using P-glycoprotein inhibitors. We assessed 57 chemically defined compounds derived from medicinal plants used in traditional Chinese medicine for their potential to inhibit P-glycoprotein. Nine phytochemicals inhibited P-glycoprotein in porcine brain capillary endothelial cells (PBCECs) and multidrug-resistant CEM/ADR5000 cells as shown by a calcein fluorescence assay. The cytotoxicity of the 57 phytochemicals was measured by a growth inhibition assay. Seven compounds inhibiting P-glycoprotein at lower doses were cytotoxic to drug-sensitive parental CCRF-CEM cells at higher doses. Of them, five were not cross-resistant to CEM/ADR5000 cells (baicalein, bufalin, glybomine B, deoxyserofendic acid, and shogaol). Bufalin was chosen as a lead compound. Of a further six bufalin-related compounds, scillarenin showed improved features in comparison to bufalin. It was cytotoxic to cancer cells at a nanomolar range. COMPARE and hierarchical cluster analyses of microarray-based mRNA expression were used to investigate determinants of sensitivity or resistance of the bufalin-related compounds downstream of P-glycoprotein. CEM/ADR5000 cells were not cross-resistant, but were collaterally sensitive towards scillarenin. Finally, scillarenin inhibited P-glycoprotein in PBCECs. Taken together, these data show that scillarenin is a potential novel candidate for P-glycoprotein inhibition at BBB, and, thereby, may improve the efficacy of therapy regimens in treating brain diseases.

17-ß-Estradiol: a powerful modulator of blood-brain barrier BCRP activity.

The ATP-driven efflux transporter, breast cancer resistance protein (BCRP), handles many therapeutic drugs, including chemotherapeutics, limiting their ability to cross the blood-brain barrier. This study provides new insight into rapid, nongenomic regulation of BCRP transport activity at the blood-brain barrier. Using isolated brain capillaries from rats and mice as an ex vivo blood-brain barrier model, we show that BCRP protein is highly expressed in brain capillary membranes and functionally active in intact capillaries. We show that nanomolar concentrations of 17-ß-estradiol (E2) rapidly reduced BCRP transport activity in the brain capillaries. This E2-mediated effect occurred within minutes and did not involve transcription, translation, or proteasomal degradation, indicating a nongenomic mechanism. Removing E2 after 1 h fully reversed the loss of BCRP activity. Experiments using agonists and antagonists for estrogen receptor (ER)α and ERß and brain capillaries from ERα and ERß knockout mice demonstrated that E2 could signal through either receptor to reduce BCRP transport function. We speculate that this nongenomic E2-signaling pathway could potentially be used for targeting BCRP at the blood-brain barrier, in brain tumors, and in brain tumor stem cells to improve chemotherapy of the central nervous system.