Björn Bauer

Transport of paclitaxel (Taxol) across the blood-brain barrier in vitro and in vivo.

Paclitaxel concentrations in the brain are very low after intravenous injection. Since paclitaxel is excluded from some tumors by p-glycoprotein (p-gp), the same mechanism may prevent entry into the brain. In vitro, paclitaxel transport was examined in capillaries from rat brains by confocal microscopy using BODIPY Fl-paclitaxel. Western blots and immunostaining demonstrated apical expression of p-gp in isolated endothelial cells, vessels, and tissue. Secretion of BODIPY Fl-paclitaxel into capillary lumens was specific and energy-dependent. Steady state luminal fluorescence significantly exceeded cellular fluorescence and was reduced by NaCN, paclitaxel, and SDZ PSC-833 (valspodar), a p-gp blocker. Leukotriene C(4) (LTC(4)), an Mrp2-substrate, had no effect. Luminal accumulation of NBDL-cyclosporin, a p-gp substrate, was inhibited by paclitaxel. In vivo, paclitaxel levels in the brain, liver, kidney, and plasma of nude mice were determined after intravenous injection. Co-administration of valspodar led to increased paclitaxel levels in brains compared to monotherapy. Therapeutic relevance was proven for nude mice with implanted intracerebral human U-118 MG glioblastoma. Whereas paclitaxel did not affect tumor volume, co-administration of paclitaxel (intravenous) and PSC833 (peroral) reduced tumor volume by 90%. Thus, p-gp is an important obstacle preventing paclitaxel entry into the brain, and inhibition of this transporter allows the drug to reach sensitive tumors within the CNS.

Modulation of P-Glycoprotein at the Blood-Brain Barrier: Opportunities to Improve Central Nervous System Pharmacotherapy

Pharmacotherapy of central nervous system (CNS) disorders (e.g., neurodegenerative diseases, epilepsy, brain cancer, and neuro-AIDS) is limited by the blood-brain barrier. P-glycoprotein, an ATP-driven, drug efflux transporter, is a critical element of that barrier. High level of expression, luminal membrane location, multispecificity, and high transport potency make P-glycoprotein a selective gatekeeper of the blood-brain barrier and thus a primary obstacle to drug delivery into the brain. As such, P-glycoprotein limits entry into the CNS for a large number of prescribed drugs, contributes to the poor success rate of CNS drug candidates, and probably contributes to patient-to-patient variability in response to CNS pharmacotherapy. Modulating P-glycoprotein could therefore improve drug delivery into the brain. Here we review the current understanding of signaling mechanisms responsible for the modulation of P-glycoprotein activity/expression at the blood-brain barrier with an emphasis on recent studies from our laboratories. Using intact brain capillaries from rats and mice, we have identified multiple extracellular and intracellular signals that regulate this transporter; several signaling pathways have been mapped. Three pathways are triggered by elements of the brains innate immune response, one by glutamate, one by xenobiotic-nuclear receptor (pregnane X receptor) interactions, and one by elevated β-amyloid levels. Signaling is complex, with several pathways sharing common signaling elements [tumor necrosis factor (TNF) receptor 1, endothelin (ET) B receptor, protein kinase C, and nitric-oxide synthase), suggesting a regulatory network. Several pathways include autocrine/paracrine elements, involving release of the proinflammatory cytokine, TNF-α, and the polypeptide hormone, ET-1. Finally, several steps in signaling are potential therapeutic targets that could be used to modulate P-glycoprotein activity in the clinic.

Diesel exhaust particles induce oxidative stress, proinflammatory signaling, and P-glycoprotein up-regulation at the blood-brain barrier

Here, we report that diesel exhaust particles (DEPs), a major constituent of urban air pollution, affect blood‐brain barrier function at the tissue, cellular, and molecular levels. Isolated rat brain capillaries exposed to DEPs showed increased expression and transport activity of the key drug efflux transporter, P‐glycoprotein (6 h EC50 was ~5 μg/ml). Upregulation of P‐glycoprotein was abolished by blocking transcription or protein synthesis. Inhibition of NADPH oxidase or pretreatment of capillaries with radical scavengers ameliorated DEP‐induced P‐glycoprotein up‐regulation, indicating a role for reactive oxygen species in signaling. DEP exposure also increased brain capillary tumor necrosis factor‐α (TNF‐α) levels. DEP‐induced P‐glycoprotein up‐regulation was abolished when TNF‐receptor 1 (TNF‐R1) was blocked and was not evident in experiments with capillaries from TNF‐R1 knockout mice. Inhibition of JNK, but not NF‐κB, blocked DEP‐induced P‐glycoprotein up‐regulation, indicating a role for AP‐1 in the signaling pathway. Consistent with this, DEPs increased phosphorylation of c‐jun. Together, our results show for the first time that a component of air pollution, DEPs, alters blood‐brain barrier function through oxidative stress and proinflammatory cytokine production. These experiments disclose a novel blood‐brain barrier signaling pathway, with clear implications for environmental toxicology, CNS pathology, and the pharmacotherapy of CNS disorders.—Hartz, A. M. S., Bauer, B., Block, M. L., Hong, J.‐S., Miller, D.‐S. Diesel exhaust particles induce oxidative stress, proinflammatory signaling, and P‐glycoprotein up‐regulation at the blood‐brain barrier. FASEB J. 22, 2723–2733 (2008)

Modulation of p-glycoprotein transport function at the blood-brain barrier

The central nervous system (CNS) effects of many therapeutic drugs are blunted because of restricted entry into the brain. The basis for this poor permeability is the brain capillary endothelium, which comprises the blood-brain barrier. This tissue exhibits very low paracellular (tight-junctional) permeability and expresses potent, multispecific, drug export pumps. Together, these combine to limit use of pharmacotherapy to treat CNS disorders such as brain cancer and bacterial or viral infections. Of all the xenobiotic efflux pumps highly expressed in brain capillary endothelial cells, p-glycoprotein handles the largest fraction of commonly prescribed drugs and thus is an obvious target for manipulation. Here we review recent studies focused on understanding the mechanisms by which p-glycoprotein activity in the blood-brain barrier can be modulated. These include (i) direct inhibition by specific competitors, (ii) functional modulation, and (iii) transcriptional modulation. Each has the potential to specifically reduce p-glycoprotein function and thus selectively increase brain permeability of p-glycoprotein substrates.

Coordinated Nuclear Receptor Regulation of the Efflux Transporter, Mrp2, and the Phase-Ii Metabolizing Enzyme, GSTπ, at the Blood—Brain Barrier

Xenobiotic efflux pumps at the blood—brain barrier are critical modulators of central nervous system pharmacotherapy. We previously found expression of the ligand-activated nuclear receptor, pregnane X receptor (PXR), in rat brain capillaries, and showed increased expression and transport activity of the drug efflux transporter, P-glycoprotein, in capillaries exposed to PXR ligands (pregnenolone-16α-carbonitrile (PCN) and dexamethasone) in vitro and in vivo. Here, we show increased protein expression and transport activity of another efflux pump, multidrug resistance-associated protein isoform 2 (Mrp2), in rat brain capillaries after in vitro and in vivo exposure to PCN and dexamethasone. The phase-II drug-metabolizing enzyme, glutathione S-transferase-π (GSTπ), was found to be expressed in brain capillaries, where it colocalized to a large extent with Mrp2 at the endothelial cell luminal plasma membrane. Like Mrp2, GSTπ protein expression increased with PXR activation. Colocalization and coordinated upregulation suggest functional coupling of the metabolizing enzyme and efflux transporter. These findings indicate that, as in hepatocytes, brain capillaries possess a regulatory network consisting of nuclear receptors, metabolizing enzymes, and efflux transporters, which modulate blood—brain barrier function.

Amyloid-β Contributes to Blood–Brain Barrier Leakage in Transgenic Human Amyloid Precursor Protein Mice and in Humans With Cerebral Amyloid Angiopathy

Background and Purpose— Cerebral amyloid angiopathy (CAA) is a degenerative disorder characterized by amyloid-&bgr; (A&bgr;) deposition in the blood–brain barrier (BBB). CAA contributes to injuries of the neurovasculature including lobar hemorrhages, cortical microbleeds, ischemia, and superficial hemosiderosis. We postulate that CAA pathology is partially due to A&bgr; compromising the BBB. Methods— We characterized 19 patients with acute stroke with “probable CAA” for neurovascular pathology based on MRI and clinical findings. Also, we studied the effect of A&bgr; on the expression of tight junction proteins and matrix metalloproteases (MMPs) in isolated rat brain microvessels. Results— Two of 19 patients with CAA had asymptomatic BBB leakage and posterior reversible encephalopathic syndrome indicating increased BBB permeability. In addition to white matter changes, diffusion abnormality suggesting lacunar ischemia was found in 4 of 19 patients with CAA; superficial hemosiderosis was observed in 7 of 9 patients. A&bgr;40 decreased expression of the tight junction proteins claudin-1 and claudin-5 and increased expression of MMP-2 and MMP-9. Analysis of brain microvessels from transgenic mice overexpressing human amyloid precursor protein revealed the same expression pattern for tight junction and MMP proteins. Consistent with reduced tight junction and increased MMP expression and activity, permeability was increased in brain microvessels from human amyloid precursor protein mice compared with microvessels from wild-type controls. Conclusions— Our findings indicate that A&bgr; contributes to changes in brain microvessel tight junction and MMP expression, which compromises BBB integrity. We conclude that A&bgr; causes BBB leakage and that assessing BBB permeability could potentially help characterize CAA progression and be a surrogate marker for treatment response.

Curcumin inhibits the activity of ABCG2/BCRP1, a multidrug resistance-linked ABC drug transporter in mice.

PurposeTo evaluate the in vivo efficacy of curcumin as an inhibitor of the multidrug-resistance-linked ATP Binding Cassette (ABC) drug transporter, ABCG2.MethodsPhotoaffinity labeling with [125I]-iodoarylazidoprazosin was used to characterize the interaction of sulfasalazine, a substrate of the mouse ABCG2, with human ABCG2. In addition, the inhibitory effect of curcumin on ABCG2 was evaluated in brain capillaries from rats. Furthermore, the effect of curcumin on absorption of orally administered sulfasalazine in wild-type and abcg2−/− mice was also determined.ResultsSulfasalazine interacted at the drug-substrate site(s) of human ABCG2. Curcumin inhibited ABCG2 activity at nanomolar concentrations at the rat blood-brain barrier in the ex vivo assay. Based on studies in wild type and abcg2−/− mice, we observed that oral curcumin increased Cmax and relative bioavailability of sulfasalazine by selectively inhibiting ABCG2 function.ConclusionsThis study validates our previous in vitro results with human ABCG2 (Chearwae et al., Mol. Cancer Ther. 5:1995–2006, 2006) and provides the first in vivo evidence for the inhibition by curcumin of ABCG2-mediated efflux of sulfasalazine in mice. Based on these studies, we propose that non-toxic concentrations of curcumin may be used to enhance drug exposure when the rate-limiting step of drug absorption and/or tissue distribution is impacted by ABCG2.

Prevention of seizure-induced up-regulation of endothelial P-glycoprotein by COX-2 inhibition

In the epileptic brain, seizure activity induces expression of the blood-brain barrier efflux transporter, P-glycoprotein, thereby limiting brain penetration and therapeutic efficacy of antiepileptic drugs. We recently provided the first evidence that seizures drive P-glycoprotein induction through a pathway that involves glutamate-signaling through the NMDA receptor and cyclooxygenase-2 (COX-2). Based on these data, we hypothesized that selective inhibition of COX-2 could prevent seizure-induced P-glycoprotein up-regulation. In the present study, we found that the highly selective COX-2 inhibitors, NS-398 and indomethacin heptyl ester, blocked the glutamate-induced increase in P-glycoprotein expression and transport function in isolated rat brain capillaries. Importantly, consistent with this, the COX-2 inhibitor, celecoxib, blocked seizure-induced up-regulation of P-glycoprotein expression in brain capillaries of rats in vivo. To explore further the role of COX-2 in signaling P-glycoprotein induction, we analyzed COX-2 protein expression in capillary endothelial cells in brain sections from rats that had undergone pilocarpine-induced seizures and in isolated capillaries exposed to glutamate and found no change from control levels. However, in isolated rat brain capillaries, the COX-2 substrate, arachidonic acid, significantly increased P-glycoprotein transport activity and expression indicating that enhanced substrate flux to COX-2 rather than increased COX-2 expression drives P-glycoprotein up-regulation. Together, these results provide the first in vivo proof-of-principle that specific COX-2 inhibition may be used as a new therapeutic strategy to prevent seizure-induced P-glycoprotein up-regulation at the blood-brain barrier for improving pharmacotherapy of drug-resistant epilepsy.

17-β-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-β-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 ERβ and brain capillaries from ERα and ERβ 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.

ABC Transporters and the Alzheimer’s Disease Enigma

Alzheimer’s disease (AD) is considered the “disease of the twenty-first century.” With a 10-fold increase in global incidence over the past 100 years, AD is now reaching epidemic proportions and by all projections, AD patient numbers will continue to rise. Despite intense research efforts, AD remains a mystery and effective therapies are still unavailable. This represents an unmet need resulting in clinical, social, and economic problems. Over the last decade, a new AD research focus has emerged: ATP-binding cassette (ABC) transporters. In this article, we provide an overview of the ABC transporters ABCA1, ABCA2, P-glycoprotein (ABCB1), MRP1 (ABCC1), and BCRP (ABCG2), all of which are expressed in the brain and have been implicated in AD. We summarize recent findings on the role of these five transporters in AD, and discuss their potential to serve as therapeutic targets.