Dynamic Regulation of P-glycoprotein in Human Brain Capillaries

Article pubs.acs.org/molecularpharmaceutics

Dynamic Regulation of P‑glycoprotein in Human Brain Capillaries Janine Avemary,† Josephine D. Salvamoser,† Aurelia Peraud,‡ Jan Rémi,§ Soheyl Noachtar,§ Gert Fricker,∥ and Heidrun Potschka*,† †

Institute of Pharmacology, Toxicology, and Pharmacy, Ludwig-Maximilians-University, 80539 Munich, Germany Department of Neurosurgery, University of Munich, 81377 Munich, Germany § Department of Neurology, University of Munich, 81377 Munich, Germany ∥ Institute of Pharmacy and Molecular Biotechnology, Ruprecht-Karls-University, 69117 Heidelberg, Germany ‡

ABSTRACT: Considering its role as a major blood−brain barrier gatekeeper, the dynamic regulation of the efflux transporter Pglycoprotein is of considerable functional relevance. In particular, disease-associated alterations in transport function might affect central nervous system drug efficacy. Thus, targeting regulatory signaling cascades might render a basis for novel therapeutic approaches. Using capillaries freshly prepared from patient tissue resected during epilepsy surgery, we demonstrate dynamic regulation of P-glycoprotein in human brain capillaries. Glutamate proved to up-regulate P-glycoprotein efflux transport in a significant manner via endothelial NMDA receptors. Both inhibition of cyclooxygenase-2 and antagonism at the glycine-binding site of the NMDA receptor prevented the glutamate-mediated induction of P-glycoprotein transport function in human capillaries. In conclusion, the data argue against species differences in the signaling factors increasing endothelial P-glycoprotein transport function in response to glutamate exposure. Targeting of cyclooxygenase-2 and of the NMDA receptor glycine-binding site was confirmed as an efficacious approach to control P-glycoprotein function. The findings might render a basis for translational development of add-on approaches to improve brain penetration and efficacy of drugs. KEYWORDS: multidrug transporter, blood-brain barrier, NMDA receptor, drug resistance, cyclooxygenase-2

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INTRODUCTION P-glycoprotein is considered as one of the major molecular gatekeepers at the blood−brain barrier.1 Experimental and clinical data suggest that disease-associated alterations in Pglycoprotein efflux function can affect the pathophysiology as well as the drug sensitivity in various central nervous system (CNS) diseases.2 However, so far there is limited knowledge about the signaling factors that contribute to the dynamic regulation of transport function.3 A better understanding is of major interest considering the general implications for toxic xenobiotic exposure and for drug therapy. In the epileptic brain, seizure-associated up-regulation of blood−brain barrier P-glycoprotein efflux transport is discussed as one factor contributing to therapeutic failure.4,5 Several experimental studies indicated that P-glycoprotein overexpression caused by seizure activity can restrict brain penetration and efficacy of antiepileptic drugs.6−9 Recently, we suggested a novel approach to improve antiepileptic drug efficacy based on prevention of seizure-mediated induction of P-glycoprotein.3,10 Targets for this strategy have been identified by a series of © 2013 American Chemical Society

investigations in rodent capillaries, which revealed that glutamate released during an epileptic seizure induces Pglycoprotein expression via an endothelial N-methyl-D-aspartate (NMDA) receptor.11,12 The fact that the effect of glutamate can be blocked by pharmacological or genetic modulation of cyclooxygenase-2 (COX-2) activity as well as by antagonism at the prostaglandin E2 EP1 receptor indicated that arachidonic acid signaling mediates the intracellular effects of NMDA receptor activation.11,13 Successful prevention of glutamatemediated induction of P-glycoprotein proved to depend on transcription and translation.11 In line with this finding expression analysis indicated that the increase in transport activity relates to up-regulated protein expression levels.11 Considering that pharmacological blocking resulted in control of P-glycoprotein at baseline levels despite exposure to Received: Revised: Accepted: Published: 3333

February 27, 2013 May 16, 2013 July 18, 2013 July 18, 2013 dx.doi.org/10.1021/mp4001102 | Mol. Pharmaceutics 2013, 10, 3333−3341

Molecular Pharmaceutics

Article

Figure 1. Experimental protocol for transport assays with human capillaries. Capillaries from one preparation are split into separate samples for the different experimental and exposure protocols. At time point “zero” glutamate is added to the capillaries in experimental set-ups two and three. Fifteen minutes before the start of glutamate exposure (−15 min), one of the test compounds (celecoxib or L-701,324) is administered to the capillaries in sample three. Following glutamate exposure all capillary preparations are thoroughly washed. 210 min following the start of glutamate exposure, PSC-833 is added to the capillaries in sample four. The fluorescent dye NBD-CSA is added four hours following addition of glutamate to all samples. The image acquisition at the confocal microscope was started five hours following glutamate addition. Please note that for mouse capillaries the same experimental setup was used, except for the time point of image acquisition, which was started one hour later.

function in human brain capillaries and confirming the glycine binding site as a novel target to control P-glycoprotein. Studies were performed in capillaries freshly isolated from tissue dissected during epilepsy surgery in patients with drug-resistant temporal lobe epilepsy as well as in mouse capillaries.

glutamate indicates that the NMDA receptor, COX-2, and the EP1 receptor are the main factors mediating glutamate’s effects and that there are no parallel signaling cascades linking glutamate exposure to P-glycoprotein induction.11,13 The link between NMDA receptor activation and arachidonic signaling has not been studied in detail. However, it is known that calcium influx mediated by NMDA receptors can activate phospholipase A2 releasing arachidonic acid in neurons.14 Thus, NMDA receptor mediated calcium influx is a likely factor triggering arachidonic acid signaling in endothelial cells. The events linking EP1 receptor activation to transcriptional activation of the P-glycoprotein encoding gene have also not been elucidated yet. The targeting approach has been confirmed using cyclooxygenase-2 inhibitors, demonstrating that a blockade of the signaling events can control P-glycoprotein up-regulation and expression levels, enhance antiepileptic drug brain penetration, and restore antiepileptic drug efficacy in rodent epilepsy models.13,15−17 Translational development of the novel therapeutic concept is based on the assumption that human P-glycoprotein is affected by comparable or identical regulatory pathways. Therefore we now set out to identify factors that regulate P-glycoprotein in freshly prepared human brain capillaries isolated from surgical specimen of patients with drug-resistant epilepsy. Considering tolerability issues of approaches interfering with arachidonic acid signaling, the current study also aimed to identify and validate an alternate target in the signaling cascade. In addition to cyclooxygenase-2, the NMDA receptor has been suggested as a target.11,12 However, targeting the NMDA receptor complex in epilepsy therapy requires careful tolerability considerations as epileptic animals and patients seem to be more sensitive to adverse effects.18 In contrast to compounds acting as competitive antagonists at the glutamate binding site and noncompetitive antagonists acting as ion channel blockers, antagonists at the modulatory coagonist glycine-binding site of the NMDA receptor seem to offer an alternate opportunity to modulate receptor function.19 The approach is also reflected by clinical use of the antiepileptic drug felbamate which at least in part conveys its effects via antagonism at the glycine binding site.20 In the present study, initial data were obtained, demonstrating a rapid dynamic modulation of P-glycoprotein transport

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EXPERIMENTAL SECTION Animals. Male NMRI mice from Janvier (Le Genest St-Isle, France) were used for isolation of brain capillaries. Animals were kept under controlled environmental conditions (24−25 °C; humidity 50−60%; 12 h dark/light cycle) with free access to water and standard feed. Mice were allowed to adapt to the new environment for at least 1 week before the start of the experiment. The sampling from animals was performed in accordance with the German Animal Welfare Act and with the Directive 2010/63/EU of the European Parliament and the Council of 22 September 2010. All efforts were made to minimize pain or discomfort of the animals used. Isolation of Capillaries. Human patients suffering from drug-resistant temporal lobe epilepsy underwent a surgical resection of the epileptic focus at the Department of Neurosurgery of the University of Munich, Germany. Each patient gave informed consent in accordance with the regulations of the approval by the ethical committee of the medical faculty of the University of Munich, respecting the Declaration of Helsinki. The neurosurgeons provided tissue samples of the temporal lobe including the amygdala and hippocampus weighing in total 2−10 g. Mice were killed by cervical dislocation and decapitation for preparation of mouse brain capillaries. Capillaries from human and mouse brain were isolated using a method modified from earlier experimental procedures.11,21,22 The isolation of capillaries was started immediately after arrival of the samples in the laboratories within one hour after tissue resection. In line with the incubation protocols (Figure 1) the assessment of transport function in human capillaries was performed 5 h later. Brains of 10 mice per preparation or a small amount of dissected human brain tissue were placed in ice cold buffer solution (referred to as isolation solution) containing: 103 mM NaCl; 4.7 mM KCl; 1.2 mM KH2PO4; 10 mM HEPES (N-(2hydroxyethyl)piperazine-N′-2-ethanesulfonic acid); 1.2 mM MgSO4; 2.5 mM CaCl2; buffered at pH 7.4 with NaOH. All subsequent steps of the capillary preparation procedure were 3334

dx.doi.org/10.1021/mp4001102 | Mol. Pharmaceutics 2013, 10, 3333−3341


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preparation) or from 4 to 13 human brain capillaries per patient tissue sample (with samples from three patients) were obtained with a Zeiss LSM 510 microscope using the 40 × 1.2 water immersion objective (Carl Zeiss GmbH, Jena, Germany). The 488 laser line, a 488 dichroic filter, and a 505 high pass filter were used for experiments with NBD-CSA. Low laser intensity was chosen to avoid photobleaching. Luminal NBD-CSA fluorescence intensity was quantified using Zeiss Image Examiner (Carl Zeiss GmbH, Jena, Germany). Data are presented as P-glycoprotein specific luminal NBD-CSA fluorescence. The specific luminal fluorescence was calculated as the difference between luminal fluorescence with each treatment and fluorescence in the presence of the Pglycoprotein-specific inhibitor PSC-833 (Figure 3A−C). Immunohistochemistry of Isolated Capillaries. Isolated capillaries were mounted on microscope slides (HistoBond adhesion slides; Marienfeld, Lauda-Koenigshofen, Germany) and fixed with 4% (w/v) paraformaldehyde in PBS for 15 min. Subsequently, paraformaldehyde was removed by washing with PBS. To block unspecific binding sites, the capillaries were incubated for 2 h at room temperature in blocking buffer, containing PBS, 0.5% (v/v) Triton X-100 (AppliChem GmbH, Darmstadt, Germany) and 10% (v/v) bovine serum albumin (Sigma-Aldrich, Taufkirchen, Germany). Incubation with the primary antibodies diluted in blocking buffer was carried out overnight in a cover-plate setup at 4 °C. The following primary antibodies were used: mouse monoclonal anti-P-glycoprotein, C219 (final dilution 1:50; Calbiochem, Darmstadt, Germany), monoclonal mouse anti-NR1, CT (NR1 subunit of the NMDA receptor; final dilution 1:100; Millipore, Eschborn, Germany), and rabbit polyclonal Glut1 (Glucose transporter 1; final dilution 1:500; Millipore, Eschborn, Germany). Incubation with secondary antibodies was carried out for 2 h at room temperature. The secondary antibodies used were: 488-donkey antigoat (final dilution 1:200; Biomol, Hamburg, Germany), Cy2-donkey antimouse (final dilution 1:200; Dianova, Hamburg, Germany), Cy3-donkey antimouse (final dilution 1:200; Dianova, Hamburg, Germany), Cy3-donkey antirabbit (final dilution 1:200; Dianova, Hamburg, Germany). Finally, the microscope slides were washed, and the capillaries were coverslipped with Entellan (Merck, Darmstadt, Germany). Negative controls were incubated in the absence of a primary antibody. Each immunohistochemical detection procedure was performed with tissue from at least two different human patients. The stainings were assessed by confocal fluorescence microscopy using a Zeiss LSM 510 microscope (Carl Zeiss GmbH, Jena, Germany). Double-labeling of NR1 with Glut1 was confirmed by analysis of a confocal z-series. The images shown were acquired using a 63 × 1.4 oil immersion objective. Statistical Analysis. Results are presented as means ± standard error of the mean (SEM). For transport assays fluorescence intensity of 11−18 mouse capillaries of n (at least two) different preparations and 4−13 human capillaries of individual human patients was measured. For comparisons of untreated capillaries with capillaries incubated in glutamate or in presence of glutamate and different inhibitors, a Student’s ttest was performed. All statistical tests were performed twotailed. Differences between means were considered to be statistically significant when P < 0.05.

carried out on ice. Whole brains (mice) or samples of brain tissue (human) were homogenized, and capillaries were separated from myelin by density centrifugation in dextran 30% (w/v) at 6500 g for 12 min at 4 °C. The capillary pellet was diluted in BSA-containing buffer (isolation solution supplemented with 0.5% (w/v) bovine serum albumin, 10 mM glucose, 25 mM NaHCO3, and 1 mM sodium pyruvate (pH 7.4). The capillary suspension was filtered through a 210 μm nylon mesh and passed over a glass-bead column for further purification. Capillaries adherent to the glass-beads were washed with BSA-containing buffer and then collected by gentle agitation in isolation solution. The capillary pellet obtained by centrifugation at 6500 g for 12 min at 4 °C was resolved in incubation solution (isolation solution supplemented with 10 mM glucose). Capillaries showed active transport for at least six hours, which has been demonstrated previously for rodent capillaries.11 Capillaries were then used for transport assays or immunohistochemical staining procedures. Considering the limitations in resected tissue available per patient, it proved to be impossible to quantify Pglycoprotein expression in isolated capillaries by Western blot analysis. Thus, the experimental planning was focused on the analysis of transport function, which renders more interesting data anyway, allowing functional conclusions. P-glycoprotein Transport Assay. The transport activity of P-glycoprotein was studied in freshly isolated brain capillaries by adding the fluorescent P-glycoprotein substrate NBD-CSA (NBD-CSA: [N-ε(4-nitrobenzofurazan-7-yl)-D-Lys8]-cyclosporine A) to the incubation solution and subsequent quantification of its accumulation in the capillary lumen based on measurement of fluorescence intensity.21 Capillaries were therefore transferred to imaging chambers (Ibidi, Martinsried, Germany) and incubated for 1 h (human capillaries) or 2 h (mouse capillaries) with the fluorescent Pglycoprotein substrate NBD-CSA (2 μM) as described in detail previously.23,24 NBD-CSA was synthesized by Roland Wenger. Capillaries were preincubated in the presence of different compounds according to the experimental design testing for up-regulation of P-glycoprotein transporter function as well as its inhibition (Figure 1): Fifteen minutes before start of glutamate (100 μM) exposure (setup two and three), one of the test compounds, that is, celecoxib (1 μM) or L-701,324 (1 μM), was administered to the capillaries (sample three). Capillaries were then exposed to glutamate for 30 min. PSC833 was added after 3.5 h, and the fluorescent P-glycoprotein substrate NBD-CSA was added four hours following start of glutamate exposure to all samples. The concentration of glutamate and the exposure time were chosen based on data indicating that these conditions reflect the extent of glutamate release associated with a single generalized seizure25,26 and are in line with previously used protocols.11,27 In one sample per experiment the P-glycoprotein modulator PSC-833 (5 μM) was added to the capillaries 30 min prior to NBD-CSA to control for unspecific accumulation remaining after transport inhibition (both performed ex vivo). PSC-833 was purchased from TebuBio (Offenbach, Germany). Glutamate, ceclcoxib, and L701,324 were purchased from Sigma Aldrich (Taufkirchen, Germany). Transport of NBD-CSA in human and mouse capillaries was measured 5 or 6 h after glutamate stimulation, respectively. Figure 1 gives an overview of chronological substance administration and incubation times. For each experimental protocol, confocal images of 11−18 capillaries from at least two different mouse preparations (of 10 mice per

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RESULTS Brain tissue samples from eight patients with epilepsy were used during the study. Clinical data of these patients are 3335



a

immunohistochemistry 8

sz, seizure; LTG, lamotrigine; LEV, levetiracetam; LCM, lacosamide; VPA, valproic acid; CBZ, carbamazepine; OXC, oxcarbazepine; PHT, phenytoin; TPM, topiramate; PHB, phenobarbital; ZNS, zonisamide; TLE, temporal lobe epilepsy; AED, antiepileptic drug.

LTG OXC LEV TPM one per month automotor sz → left versive sz → generalized tonic-clonic sz 4

two per week 3 47 immunohistochemistry 7

46

CBZ LEV LCM ZNS clonazepam

CBZ TPM LEV VPA OXC two per month immunohistochemistry 6

61

29

abdominal aura/unspecific aura → automotor sz → generalized tonic-clonic sz automotor sz/dialeptic sz

CBZ OXC LEV LCM 3−7 per month, 10 per day 39 transport assay 5

45

25 transport assay 4

27

6 transport assay 3

22

transport assay 2

22

20

abdominal aura → automotor sz/dialeptic sz

VPA CBZ OXC PHT TPM LEV LTG PHB, directly before surgery: VPA + clonazepam LCM LEV LTG, directly before surgery: LTG + LCM LEV CBZ LCM VPA

medication seizure frequency

10−12 aura per day, with clusters every two weeks 20 aura per day, every 1−2 months generalized tonic-clonic sz highest: 30 per day, normal: 10−20 per month up to three per hour 11−18 per month

seizure types

with AED only visual and cephalic aura → generalized tonic-clonic sz abdominal aura → aphasic sz/automotor sz/dialeptic sz → right versive sz → generalized tonic-clonic sz abdominal aura → automotor sz → generalized tonic-clonic sz abdominal aura → automotor sz generalized tonic-clonic sz 5 34 transport assay 1

duration of epilepsy age at surgery method patient

Table 1. Clinical Dataa 3336

LEV LTG LCM, directly before surgery: LEV

TLE

diagnosis

summarized in Table 1. All patients had a history of chronic drug-resistant epilepsy. The mean age of all patients was 38 years. Immunohistochemical Analyses of P-glycoprotein and the NMDA Receptor Subunit NR1 in Human Brain Capillaries. Immunohistochemical staining of brain capillaries from human tissue demonstrated the presence of the glucose transporter 1 (Glut1) in endothelial cells. As expected, colocalization of Glut1 with P-glycoprotein confirmed the expression of the efflux transporter in human brain capillaries (Figure 2A). Aiming to explore a putative role of NMDA receptors in the regulation of human P-glycoprotein, we addressed the question whether the NMDA receptor subunit NR1 is expressed in capillaries isolated from human specimen. Co-localization of the endothelial cell marker Glut1 with NR1 gave evidence that the NMDA receptor subunit NR1 is expressed in human brain capillaries (Figure 2B). Taking into consideration that the NR1 subunit carries the glycine-binding site as an important regulator of glutamate NMDA receptor signaling, the data support the hypothesis that the glycine-binding site might serve as a target for control of endothelial P-glycoprotein. Dynamic Regulation of P-glycoprotein Transport Activity in Human Brain Capillaries: Role of Glutamate and Cyclooxygenase-2. The transport function of Pglycoprotein was assessed in capillaries from five different patients with drug resistant temporal lobe epilepsy. Capillaries were either incubated under control conditions or exposed to glutamate, with and without inhibitors of the glutamate signaling cascade. Subsequently, transport activity was quantified based on accumulation of the fluorescent Pglycoprotein substrate NBD-CSA in the capillary lumen. Figure 3A shows isolated human brain capillaries with intraluminal NBD-CSA accumulation. Exposing capillaries to PSC-833 (5 μM), an inhibitor of P-glycoprotein, strongly reduced transport activity by 30% in human capillaries (n = 4) (Figure 3B). As described above, data from all experimental setups were corrected to the P-glycoprotein-specific transport rate by subtracting the unspecific accumulation in presence of PSC-833 (as indicated in Figure 3C). Considering interstitial concentrations that glutamate reaches following its release from neurons during seizures25,26 and based on earlier studies in rodent capillaries,11 we used 100 μM glutamate to assess the impact of the neurotransmitter on Pglycoprotein function. Exposure of capillaries to glutamate (30 min) induced a 3.5-fold increase of the luminal NBD-CSA fluorescence intensity in human capillaries, reflecting enhanced P-glycoprotein transport activity (Figure 3B). When interindividual data were compared regarding the impact of glutamate on P-glycoprotein transport function (Figure 4B,C, Figure 5C,D), the glutamate-induced increase in P-glycoprotein function was evident in capillaries from four patients. As an exception the impact of glutamate on luminal NBD-CSA accumulation failed to reach significance in capillaries from a female 22 year old patient (patient no. 3) with a high seizure density reaching up to 10−20 seizures per month (Figure 4D). The COX-2 Inhibitor Celecoxib Prevents GlutamateInduced Up-Regulation of P-glycoprotein Function in Isolated Human Brain Capillaries. Using the cyclooxygenase-2 inhibitor celecoxib we investigated the contribution of cyclooxygenase-2 to the glutamate-induced upregulation of P-glycoprotein transport activity. The exper-

TLE with mesial temporal sclerosis TLE with mesial temporal sclerosis TLE with mesial temporal sclerosis TLE with mesial temporal sclerosis TLE, histology unspecific gliosis TLE, histology unspecific gliosis TLE with mesial temporal sclerosis

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Figure 2. Immunohistochemical staining of freshly isolated human brain capillaries. (A) Immunostaining of P-glycoprotein (green) and the endothelial cell marker Glut1 (red). The image on the right shows an overlay of P-glycoprotein and Glut1 labeling. (B) Immunostaining of the NR1 subunit of NMDA receptors (green) and the endothelial cell marker Glut1 (red). The image on the right shows an overlay of NR1 and Glut1 labeling. For all staining procedures negative controls (without primary antibody) were performed confirming the specificity of the methods. (C) Co-localization of the NMDA receptor subunit NR1 with Glut1 confirmed by confocal z-series. Scale bar: 10 μm in A−B and 5 μm in C.

Figure 3. Transport assays in freshly isolated human capillaries with the fluorescent dye NBD-CSA. (A) Representative image showing NBD-CSA accumulation in isolated human brain capillaries under control conditions (left), following stimulation with glutamate (100 μM; middle) and following inhibition with PSC-833 (5 μM; right); scale bars = 10 μm. (B) Quantification of NBD-CSA accumulation. Stimulation of capillaries with glutamate 100 μM leads to a significant increase in NBD-CSA accumulation. Inhibition of P-glycoprotein with PSC-833 (5 μM) reduced NBD-CSA accumulation (summary of five separate transport assays with capillaries prepared from four patient specimen). (C) P-glycoprotein-mediated transport = data calculated from graph B. The P-glycoprotein specific transport rate is calculated by subtracting the accumulation remaining after blockade of P-glycoprotein transport with PSC-833. Bars represent means ± SEM, * P < 0.05 significant difference versus control.

imental protocols were based on our previous findings in which we demonstrated cyclooxygenase-2 as one downstream factor in endothelial glutamate signaling in rodent capillaries. Co-incubation with celecoxib controlled P-glycoprotein transport function during exposure of glutamate so that an increase in NBD-CSA accumulation was efficaciously prevented (Figure 4A). When comparing data from individual patients, celecoxib kept the transport activity at control level in capillaries from patient no. 4 and patient no. 5 (Figure 4B,C). In contrast, in

capillaries from patient no. 3, celecoxib reduced the transport activity below control level (Figure 4D). L-701,324 Prevents Glutamate-Induced Up-Regulation of P-Glycoprotein Function in Isolated Mouse and Human Brain Capillaries. Based on our findings regarding NR1 expression and regarding glutamate’s effects on human brain capillary endothelial cells, we next tested the hypothesis whether the glutamate-mediated up-regulation of P-glycoprotein can be prevented by antagonism at the NMDA receptor glycine binding site. 3337



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Figure 4. Effects of celecoxib in freshly isolated human capillaries. (A) Summary of three transport assays in human brain capillaries derived from three patients (patients no. 3, 4, and 5). Stimulation with glutamate (100 μM) increased the specific luminal NBD-CSA accumulation reflecting increased P-glycoprotein transport activity. Co-incubation with celecoxib (1 μM) prevented the glutamate-mediated up-regulation. (B−D) Transport data for individual patients (B = patient no. 4: 9−13 capillaries; C = patient no. 5: 9−10 capillaries; D = patient no. 3: 7−10 capillaries). Bars represent means ± SEM, * P < 0.05 significant difference versus control.

Figure 5. Effects of the glycine binding site antagonist L-701,324. Transport assays in freshly isolated mouse (A) and human (B−E) capillaries with the fluorescent dye NBD-CSA. (A) Summary of transport data from mouse capillaries. Stimulation with glutamate (100 μM) increased the specific luminal NBD-CSA accumulation reflecting enhanced P-glycoprotein transport function. L-701,324 (1 μM) prevented the glutamate-mediated increase in the accumulation of the fluorescent substrate. For each preparation brains of 10 mice were used. Each transport assay was performed with 11−18 capillaries. (B) Summary of three transport assays in human brain capillaries derived from three patients (patient no. 1, 2, and 4). In line with the data from mouse capillaries L-701,324 (1 μM) prevented the glutamate-induced increase of P-glycoprotein transport activity. (C−E) Transport data for individual patients (C = patient no. 1: 5−13 capillaries; D = patient no. 4: 9−13 capillaries; E = patient no. 2: 4−9 capillaries). Bars represent means ± SEM, * P < 0.05 significant difference versus control.

glutamate-induced increase of P-glycoprotein mediated efflux activity (Figure 5A). To rule out species differences, transport assay experiments were performed using capillaries from three patients (Figure 5B). Confirming data obtained with mouse capillaries, L701,324 efficaciously blocked the impact of glutamate on Pglycoprotein transport activity in human capillaries.

Pilot experiments were performed in mouse capillaries. Coincubation of mouse capillaries with 1 μM L-701,324, a selective antagonist at the glycine binding site of NMDA receptors, counteracted the glutamate-induced P-glycoprotein up-regulation in capillaries. Transport assay experiments revealed a 1.9 fold up-regulation of P-glycoprotein transport activity as a result of previous glutamate stimulation, whereas coincubation with L-701,324 (1 μM) completely prevented the 3338



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When comparing individual data, L-701,324 proved to exert effects in capillaries from all patients. Whereas P-glycoprotein activity was kept at control levels in two patients (patient no. 1 and no. 4) (Figure 5C,D) by L-701,324, activity of Pglycoprotein was reduced below basal levels in another patient (patient no. 2) (Figure 5E).

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However, the transport analysis is anyway more relevant, allowing functional conclusions. The data obtained also demonstrate that P-glycoprotein function can by rapidly modulated in a highly dynamic manner at the human blood−brain barrier. P-glycoprotein has been described to serve an important protective function especially limiting brain penetration of harmful xenobiotics.37 Thus, the capability of human capillaries to up-regulate the efflux transport system might critically contribute to the adaptive alterations in the protective features of the human blood−brain barrier in response to changing environmental conditions. So far data on regulation of human P-glycoprotein have been limited to recent findings in an immortalized human cerebral microvessel cell culture system, which demonstrated a regulation of human P-glycoprotein expression by orphan nuclear receptors.38 However, it is well-known that cell culture conditions might even affect basal expression levels,39 and thus, it is questionable to what extent data obtained can be extrapolated to intact capillaries. Thus, freshly isolated capillaries might render more reliable data; however, conclusions also need to take into consideration that the preparation procedure and associated metabolic changes might also have affected data in the ex vivo setup used in the present study. For instance decreasing energy metabolism and in particular ATP concentrations might result in an underestimation of P-glycoprotein transport function. As P-glycoprotein does not distinguish between harmful xenobiotics and beneficial therapeutics, enhanced efflux function might also limit brain penetration and therapeutic efficacy of several CNS active drugs.2 In the epileptic brain seizure-associated up-regulation of P-glycoprotein is discussed as a factor contributing to therapeutic failure and drug resistance. Evidence exists that human P-glycoprotein can affect brain penetration of several antiepileptic drugs including lamotrigine, levetiracetam, phenobarbital, phenytoin, topiramate, oxcarbazepine, and eslicarbazepine acetate as well as active metabolites of carbamazepine and oxcarbazepine.4,40 Controversial data existing for some of the compounds might be attributed to the fact that specific experimental setups are necessary to detect active transport of P-glycoprotein substrates with a high passive permeability.4 Considering the putative impact on brain access of antiepileptic drugs, it is of specific interest to develop and validate novel strategies targeting P-glycoprotein transport function. Whereas chronic inhibition of P-glycoprotein might have detrimental consequences due to the restriction of the protective function, preventing seizure-associated up-regulation of P-glycoprotein might control efflux function while preserving basal transport levels. Here, we report that inhibition of cyclooxygenase-2 counteracts glutamate-associated induction of P-glycoprotein function in human capillaries. The data confirm cyclooxygenase-2 as a downstream signaling factor in response to activation of endothelial NMDA receptors being in line with recent findings in rodent capillaries and rodent models.11,15,16 Studies in rodent capillaries have demonstrated that glutamate rapidly up-regulates P-glycoprotein expression, which results in enhanced efflux function, and that this induction can be prevented by cyclooxygenase-2 inhibition or its genetic deficiency by interference with the intracellular signaling mechanisms.11 It is highly likely that glutamate-mediated increases in expression and its prevention by cyclooxygenase2 inhibition also underlie the impact of these factors on Pglycoprotein transport function in human capillaries. Unfortu-

DISCUSSION

To our knowledge this represents the first study demonstrating P-glycoprotein efflux function and its dynamic regulation in intact brain capillaries isolated from human tissue. Glutamate, the NMDA receptor, and cyclooxygenase-2 were identified as factors modulating P-glycoprotein function at the human blood−brain barrier. Targeting cyclooxygenase-2 or the glycine-binding site of endothelial NMDA receptors was confirmed as a novel strategy to control human P-glycoprotein. This approach might optimize therapeutic outcome in central nervous system (CNS) diseases which are characterized by excessive neuronal glutamate release such as epilepsy and brain ischemia.3 There is an ongoing controversial discussion about the relative contribution of P-glycoprotein to efflux function at the human blood−brain barrier.1 Our studies in capillaries isolated from surgical tissue confirmed P-glycoprotein expression and more importantly revealed that P-glycoprotein modulation significantly reduces luminal accumulation of a fluorescent transporter substrate. Thus, the data indicate that Pglycoprotein must be considered as a major molecular component of the human blood−brain barrier, which seems to exert an even more pronounced barrier function once an upregulation occurs in disease states. Rodent studies indicated that seizure activity is the main factor driving P-glycoprotein expression in the epileptic brain.28−30 Both, studies in isolated rodent brain capillaries as well as in vivo studies in rats with intrahippocampal microinjections of glutamate suggested that glutamate released in high concentrations during an epileptic seizure serves as the factor initiating a signaling cascade that up-regulates Pglycoprotein by activation of endothelial NMDA receptors.11 The identification of the NR1 subunit in human brain capillaries now confirmed that NMDA receptors might also serve an important regulatory function at the human blood− brain barrier. In line with this hypothesis, glutamate significantly up-regulated P-glycoprotein transport function in freshly isolated human capillaries. Therefore, it is likely that glutamate is the main factor that contributes to the upregulation of P-glycoprotein described in human epileptic tissue in a series of immunohistological studies.31−36 Considering recent11 and present findings, we have so far confirmed the role of glutamate in rat, mouse, and human capillaries. Thus, the data argue against species differences and suggest that key mechanisms in the regulation of the efflux transport system are preserved among different species. Comparison between data from mouse and human capillaries indicates that the extent of up-regulation might even be more pronounced at the human blood−brain barrier. However, conclusions need to take differences in the protocol into account which were necessary to optimize the experimental protocol for capillaries from different species. Moreover, the human tissue-specimens are limited in size based on the surgical resection so that it was impossible to compare the findings at the expression level by Western blot analysis. 3339



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diseases with enhanced glutamate release including ischemic and traumatic brain injury.

nately, it is impossible to additionally analyze the effects on protein expression in human capillaries due to the limited amount of tissue available from surgical specimen. Taking into consideration that cyclooxygenase-2 inhibition helped to control P-glycoprotein, to enhance antiepileptic drug efficacy, and to overcome drug resistance in rodent epilepsy models,15−17 the data suggest that the add-on of cyclooxygenase-2 inhibitors might indeed serve as a novel therapeutic approach to overcome transporter-associated drug resistance. Translational development needs to take adverse effects of cyclooxygenase-2 inhibitors into account including the increased risk for cardiovascular and cerebrovascular events in patients with a predisposition.41,42 Thus, it is of particular interest to identify alternate targets in the signaling cascade affecting P-glycoprotein in response to seizure activity. The NMDA receptor seems to serve as an obvious target.11,12 However, it is well-known that epilepsy predisposes to the adverse effects of noncompetitive and competitive NMDA receptor antagonists.18 Whereas compeẗ rats and healthy itive antagonists are well-tolerated in naive human volunteers, the compounds cause a behavioral syndrome and severe ataxia in epileptic rodents as well as patients. Antagonists at the glycine binding site of the NMDA receptor seem to offer advantages regarding these tolerability issues.43 Therefore we evaluated the efficacy of the highly selective glycine receptor antagonist L-701,324. The data confirmed targeting of the NMDA receptor glycine binding site as an alternate approach for the control of P-glycoprotein function in mouse and human capillaries exposed to glutamate. As already mentioned, one major advantage of the strategy might be the preservation of basal transport function. However, assessment of individual data obtained with capillaries from different patients indicated that down-regulation below basal transport levels is possible in individual patients. It needs to be further studied which factors contribute to interindividual differences especially considering seizure density and time from last seizure. As capillaries were from epileptic patients, signaling might still be induced due to recent seizure activity affecting transport function in the control condition without addition of glutamate to the capillary preparation. Under these circumstances an intervention with the glutamate/NMDA receptor/ cycloxygenase-2 signaling cascade would even cause effects on the transport level observed in the control condition. Frequent seizures in the period before surgery with an almost maximum up-regulation of P-glycoprotein might also have resulted in the failure to detect a significant effect of glutamate on Pglycoprotein function in one patient which exhibited a high seizure density with 10−20 seizures per month also occurring in clusters with up to three seizures per hour. In conclusion, the transport studies in patient capillaries demonstrate dynamic regulation of human P-glycoprotein. Glutamate, the NMDA receptor, and cyclooxygenase-2 were confirmed as signaling factors driving an increased Pglycoprotein transport function at the human blood−brain barrier. Taken together, data from rodent capillaries and the present findings with human capillaries rather argue against any parallel pathways linking glutamate’s effects during seizures to P-glycoprotein induction. Targeting of the NMDA receptor glycine-binding site and of cyclooxygenase-2 was substantiated as an efficacious approach to control P-glycoprotein function. These findings might render a basis for translational development of add-on approaches to improve brain penetration and efficacy of CNS drugs in the epileptic brain and other CNS

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AUTHOR INFORMATION

Corresponding Author

*Institute of Pharmacology, Toxicology, and Pharmacy, Ludwig-Maximilians-University, Koeniginstr. 16, D-80539 Munich, Germany; phone: +49-89-21802662; fax: +49-89218016556; e-mail: [email protected]. Author Contributions

J.A. and J.D.S. contributed equally to this work. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS Angela Vicidomini and Andrea Wehmeyer are acknowledged for their excellent technical assistance. We thank Sarah Fischborn, Ariane Rode, and Anton Pekcec for efforts made in the early phase of establishing methods and Björn Bauer and Anika Hartz for discussion of technical issues in the early phase of establishing the capillary isolation and transport assay. The authors wish to thank Roland Wenger for the synthesis of NBD-CSA.

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REFERENCES

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Dynamic Regulation of P-glycoprotein in Human Brain Capillaries

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