Blood-brain Barrier Efflux Transporter Research Articles

Gabapentin and Diclofenac Reduce Opioid Consumption in Patients Undergoing Tonsillectomy: A Result of Altered CNS Drug Delivery?

Dear Editor, We have read with great interest the article by Mogadam and colleagues on utilization of gabapentin and diclofenac for management of post-operative pain in patients undergoing tonsillectomy (1). Perhaps the most intriguing aspect of this study was the observation that pre-operative administration of gabapentin or diclofenac resulted in reduced post-operative utilization of meperidine, an opioid analgesic. Optimal therapeutic efficacy of opioids requires that they cross the blood-brain barrier (BBB) and attain effective concentrations in the CNS (2). CNS delivery of opioids is determined by putative membrane transporters localized to the BBB endothelium (3, 4). Both pathophysiological factors (i.e., inflammatory signaling in pain) and pharmacological factors (i.e., use of ancillary pain medications) can modulate mechanisms involved in BBB opioid transport, an effect that can cause profound changes in CNS delivery of traditional opioids (i.e., morphine, meperidine). In fact, our research group has demonstrated that painful and/or inflammatory stimuli in the periphery can significantly change transport mechanisms for opioids at the BBB such as the drug efflux transporter P-glycoprotein (P-gp) (5) and the drug influx transporter organic anion transporting polypeptide 1a4 (6). Of particular note, we have also shown that diclofenac itself can attenuate pain-induced changes in BBB transporter activity (6). Our data suggest that a modulation in post-operative meperidine efficacy may, in part, be the result of altered CNS opioid delivery induced by diclofenac. An additional factor to consider is that some ancillary pain medications may act as chemical inhibitors of the same BBB transporters. In the context of the study by Mogadam and colleagues, this effect may involve the critical BBB efflux transporter P-gp. Specifically, gabapentin is a known P-gp inhibitor (7) while in vitro studies have suggested that meperidine is a P-gp transport substrate (8). Although in vivo studies in mdr1a knockout mice showed no difference in meperidine antinociception (9), a direct analysis of P-gp-mediated transport of meperidine in intact laboratory animals has not been undertaken. Furthermore, this study measured analgesic efficacy using only tail-pinch, a technique that may not have been sensitive enough to detect differences in meperidine analgesia between P-gp-deficient and P-gp-competent mice. Nonetheless, it is highly plausible that gabapentin blocked P-gp-mediated efflux transport at the BBB, an effect that effectively increased CNS meperidine delivery with the clinical manifestation of reduced opioid consumption in the post-operative period. Overall, results obtained from basic science studies of our laboratory and others point towards an explanation of modified post-operative opioid efficacy in terms of altered BBB transport and/or CNS delivery of meperidine induced by ancillary pain medications. Additionally, the paper by Mogadam and colleagues emphasizes the absolute importance of novel translational studies aimed at understanding specific mechanisms involved in CNS opioid delivery, opioid efficacy, and/or drug-opioid interactions.

SAHA Enhances Synaptic Function and Plasticity In Vitro but Has Limited Brain Availability In Vivo and Does Not Impact Cognition

Suberoylanilide hydroxamic acid (SAHA) is an inhibitor of histone deacetylases (HDACs) used for the treatment of cutaneous T cell lymphoma (CTCL) and under consideration for other indications. In vivo studies suggest reducing HDAC function can enhance synaptic function and memory, raising the possibility that SAHA treatment could have neurological benefits. We first examined the impacts of SAHA on synaptic function in vitro using rat organotypic hippocampal brain slices. Following several days of SAHA treatment, basal excitatory but not inhibitory synaptic function was enhanced. Presynaptic release probability and intrinsic neuronal excitability were unaffected suggesting SAHA treatment selectively enhanced postsynaptic excitatory function. In addition, long-term potentiation (LTP) of excitatory synapses was augmented, while long-term depression (LTD) was impaired in SAHA treated slices. Despite the in vitro synaptic enhancements, in vivo SAHA treatment did not rescue memory deficits in the Tg2576 mouse model of Alzheimer’s disease (AD). Along with the lack of behavioral impact, pharmacokinetic analysis indicated poor brain availability of SAHA. Broader assessment of in vivo SAHA treatment using high-content phenotypic characterization of C57Bl6 mice failed to demonstrate significant behavioral effects of up to 150 mg/kg SAHA following either acute or chronic injections. Potentially explaining the low brain exposure and lack of behavioral impacts, SAHA was found to be a substrate of the blood brain barrier (BBB) efflux transporters Pgp and Bcrp1. Thus while our in vitro data show that HDAC inhibition can enhance excitatory synaptic strength and potentiation, our in vivo data suggests limited brain availability may contribute to the lack of behavioral impact of SAHA following peripheral delivery. These results do not predict CNS effects of SAHA during clinical use and also emphasize the importance of analyzing brain drug levels when interpreting preclinical behavioral pharmacology.

Inflammation-induced dysfunction of the low-density lipoprotein receptor-related protein-1 at the blood–brain barrier: Protection by the antioxidant N-acetylcysteine

Impairment in two blood-brain barrier (BBB) efflux transporters, p-glycoprotein (Pgp) and low-density lipoprotein receptor-related protein-1 (LRP-1) are thought to contribute to the progression of Alzheimer's disease (AD) by resulting in the brain accumulation of their substrate amyloid beta peptide (Aβ). The initial cause of impaired efflux, however, is unknown. We have shown that induction of systemic inflammation by intraperitoneal administration of lipopolysaccharide impairs the efflux of Aβ from the brain, suggesting that systemic inflammation could be one such initiator. In this study, we determined whether pre-administration of the antioxidant N-aceytlcysteine (Nac) has a protective effect against LPS-induced Aβ transporter dysfunction. Our findings were that Nac protected against LPS-induced Aβ transport dysfunction at the BBB through an LRP-1-dependent and Pgp-independent mechanism. This was associated with Nac exerting antioxidant effects in the periphery but not the brain, despite an increased rate of entry of Nac into the brain following LPS. We also found that Nac pre-administration resulted in lower blood levels of the cytokines and chemokines interferon-γ, interleukin-10, CCL2, CCL4, and CCL5, but only lowered CCL4 in the cerebral cortex and hippocampus. Finally, we observed that hippocampal cytokine responses to LPS were decreased compared to cortex. These findings demonstrate a novel mechanism by which antioxidants prevent Aβ accumulation in the brain caused by inflammation, and therefore protect against AD.

In-vitro evaluation of the P-glycoprotein interactions of a series of potentially CNS-active Amaryllidaceae alkaloids

Drug compounds interacting with the blood-brain barrier efflux transporter P-glycoprotein (P-gp) might have limited access to brain tissue. The aim of the present study was to evaluate whether nine potentially CNS-active Amaryllidaceae alkaloids of the crinine, lycorine and galanthamine types interact with P-gp. Alkaloids with inhibitory activity towards either the serotonin reuptake transporter or acetylcholinesterase, or both, were investigated using the calcein-AM efflux assay in Madin-Darby canine kidney cells transfected with human multidrug resistance transporter 1. Powelline and 6-hydroxycrinamine showed an interaction with P-gp, with IC50 values between 300 and 500 µM. 3-O-Acetylhamayne showed a weaker interaction, with an IC50 value above 3 mM. Epibuphanisine, lycorine, 1-epi-deacetylbowdenisine, papyramine and galanthamine all showed weak or no interaction with P-gp. There was no observed correlation between alkaloid type and P-gp interaction. Structurally similar compounds such as crinine and epibuphanisine showed very different P-gp interactions, highlighting the difficulty in predicting P-gp interactions. Epibuphanisine has previously shown activity in the serotonin reuptake transporter assay and may therefore serve as a lead for serotonin reuptake transporter active compounds. The most potent compound in the acetylcholinesterase assay, the marketed drug compound galanthamine (Reminyl), showed no interaction with P-gp.

11C]Flumazenil brain uptake is influenced by the blood-brain barrier efflux transporter P-glycoprotein

Background[11C]Flumazenil and positron emission tomography (PET) are used clinically to assess gamma-aminobutyric acid (GABA)-ergic function and to localize epileptic foci prior to resective surgery. Enhanced P-glycoprotein (P-gp) activity has been reported in epilepsy and this may confound interpretation of clinical scans if [11C]flumazenil is a P-gp substrate. The purpose of this study was to investigate whether [11C]flumazenil is a P-gp substrate.Methods[11C]Flumazenil PET scans were performed in wild type (WT) (n = 9) and Mdr1a/1b, (the genes that encode for P-gp) double knockout (dKO) (n = 10) mice, and in naive rats (n = 10). In parallel to PET scanning, [11C]flumazenil plasma concentrations were measured in rats. For 6 of the WT and 6 of the dKO mice a second, [11C]flumazenil scan was acquired after administration of the P-gp inhibitor tariquidar. Cerebral [11C]flumazenil concentrations in WT and Mdr1a/1b dKO mice were compared (genetic disruption model). Furthermore, pre and post P-gp-blocking cerebral [11C]flumazenil concentrations were compared in all animals (pharmacological inhibition model).ResultsMdr1a/1b dKO mice had approximately 70% higher [11C]flumazenil uptake in the brain than WT mice. After administration of tariquidar, cerebral [11C]flumazenil uptake in WT mice increased by about 80% in WT mice, while it remained the same in Mdr1a/1b dKO mice. In rats, cerebral [11C]flumazenil uptake increased by about 60% after tariquidar administration. Tariquidar had only a small effect on plasma clearance of flumazenil.ConclusionsThe present study showed that [11C]flumazenil is a P-gp substrate in rodents. Consequently, altered cerebral [11C]flumazenil uptake, as observed in epilepsy, may not reflect solely GABAA receptor density changes but also changes in P-gp activity.

Blood–brain barrier P-glycoprotein function in healthy subjects and Alzheimer's disease patients: effect of polymorphisms in the ABCB1 gene

BackgroundP-glycoprotein is a blood–brain barrier efflux transporter involved in the clearance of amyloid-beta from the brain and, as such, might be involved in the pathogenesis of Alzheimer's disease. P-glycoprotein is encoded by the highly polymorphic ABCB1 gene. Single-nucleotide polymorphisms in the ABCB1 gene have been associated with altered P-glycoprotein expression and function. P-glycoprotein function at the blood–brain barrier can be quantified in vivo using the P-glycoprotein substrate tracer (R)-[11C]verapamil and positron emission tomography (PET). The purpose of this study was to assess the effects of C1236T, G2677T/A and C3435T single-nucleotide polymorphisms in ABCB1 on blood–brain barrier P-glycoprotein function in healthy subjects and patients with Alzheimer's disease.MethodsThirty-two healthy subjects and seventeen patients with Alzheimer's disease underwent 60-min dynamic (R)-[11C]verapamil PET scans. The binding potential of (R)-[11C]verapamil was assessed using a previously validated constrained two-tissue plasma input compartment model and used as outcome measure. DNA was isolated from frozen blood samples and C1236T, G2677T/A and C3435T single-nucleotide polymorphisms were amplified by polymerase chain reaction.ResultsIn healthy controls, binding potential did not differ between subjects without and with one or more T present in C1236T, G2677T and C3435T. In contrast, patients with Alzheimer's disease with one or more T in C1236T, G2677T and C3435T had significantly higher binding potential values than patients without a T. In addition, there was a relationship between binding potential and T dose in C1236T and G2677T.ConclusionsIn Alzheimer's disease patients, C1236T, G2677T/A and C3435T single-nucleotide polymorphisms may be related to changes in P-glycoprotein function at the blood–brain barrier. As such, genetic variations in ABCB1 might contribute to the progression of amyloid-beta deposition in the brain.

OT2-05-01: An Open-Label Positron Emission Tomography Study To Investigate and Quantify Brain and Tumor Penetration of Carbon-11-Labeled Lapatinib in Patients with HER2−Overexpressing Advanced or Metastatic Breast Cancer.

Abstract Background: Patients with HER2−overexpressing (HER2+) breast cancer tend to develop visceral metastases. About a third will develop brain metastases in the course of their disease. Several anticancer agents including trastuzumab are not believed to readily cross the blood-brain barrier (BBB); therefore, central nervous system (CNS) disease progression is emerging as an important clinical problem in this patient population. Although preclinical data indicate that lapatinib brain concentrations are low in healthy animals, probably due to the BBB efflux transporters, observations from clinical studies suggest lapatinib treatment might be associated with reduced CNS tumor growth. One hypothesis is that disruption of the BBB by tumors circumvents the usual protective function of transporters. Alternatively, inhibition of efflux transporters by lapatinib enhances its own accumulation with repeat dosing. The role of lapatinib in prevention of brain metastases is currently being explored in a phase III trial, EGF 111438 (CEREBEL). Methods: To directly assess if lapatinib penetrates the brain and brain metastases, an open-label non-randomized study using carbon-11-labeled [11C] lapatinib is enrolling patients with HER2+ metastatic breast cancer. A minimum of 10 patients with an ECOG of <3 are grouped into 2 cohorts: with at least one 1-cm diameter brain metastasis or without brain metastases. Two positron emission tomography (PET) scans will be performed in each patient following intravenous administration of a microdose of [11C] lapatinib: in lapatinib-naïve patients and at steady-state lapatinib, after treatment with unlabelled oral lapatinib 1500 mg once daily for 8 days. [11C] lapatinib time-activity curves will be generated for normal brain and brain metastases, the PET volume of distribution in the brain calculated and penetration of [11C] lapatinib into the brain quantified. Analysis of brain PET data is explorative as [11C] lapatinib is a new tracer. Therefore, this study investigates both the difference in lapatinib brain penetration between patients with and without brain metastases, as well as the effect of low and high concentrations of lapatinib on the BBB efflux system. The study is currently recruiting in one center in the United Kingdom. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr OT2-05-01.

An open-label positron emission tomography (PET) study to investigate and quantify brain and tumor penetration of carbon-11 labeled lapatinib in patients with HER2-overexpressing (HER2+) advanced or metastatic breast cancer (MBC).

TPS107 Background: Patients with HER2+ breast cancer tend to develop visceral metastases. Several anti-cancer agents including trastuzumab are not believed to readily cross the blood-brain barrier (BBB); hence, central nervous system (CNS) disease progression is emerging as an important clinical problem in this patient population. Although preclinical data suggest that lapatinib brain concentrations are low in healthy animals, probably due to the BBB efflux transporters, observations from clinical studies suggest lapatinib treatment might be associated with reduced CNS tumor growth. One hypothesis is that disruption of the BBB by tumors circumvents the usual protective function of transporters or that inhibition of efflux transporters by lapatinib enhances accumulation upon repeat dosing. The role of lapatinib in prevention of brain metastases is currently explored in a Phase III trial, EGF 111438 (CEREBREL). Methods: In order to directly assess if lapatinib penetrates into the brain and brain metastases,...

Transport characteristics of guanidino compounds at the blood-brain barrier and blood-cerebrospinal fluid barrier: relevance to neural disorders

Guanidino compounds (GCs), such as creatine, phosphocreatine, guanidinoacetic acid, creatinine, methylguanidine, guanidinosuccinic acid, γ-guanidinobutyric acid, β-guanidinopropionic acid, guanidinoethane sulfonic acid and α-guanidinoglutaric acid, are present in the mammalian brain. Although creatine and phosphocreatine play important roles in energy homeostasis in the brain, accumulation of GCs may induce epileptic discharges and convulsions. This review focuses on how physiologically important and/or neurotoxic GCs are distributed in the brain under physiological and pathological conditions. Transporters for GCs at the blood-brain barrier (BBB) and the blood-cerebrospinal fluid (CSF) barrier (BCSFB) have emerged as substantial contributors to GCs distribution in the brain. Creatine transporter (CRT/solute carrier (SLC) 6A8) expressed at the BBB regulates creatine concentration in the brain, and represents a major pathway for supply of creatine from the circulating blood to the brain. CRT may be a key factor facilitating blood-to-brain guanidinoacetate transport in patients deficient in S-adenosylmethionine:guanidinoacetate N-methyltransferase, the creatine biosynthetic enzyme, resulting in cerebral accumulation of guanidinoacetate. CRT, taurine transporter (TauT/SLC6A6) and organic cation transporter (OCT3/SLC22A3) expressed at the BCSFB are involved in guanidinoacetic acid or creatinine efflux transport from CSF. Interestingly, BBB efflux transport of GCs, including guanidinoacetate and creatinine, is negligible, though the BBB has a variety of efflux transport systems for synthetic precursors of GCs, such as amino acids and neurotransmitters. Instead, the BCSFB functions as a major cerebral clearance system for GCs. In conclusion, transport of GCs at the BBB and BCSFB appears to be the key determinant of the cerebral levels of GCs, and changes in the transport characteristics may cause the abnormal distribution of GCs in the brain seen in patients with certain neurological disorders.

Ibogaine labeling with 99mTc-tricarbonyl: Synthesis and transport at the mouse blood–brain barrier

The 99mTc-tricarbonyl core may be used as an ideal tool for gamma-labeling ligands in noninvasive SPECT imaging. However, most 99mTc-tricarbonyl-labeled agents have difficulty crossing the blood–brain barrier (BBB). We radiolabeled the neuroactive indole ibogaine with 99mTc-tricarbonyl and measured its transport into the mouse brain by in situ brain perfusion. We measured the interactions of [99mTc(CO)3-ibogaine]+ and 99mTc-tricarbonyl with the main BBB efflux transporters P-gp and BCRP in vitro and in vivo. Ibogaine was radiolabeled (yield: over 95%). [99mTc(CO)3-ibogaine]+ entered the brain (Kin) poorly (0.18 µL/g/s), at about the same rate as 99mTc-tricarbonyl (0.16 µL/g/s) and [99mTc-sestamibi]+ (0.10 µL/g/s). The CNS tracer [99mTc-HMPAO]0 entered the brain ∼70-times higher than [99mTc(CO)3-ibogaine]+. In vitro studies revealed that neither [99mTc(CO)3-ibogaine]+ nor 99mTc-tricarbonyl ion were substrates for P-gp or BCRP. But lowering the membrane dipole potential barrier with phloretin enhanced the brain transport of [99mTc(OH2)3(CO)3]+ ∼3-fold. Thus, ibogaine directly labeled with 99mTc-tricarbonyl is not suitable for CNS imaging because of its poor uptake. Brain transport is not restricted by efflux transporters but is reduced by its lipophilicity and interaction with the membrane-positive dipole potential.

COX-2 inhibition controls P-glycoprotein expression and promotes brain delivery of phenytoin in chronic epileptic rats

Epileptic seizures drive expression of the blood-brain barrier efflux transporter P-glycoprotein via a glutamate/cyclooxygenase-2 mediated signalling pathway. Targeting this pathway may represent an innovative approach to control P-glycoprotein expression in the epileptic brain and to enhance brain delivery of antiepileptic drugs. Therefore, we tested the effect of specific cyclooxygenase-2 inhibition on P-glycoprotein expression in two different status epilepticus models. Moreover, the impact of a cyclooxygenase-2 inhibitor on expression of the efflux transporter and on brain delivery of an antiepileptic drug was evaluated in rats with recurrent spontaneous seizures. The highly selective cyclooxygenase-2 inhibitors SC-58236 and NS-398 both counteracted the status epilepticus-associated increase in P-glycoprotein expression in the parahippocampal cortex and the ventral hippocampus. In line with our working hypothesis, a sub-chronic 2-week treatment with SC-58236 in the chronic epileptic state kept P-glycoprotein expression at control levels. As described previously, enhanced P-glycoprotein expression in chronic epileptic rats was associated with a significant reduction in the brain penetration of the antiepileptic drug phenytoin. Importantly, the brain delivery of phenytoin was significantly enhanced by sub-chronic cyclooxygenase-2 inhibition in rats with recurrent seizures. In conclusion, the data substantiate targeting of cyclooxygenase-2 in the chronic epileptic brain as a promising strategy to control the expression levels of P-glycoprotein despite recurrent seizure activity. Cyclooxygenase-2 inhibition may therefore help to increase concentrations of antiepileptic drugs at the target sites in the epileptic brain. It needs to be further evaluated whether the approach also enhances efficacy.

Targeting Prostaglandin E2 EP1 Receptors Prevents Seizure-Associated P-glycoprotein Up-Regulation

Up-regulation of the blood-brain barrier efflux transporter P-glycoprotein in central nervous system disorders results in restricted brain access and limited efficacy of therapeutic drugs. In epilepsies, seizure activity strongly triggers expression of P-glycoprotein. Here, we identified the prostaglandin E2 receptor, EP1, as a key factor in the signaling pathway that mediates seizure-induced up-regulation of P-glycoprotein at the blood-brain barrier. In the rat pilocarpine model, status epilepticus significantly increased P-glycoprotein expression by 92 to 197% in the hippocampal hilus and granule cell layer as well as the piriform cortex. The EP1 receptor antagonist 8-chlorodibenz[b,f][1,4]oxazepine-10(11H)-carboxylic acid, 2-[1-oxo-3-(4-pyridinyl)propyl]hydrazide hydrochloride (SC-51089) abolished seizure-induced P-glycoprotein up-regulation and retained its expression at the control level. The control of P-glycoprotein expression despite prolonged seizure activity suggests that EP1 receptor antagonism will also improve antiepileptic drug efficacy. Preliminary evidence for this concept has been obtained using a massive kindling paradigm during which animals received a subchronic SC-51089 treatment. After withdrawal of the EP1 receptor antagonist, a low dose of the P-glycoprotein substrate phenobarbital resulted in an anticonvulsant effect in this pretreated group, whereas the same dosage of phenobarbital did not exert a significant effect in the respective control group. In conclusion, our data demonstrate that EP1 is a key signaling factor in the regulatory pathway that drives P-glycoprotein up-regulation during seizures. These findings suggest new intriguing possibilities to prevent and interrupt P-glycoprotein overexpression in epilepsy. Future studies are necessary to further evaluate the appropriateness of the strategy to enhance the efficacy of antiepileptic drugs.

Amyloid and tau accumulation precede CSF production decline in normal aging

Background It has been shown that there is an age-related decrease in CSF production in the Fischer rat brain, and that this decrease follows the CNS deposition of amyloid rather than preceding it [1]. We have postulated that amyloid and Tau accumulation in normal aging is multi-factorial, combining early defects in the blood-brain barrier (BBB) efflux transport of amyloid beta-peptide (A-beta) with later changes in CSF production and turnover, as well as up-regulation of A-beta influx transport.

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.

Signaling to P-glycoprotein - A new therapeutic target to treat drug-resistant epilepsy

Epilepsy affects more than 60 million people worldwide. While most patients can be treated with antiepileptic drugs, up to 40% of patients respond poorly to pharmacotherapy. This drug resistance is not well understood and presents a major clinical problem. In this short review we provide background information on one potential cause of antiepileptic drug resistance, namely, upregulation of the drug efflux transporter P-glycoprotein at the blood-brain barrier. We summarize recent findings that connect antiepileptic drug resistance with P-glycoprotein upregulation and show a mechanistic link between seizures and upregulation of this transporter. We provide an overview of results demonstrating that glutamate released during seizures signals through N-methyl-Daspartate (NMDA) receptor and cyclooxygenase-2 (COX-2) to increase P-glycoprotein. In this context we discuss the NMDA receptor and COX-2 as potential therapeutic targets and provide information on current clinical trials on drugresistant epilepsy involving blood-brain barrier efflux transporters. Finally, we provide a perspective on future research that could help improve the treatment of drug-resistant epilepsy.

Over-expression of P-glycoprotein in the canine brain following spontaneous status epilepticus

Over-expression of blood-brain barrier (BBB) efflux transporters following a status epilepticus has been described in one case report as well as in animal models with electrical, or chemical induction of status epilepticus. As an influence of stimulation cannot be excluded in the status epilepticus models, it is of specific interest to quantitatively determine the consequences of seizure activity with spontaneous onset. Therefore, we immunohistologically studied expression of the major BBB efflux transporter P-glycoprotein (Pgp) in brain tissue sampled from epileptic dogs following spontaneous seizure clusters or status epilepticus. Data were compared with tissue from control dogs that were euthanized due to non-CNS diseases. Following a status epilepticus, a significant up-regulation of endothelial Pgp expression by 87-166% was revealed in the hilus and the granule cell layer of the dentate gyrus as well as in the parietal cortex. In view of the suggested role of Pgp in drug-refractoriness its up-regulation in response to spontaneous prolonged seizure activity may be of specific relevance for subsequent therapeutic outcome. Moreover, our findings indicate that molecular changes in dogs with refractory epilepsy reflect those in human epileptic patients, thereby suggesting epileptic dogs as a suitable model of pharmacoresistant epilepsy. Clinical studies with Pgp modulating compounds are currently envisaged in canine epilepsy.

Labelling and biological evaluation of [11C]methoxy-Sch225336: a radioligand for the cannabinoid-type 2 receptor

The cannabinoid type 2 receptor (CB(2) receptor) is part of the endocannabinoid system and has been suggested as mediator of a number of central and peripheral inflammatory processes. In the present study, we have synthesized N-[(1s)-1-[4-[[4-methoxy-2-[(4-[(11)C]methoxyphenyl)sulfonyl)-phenyl]sulfonyl] phenyl]ethyl]methanesulfonamide ([(11)C]methoxy-Sch225336) and evaluated this new tracer agent as a potential positron emission tomography radioligand for the in vivo visualization of CB(2) receptors. Sch225336 was demethylated and the resulting phenol precursor was radiolabelled with a carbon-11 methyl group by methylation using [(11)C]methyl iodide, followed by purification by high-performance liquid chromatography. The log P of [(11)C]methoxy-Sch225336 and its biodistribution in normal mice were determined. Enhancement of brain uptake by inhibition of blood-brain barrier (BBB) efflux transporters was studied. Mouse plasma was analysed to quantify the formation of radiometabolites. The affinity of Sch225336 for the human cannabinoid type 1 and type 2 receptor was determined. [(11)C]methoxy-Sch225336 was obtained with a decay corrected radiochemical yield of about 30% and a specific activity of 88.8 GBq/mumol (end of synthesis). After intravenous injection in mice, the compound is rapidly cleared from the blood through the hepatobiliary pathway and does not show particular retention in any of the major organs. Polar metabolites were found in mouse plasma. Brain uptake was low despite the favourable log P value of 2.15, which is partly due to efflux by BBB pumps. [(11)C]methoxy-Sch225336 is a good candidate for in vivo imaging of the CB(2) receptor, although the low blood-brain barrier penetration limits its potential for central nervous system imaging.

A critical overview of the influence of inflammation and infection on P-glycoprotein expression and activity in the brain

Background: P-glycoprotein is a blood–brain barrier efflux transporter that limits drug accumulation in the brain. Objective: To review recent in vivo and in vitro investigations that examined the influence of inflammation, infection and related clinical neuroinflammatory disorders on P-glycoprotein expression and activity in the brain. Methods: Critical overview of English-language studies. Results/conclusions: Inflammation and infection produce dynamic changes in P-glycoprotein expression and activity in the blood–brain barrier. In vitro, blood–brain barrier P-glycoprotein activity is downregulated after short-term exposure to inflammatory mediators whereas its activity is upregulated following more prolonged exposure. In vivo studies in both humans and animals have linked CNS inflammation, peripheral inflammation and related clinical neuroinflammatory disorders with alterations in the expression and activity of blood–brain barrier P-glycoprotein. The direction and degree of change in P-glycoprotein activity depends on the in vivo or in vitro model examined, the cell type examined (e.g., endothelial or glial), the inflammatory mediator utilized, the anatomic site in which the inflammatory response was first generated, the time points chosen for observation and the substrates analyzed. Alterations in P-glycoprotein activity affect drug activity in the CNS and seem clinically important.

Blood–Brain Barrier Efflux Transport of Pyrimidine Nucleosides and Nucleobases in the Rat

The brain efflux index (BEI), a measurement of blood-brain barrier (BBB) efflux transport, was estimated at 15 s, 30 s, 1 min, 3 min and 10 min after intracerebral injection of [14C]pyrimidines. An initial steep increase of the BEI values over time was observed for [14]uracil and [14C]thymine, followed by a more moderate increase after 1 min. For the corresponding nucleosides, [14C]uridine and [14C]thymidine, the increase of BEI values over time was less steep and linear between 30 s and 3 min. The apparent BBB efflux clearances for [14C]uridine, [14C]thymidine, [14C]uracil and [14C]thymine were (microl/min/g): 95.2 +/- 12.1, 125.3 +/- 18.4, 290.4 +/- 28 and 358.5 +/- 32.5, respectively, which is at least several folds higher than the predicted BBB influx clearances of uridine, uracil and thymidine. Quick depletion of brain parenchyma from brain microvasculature has revealed that [14C] radioactivity accumulated in brain microvessels after injection of nucleosides [14C]thymidine and [14C]uridine, but that was not observed when nucleobases, [14C]thymine and [14C]uracil, were injected. Reverse transcriptase-PCR revealed that the rat brain and liver (positive control) express dihydropyrimidine dehydrogenase, a key enzyme in pyrimidine nucleobase catabolism. Two bands representing spliced variants have been detected with the relative density of the bands (expressed relative to the density of glyceraldehyde3-phosphate dehydrogenase bands, mean +/- SEM from 3 separate samples) 0.16 +/- 0.06 and 0.04 +/- 0.01 (brain) and 0.49 +/- 0.1 and 0.07 +/- 0.01 (liver). Overall, these results indicate that the net direction of pyrimidine BBB transport is the efflux transport; rapid BBB efflux transport and metabolic breakdown of pyrimidine nucleobases appear to be important for brain homeostasis.

Neuroinflammation Activates Mdr1b Efflux Transport Through NFκB: Promoter Analysis in BBB Endothelia

Background/aims: Although it is known that drug delivery across the blood-brain barrier (BBB) may be hampered by efflux transport activity of the multidrug resistance (mdr) gene product P-glycoprotein, it is not clear how inflammation regulates efflux transporters. In rat brain endothelial (RBE4) cells of BBB origin, the proinflammatory cytokine TNF mainly induced transcriptional upregulation of mdr1b, and to a lesser extent mdr1a, resulting in greater efflux of the substrates. This study further determines the mechanisms by which TNF activates mdr1b promoter activity. Methods/Results: Luciferase reporter assays and DNA binding studies show that (1) maximal basal promoter activity was conferred by a 476 bp sequence upstream to the mdr1b transcriptional initiation site; (2) TNF induced upregulation of promoter activity by NFκB nuclear translocation; and (3) the NFκB binding site of the mdr1b promoter was solely responsible for basal and TNF-activated gene transcription, whereas the p53 binding site was not involved. Binding of the p65 subunit of NFκB to nuclear DNA from RBE4 cells was shown by electrophoretic mobility shift assay and chromatin immunoprecipitation assays. Conclusion: NFκB mediates TNF-induced upregulation of mdr1b promoter activity, illustrating how inflammation activates BBB efflux transport.