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Expression of Multidrug Transporters MRP1, MRP2, and BCRP Shortly after Status Epilepticus, during the Latent Period, and in Chronic Epileptic Rats
Abstract
Overexpression of multidrug transporters may play a role in the development of pharmacoresistance by decreasing extracellular drug levels in the brain. However, it is not known whether overexpression is due to an initial insult or evolves more gradually because of recurrent spontaneous seizures. In the present study, we investigated the expression of different multidrug transporters during epileptogenesis in the rat. In addition, we determined whether these transporters affected phenytoin (PHT) distribution in the brain.
Expression of multidrug resistance-associated proteins MRP1 and MRP2 and breast cancer-resistance protein (BCRP) was examined after electrically induced status epilepticus (SE) by immunocytochemistry and Western blot analysis. Brain/blood PHT levels were determined by high-performance liquid chromatography (HPLC) analysis in the presence and absence of the MRP inhibitor probenecid.
Shortly after SE, MRP1, MRP2, and BCRP were upregulated in astrocytes within several limbic structures, including hippocampus. In chronic epileptic rats, these proteins were overexpressed in the parahippocampal cortex, specifically in blood vessels and astrocytes surrounding these vessels. Overexpression was related to the occurrence of SE and was present mainly in rats with a high seizure frequency. Brain PHT levels were significantly lower in epileptic rats compared with control rats, but pharmacologic inhibition of MRPs increased the PHT levels.
Overexpression of MRP and BCRP was induced by SE as well as recurrent seizures. Moreover, overexpression was associated with lower PHT levels in the brain, which was reversed through inhibition of MRPs. These data suggest that administration of antiepileptic drugs in combination with specific inhibitors for multidrug transporters may be a promising therapeutic strategy in pharmacoresistant patients.
... • MS: ABCC1 expression was increased in reactive astrocytes in the brain of MS patients. [127,199,[220][221][222][223] • Neuroinflammation: Abcc2 transport activity and protein expression were increased in rat or mouse brain capillaries in response to activation of Nrf2. • Epilepsy: ABCC2 overexpression and increased activity induced by status epilepticus was associated with reduced anti-epileptic drugs levels in the brain and reduced efficacy. ...
... • Epilepsy: ABCC2 overexpression and increased activity induced by status epilepticus was associated with reduced anti-epileptic drugs levels in the brain and reduced efficacy. [137,199,222] ABCC4 (MRP4) Luminal (apical) and basolateral Efflux • AD: ABCC4 increased protein expression was detected at in the hippocampal sections from AD brain. [207,222] Abbreviation: Aβ, amyloid beta; AD, Alzheimer's disease; ALS, amyotrophic lateral sclerosis; BBB, blood-brain barrier; BCRP, breast cancer resistance protein; ENT1, equilibrative nucleoside transporter 1; FATP1, fatty acid transport protein 1; GLUT1, glucose transporter 1; GWAS, genome-wide association studies; MRP, multidrug resistance-associated protein; MS, multiple sclerosis; Nrf2: nuclear factor erythroid-derived 2-like factor; OATP, organic anion transporting polypeptides. ...
... [137,199,222] ABCC4 (MRP4) Luminal (apical) and basolateral Efflux • AD: ABCC4 increased protein expression was detected at in the hippocampal sections from AD brain. [207,222] Abbreviation: Aβ, amyloid beta; AD, Alzheimer's disease; ALS, amyotrophic lateral sclerosis; BBB, blood-brain barrier; BCRP, breast cancer resistance protein; ENT1, equilibrative nucleoside transporter 1; FATP1, fatty acid transport protein 1; GLUT1, glucose transporter 1; GWAS, genome-wide association studies; MRP, multidrug resistance-associated protein; MS, multiple sclerosis; Nrf2: nuclear factor erythroid-derived 2-like factor; OATP, organic anion transporting polypeptides. ...
The blood–brain barrier (BBB) is a highly selective and restrictive semipermeable network of cells and blood vessel constituents. All components of the neurovascular unit give to the BBB its crucial and protective function, i.e., to regulate homeostasis in the central nervous system (CNS) by removing substances from the endothelial compartment and supplying the brain with nutrients and other endogenous compounds. Many transporters have been identified that play a role in maintaining BBB integrity and homeostasis. As such, the restrictive nature of the BBB provides an obstacle for drug delivery to the CNS. Nevertheless, according to their physicochemical or pharmacological properties, drugs may reach the CNS by passive diffusion or be subjected to putative influx and/or efflux through BBB membrane transporters, allowing or limiting their distribution to the CNS. Drug transporters functionally expressed on various compartments of the BBB involve numerous proteins from either the ATP-binding cassette (ABC) or the solute carrier (SLC) superfamilies. Pathophysiological stressors, age, and age-associated disorders may alter the expression level and functionality of transporter protein elements that modulate drug distribution and accumulation into the brain, namely, drug efficacy and toxicity. This review focuses and sheds light on the influence of inflammatory conditions and diseases such as Alzheimer’s disease, epilepsy, and stroke on the expression and functionality of the BBB drug transporters, the consequential modulation of drug distribution to the brain, and their impact on drug efficacy and toxicity.
... MRP2 is another ABC transporter in the luminal surface of brain capillary endothelium. It was demonstrated that MRP2 was overexpressed in the capillary endothelial cells in patients with drug-resistant epilepsy and the epileptic foci of epileptic rats (Hoffmann et al., 2006;Liu et al., 2015;Loscher & Potschka, 2005;Potschka et al., 2003b;van Vliet et al., 2005;Yao et al., 2012). Several AEDs, such as phenytoin and carbamazepine, may be substrates of MRP2 in vitro or in vivo (Kim et al., 2010;Potschka et al., 2003a,b;van Vliet et al., 2005). ...
... It was demonstrated that MRP2 was overexpressed in the capillary endothelial cells in patients with drug-resistant epilepsy and the epileptic foci of epileptic rats (Hoffmann et al., 2006;Liu et al., 2015;Loscher & Potschka, 2005;Potschka et al., 2003b;van Vliet et al., 2005;Yao et al., 2012). Several AEDs, such as phenytoin and carbamazepine, may be substrates of MRP2 in vitro or in vivo (Kim et al., 2010;Potschka et al., 2003a,b;van Vliet et al., 2005). These observations indicate that P-gp and MRP2 may contribute to drug-resistant epilepsy (Chan et al., 2014;Kwan & Brodie, 2005;Loscher & Potschka, 2005;Zhang et al., 2011Zhang et al., , 2010Zhang et al., , 2012Zhang et al., , 2013. ...
... The expression of MRP2 was significantly increased in the hippocampus of patients with hippocampal sclerosis . MRP2 was also significantly upregulated in rat brains after electrical stimulation and pilocarpine-induced seizures (van Vliet et al., 2005;Zhang et al., 2004). In this study, we demonstrated that P-gp and MRP2 transported Hup-A in vitro, implying that P-gp and MRP2 might affect the therapeutic efficacy of Hup-A and the druggability in drug development. ...
1. More than 30% of epilepsy patients suffer pharmacoresistance. Transport of antileptic drugs by P-glycoprotein (P-gp) and MRP2 plays an important role in drug-resistant epilepsy. Huperzine A (Hup-A) is a natural compound, which might have potential in treating neurological disorders including epilepsy and Alzheimer's disease. In this study, we investigated whether human P-gp and MRP2 transport Hup-A.
2. LLC-PK1 and MDCKII cells transfected with human P-gp or MRP2 were used to establish concentration equilibrium transport assays (CETAs) and determine the transport profile of Hup-A. The expression of P-gp and MRP2 was detected by qPCR and western blotting. The transport function of P-gp and MRP2 was measured by Rho123 and CDFDA cell uptake assay.
3. In CETAs, Hup-A at concentrations of 10 ng/ml or 2 µg/ml was transported by MDR1 and MRP2 from basolateral to apical sides of the cell monolayers. P-gp and MRP2 inhibitors completely blocked the efflux of Hup-A. There was no efflux of Hup-A in LLC-PK1 or MDCKII wild-type cells.
4. We demonstrate that Hup-A is a substrate of P-gp and MRP2. These results imply the efflux of Hup-A across the BBB in vivo, suggesting potential drug resistance of Hup-A.
... Se ha reportado sobreexpresión de la P-gp y MRP´s en el endotelio de los capilares del cerebro, en la membrana perivascular de los astrocitos y neuronas de tejido cerebral de pacientes con epilepsia del lóbulo temporal (ELT) (6,62,206,211) . Además, numerosos estudios han reportado resultados similares en tejido cerebral de pacientes con epilepsia asociada a malformaciones corticales, en células mononucleares de sangre periférica en niños con epilepsia intratable y en modelos animales de epilepsia (6,11,12,110,116,132,179,180,219) . ...
... Por otro lado, la administración de probenecid (inhibidor de MRP´s) restaura los niveles extracelulares de FAEs en cerebro de ratas con epilepsia fármaco resistente (38,211) . Sin embargo, al presente no se han reportado evidencias terapéuticas del probenecid en pacientes con epilepsia intratable. ...
... Este trastorno también provoca que el sujeto no despierte en un tiempo de mañana socialmente aceptable. Teniendo esto en mente, sería interesante proponer que la Adenosina podría controlar el ciclo sueño-vigilia por tener un papel clave en la modulación de la respuesta de la luz en los fotorreceptores como sugiere Sodhi P, y sus colaboradores (211) . ...
... Se ha reportado sobreexpresión de la P-gp y MRP´s en el endotelio de los capilares del cerebro, en la membrana perivascular de los astrocitos y neuronas de tejido cerebral de pacientes con epilepsia del lóbulo temporal (ELT) (6,62,206,211) . Además, numerosos estudios han reportado resultados similares en tejido cerebral de pacientes con epilepsia asociada a malformaciones corticales, en células mononucleares de sangre periférica en niños con epilepsia intratable y en modelos animales de epilepsia (6,11,12,110,116,132,179,180,219) . ...
... Por otro lado, la administración de probenecid (inhibidor de MRP´s) restaura los niveles extracelulares de FAEs en cerebro de ratas con epilepsia fármaco resistente (38,211) . Sin embargo, al presente no se han reportado evidencias terapéuticas del probenecid en pacientes con epilepsia intratable. ...
... Este trastorno también provoca que el sujeto no despierte en un tiempo de mañana socialmente aceptable. Teniendo esto en mente, sería interesante proponer que la Adenosina podría controlar el ciclo sueño-vigilia por tener un papel clave en la modulación de la respuesta de la luz en los fotorreceptores como sugiere Sodhi P, y sus colaboradores (211) . ...
... Various multidrug resistance proteins such as multidrug resistance gene-1 (MDR1), MRP2 and MRP5 have been shown to be over expressed in animal models of epilepsy as well as in brain tissue resected from DRE patients [9][10][11][12][13]. Prior investigations have shown involvement of MRP1, BCRP, MVP and UGT1A4 in drug resistance in various neurological diseases as well as in cancer yet very few reports are available on their contribution to DRE [13][14][15][16]. ...
... Various multidrug resistance proteins such as multidrug resistance gene-1 (MDR1), MRP2 and MRP5 have been shown to be over expressed in animal models of epilepsy as well as in brain tissue resected from DRE patients [9][10][11][12][13]. Prior investigations have shown involvement of MRP1, BCRP, MVP and UGT1A4 in drug resistance in various neurological diseases as well as in cancer yet very few reports are available on their contribution to DRE [13][14][15][16]. MRP1 is a specific organic anion transporter and can cause efflux of antitumor drugs and some AEDs namely phenobarbital, carbamazepine, phenytoin, sodium valproate [12]. ...
... Reports related to the contribution of BCRP in AED resistance are contradictory. Some studies found no upregulation of BCRP in human epileptogenic brain tissue and no evidence for BCRP mediated AED transport in vitro, but other studies reported upregulation of BCRP expression in the microvasculature of epileptogenic brain tumors and in chronic epilepsy animal models [13,14]. Alterations in the expression level of transporters in the brain tissues of DRE patients is clinically relevant and need not to be extrapolated, unlike animal models. ...
Purpose:
Identifying factors involved in the development of drug resistant epilepsy (DRE) remains a challenge. Candidate gene studies have shown modulation of resistance to drugs by various multidrug resistance proteins in DRE. However the resistance to drugs in DRE could be more complex and multifactorial involving molecules in different pharmacokinetic processes. In this study for the first time we have analyzed the relative expression of four molecules with different drug resistance mechanisms in two most common DRE pathologies, mesial temporal lobe epilepsy (MTLE) and focal cortical dysplasia (FCD) with respect to each other and also with different non-epileptic controls.
Methods:
Brain tissues resected from MTLE (n=16) and FCD type I and II (n=12) patients who had undergone surgery were analysed for mRNA levels of multidrug resistance-associated protein 1(MRP1), major vault protein (MVP), breast cancer resistance protein (BCRP), and one drug metabolising enzyme (UGT1A4) as compared to non-epileptic controls which were tissues resected from tumor periphery (n=6) and autopsy tissues (n=4) by quantitative PCR.
Results:
We found significant upregulation of MVP and BCRP whereas MRP1 and UGT1A4 were unaltered in both pathologies. While upregulation of BCRP was significantly higher in MTLE (9.34±0.45; p<0.05), upregulation of MVP was significantly higher in FCD (2.94±0.65; p<0.01).
Conclusion:
We propose that upregulation of BCRP and MVP is associated with MTLE and FCD and these molecules not only may have the potential to predict pathology specific phenotypes but may also have therapeutic potential as adjunct treatment in these pathologies.
... Various multidrug resistance proteins such as multidrug resistance gene-1 (MDR1), MRP2 and MRP5 have been shown to be over expressed in animal models of epilepsy as well as in brain tissue resected from DRE patients [9][10][11][12][13]. Prior investigations have shown involvement of MRP1, BCRP, MVP and UGT1A4 in drug resistance in various neurological diseases as well as in cancer yet very few reports are available on their contribution to DRE [13][14][15][16]. ...
... Various multidrug resistance proteins such as multidrug resistance gene-1 (MDR1), MRP2 and MRP5 have been shown to be over expressed in animal models of epilepsy as well as in brain tissue resected from DRE patients [9][10][11][12][13]. Prior investigations have shown involvement of MRP1, BCRP, MVP and UGT1A4 in drug resistance in various neurological diseases as well as in cancer yet very few reports are available on their contribution to DRE [13][14][15][16]. MRP1 is a specific organic anion transporter and can cause efflux of antitumor drugs and some AEDs namely phenobarbital, carbamazepine, phenytoin, sodium valproate [12]. ...
... Reports related to the contribution of BCRP in AED resistance are contradictory. Some studies found no upregulation of BCRP in human epileptogenic brain tissue and no evidence for BCRP mediated AED transport in vitro, but other studies reported upregulation of BCRP expression in the microvasculature of epileptogenic brain tumors and in chronic epilepsy animal models [13,14]. Alterations in the expression level of transporters in the brain tissues of DRE patients is clinically relevant and need not to be extrapolated, unlike animal models. ...
... This suggests a role for inflammation in fine tuning the regulation of drug transporters in epilepsy. (Vezzani, 2014;Vezzani, French, Bartfai, & Baram, 2011). In a recent study, Enrique et al. performed a virtual screening to select compounds with simultaneous anticonvulsant and anti-inflammatory effects to treat refractory epilepsy, acquiring five candidates: caffeic acid, p-coumaric acid, γdecanolactone, sodium phenylbutyrate and sebacic acid (SA). ...
... More recently, multiple studies confirmed overexpression of P-gp, BCRP and MRPs in epilepsy (Table 1) (Dombrowski et al., 2001;Feldmann et al., 2013;Sisodiya, Lin, Harding, Squier, & Thom, 2002;van Vliet, Redeker, Aronica, Edelbroek, & Gorter, 2005). For example, Dombrowski et al., using ECs from patients with refractory epilepsy, demonstrated an overexpression of P-gp-(+134%), MRP2-(+225%) and MRP5-(+179%) coding genes and P-gp protein upregulation, when compared to controls (Dombrowski et al., 2001). ...
ABC (ATP-binding cassette) transporters represent one of the largest and most diverse superfamily of proteins in living species, playing an important role in many biological processes such as cell homeostasis, cell signaling, drug metabolism and nutrient uptake. Moreover, using the energy generated from ATP hydrolysis, they mediate the efflux of endogenous and exogenous substrates from inside the cells, thereby reducing their intracellular accumulation. At present, 48 ABC transporters have been identified in humans, which were classified into 7 different subfamilies (A to G) according to their phylogenetic analysis. Nevertheless, the most studied members with importance in drug therapeutic efficacy and toxicity include P-glycoprotein (P-gp), a member of the ABCB subfamily, the multidrug-associated proteins (MPRs), members of the ABCC subfamily, and breast cancer resistance protein (BCRP), a member of the ABCG subfamily. They exhibit ubiquitous expression throughout the human body, with a special relevance in barrier tissues like the blood-brain barrier (BBB). At this level, they play a physiological function in tissue protection by reducing or limiting the brain accumulation of neurotoxins. Furthermore, dysfunction of ABC transporters, at expression and/or activity level, has been associated with many neurological diseases, including epilepsy, multiple sclerosis, Alzheimer's disease, and amyotrophic lateral sclerosis. Additionally, these transporters are strikingly associated with the pharmacoresistance to central nervous system (CNS) acting drugs, because they contribute to the decrease in drug bioavailability. This article reviews the signaling pathways that regulate the expression and activity of P-gp, BCRP and MRPs subfamilies of transporters, with particular attention at the BBB level, and their mis-regulation in neurological disorders.
... In another study, Bankstahl and Löscher showed overexpression of P-gp protein in brain capillary endothelial cells 48 h after SE in two rat models, the lithium/pilocarpine model and the basolateral amygdala electrical stimulation model (125). van Vliet et al. (126) also reported increased MRP1, MRP2, and BCRP protein expression levels in rat astrocytes and cerebral blood vessels after acute SE and in chronic epilepsy. Similar to the finding with P-gp, overexpression of these transporters was greater in chronic epileptic rats that demonstrated progression of epilepsy (126). ...
... van Vliet et al. (126) also reported increased MRP1, MRP2, and BCRP protein expression levels in rat astrocytes and cerebral blood vessels after acute SE and in chronic epilepsy. Similar to the finding with P-gp, overexpression of these transporters was greater in chronic epileptic rats that demonstrated progression of epilepsy (126). Recent research in the field has postulated two main mechanisms leading to efflux transporter overexpression in the brain in epilepsy: (1) ASDmediated induction of efflux transporters via nuclear receptors and (2) seizure-induced signaling causing efflux transporter overexpression. ...
Epilepsy is a common neurological disorder that affects over 70 million people worldwide. Despite the recent introduction of new antiseizure drugs (ASDs), about one-third of patients with epilepsy have seizures refractory to pharmacotherapy. Early identification of patients who will become refractory to ASDs could help direct such patients to appropriate non-pharmacological treatment, but the complexity in the temporal patterns of epilepsy could make such identification difficult. The target hypothesis and transporter hypothesis are the most cited theories trying to explain refractory epilepsy, but neither theory alone fully explains the neurobiological basis of pharmacoresistance. This review summarizes evidence for and against several major theories, including the pharmacokinetic hypothesis, neural network hypothesis, intrinsic severity hypothesis, gene variant hypothesis, target hypothesis, and transporter hypothesis. The discussion is mainly focused on the transporter hypothesis, where clinical and experimental data are discussed on multidrug transporter overexpression, substrate profiles of ASDs, mechanism of transporter upregulation, polymorphisms of transporters, and the use of transporter inhibitors. Finally, future perspectives are presented for the improvement of current hypotheses and the development of treatment strategies as guided by the current understanding of refractory epilepsy.
... The best-known efflux transporters are the ATP-binding cassette (ABC) transporters, including P-glycoprotein (P-gp/ABCB1), multidrug resistance associated protein 1 (MRP1/ABCC1), and breast cancer resistance protein (BCRP/ABCG2). Within the brain, P-gp and MRP1 are not only expressed at the BBB but also on the brain cells [169], whereas BCRP is primarily expressed on the endothelial cells that form the BBB [170][171][172][173]. ...
Neurotropic viruses may cause meningitis, myelitis, encephalitis, or meningoencephalitis. These inflammatory conditions of the central nervous system (CNS) may have serious and devastating consequences if not treated adequately. In this review, we first summarize how neurotropic viruses can enter the CNS by (1) crossing the blood-brain barrier or blood-cerebrospinal fluid barrier; (2) invading the nose via the olfactory route; or (3) invading the peripheral nervous system. Neurotropic viruses may then enter the intracellular space of brain cells via endocytosis and/or membrane fusion. Antiviral drugs are currently used for different viral CNS infections, even though their use and dosing regimens within the CNS, with the exception of acyclovir, are minimally supported by clinical evidence. We therefore provide considerations to optimize drug treatment(s) for these neurotropic viruses. Antiviral drugs should cross the blood–brain barrier/blood cerebrospinal fluid barrier and pass the brain cellular membrane to inhibit these viruses inside the brain cells. Some antiviral drugs may also require intracellular conversion into their active metabolite(s). This illustrates the need to better understand these mechanisms because these processes dictate drug exposure within the CNS that ultimately determine the success of antiviral drugs for CNS infections. Finally, we discuss mathematical model-based approaches for optimizing antiviral treatments. Thereby emphasizing the potential of CNS physiologically based pharmacokinetic models because direct measurement of brain intracellular exposure in living humans faces ethical restrictions. Existing physiologically based pharmacokinetic models combined with in vitro pharmacokinetic/pharmacodynamic information can be used to predict drug exposure and evaluate efficacy of antiviral drugs within the CNS, to ultimately optimize the treatments of CNS viral infections.
... On the other hand, recent studies investigated that the increase of internal antioxidants and the prescription of external antioxidants such as lipoic acid, maxidol, tocopherol, melatonin, resveratrol, and vitamins C and E have a protective effect against some damages induced by epilepsy on the brain [15,16]. ...
Background and Aim: Epilepsy as a neurological disorder is characterized by unpredictable and periodic seizures. In the present study, the anticonvulsant effect of different doses of salep hydroalcoholic extract on pentylenetetrazole (PTZ) induced seizures was investigated. Methods and Materials/Patients: The animal model of seizure was established by the intraperitoneal injection of pentylenetetrazole (PTZ- 85 mg/kg). Hydroalcoholic extract of salep was administered to the animals in 3 doses (80, 160, and 320 mg/kg). The parameters of the onset and duration of tonic-clonic seizure, total seizure time, balance (falling), and jumping during seizure mortality rate were measured. The results were analyzed by ANOVA test at a significant level of p≤0.05. Results: The results of the current study indicated that salep extract increased the delay in the onset of seizure in the PTZ + extract group in comparison with the PTZ group. The duration of tonic-clonic seizure and the number of falling, jumping, and total seizure times were significantly decreased by salep extract compared to the PTZ group. In the seizure + salep extract, the number and percentage of 24-hour mortality among animals decreased with increasing dosage. All changes were dose-dependent and 320 mg/kg showed the most effect. Conclusion: Extract of salep can probably have anticonvulsant properties, however, further studies are needed to clarify the exact mechanisms.
... Breast cancer resistance protein (BCRP; ABCG2), is considered one of the major BBB efflux transporters, where it controls brain diffusion of many lipophilic xenobiotics for brain protection (König et al. 2013). Despite the evidence of its potential role in restricting several drugs' brain entry (Mahringer and Fricker 2016;Saunders et al. 2016), reports related to its contribution to DRE are still scarce van Vliet et al. 2005). ...
Blood–brain barrier (BBB) efflux transporters' overexpression hinders antiepileptic drug brain entry. Breast cancer resistance protein (BCRP) is a major BBB efflux transporter. In the present work, BCRP's role as a mechanism that might contribute to drug-resistant epilepsy (DRE) in a mouse model of acute seizures was studied with further assessment of the effect of its inhibition by ko143 and metformin (MET) on lamotrigine (LTG) bioavailability and efficacy. 42 male mice divided into 6 groups: G1: Normal control, G2: LTG-injected healthy mice: LTG 20 mg/kg i.p., G3: Acute seizures (A.S) mice: Pentylenetetrazole (PTZ) 50 mg/kg i.p., G4: LTG-treated A.S mice: LTG 20 mg/kg + PTZ 50 mg/kg i.p., G5: Ko143 + LTG treated A.S mice: Ko143 15 mg/kg i.p. before LTG + PTZ, G6: MET + LTG treated A.S mice: MET 200 mg/kg i.p. before LTG + PTZ. Seizures severity, serum, brain LTG, and brain BCRP were assessed. PTZ group experienced the highest seizure frequency and brain BCRP expression. Ko143 and MET groups showed a significant decrease in brain BCRP with subsequent improvement in brain LTG level and better seizure control. BCRP has a significant role in epilepsy resistance and its inhibition with ko143 or MET adds value to DRE management.
... Extracellular glutamate upregulates Abcb1 mRNA and associated protein levels in rat brain capillary endothelial cells in vitro and proposed that glutamate activates the NMDA receptor and triggers a signaling cascade that increases Abcb1 expression at the blood-brain barrier (Zhu and Liu, 2004). At the same time, efflux of anticonvulsive drugs by ABC transporters at the blood-brain barrier was considered as one of the main causes of refractory epilepsy (van Vliet et al., 2005). ...
The drug efflux transporters ABCB1 and ABCG2 at the blood-brain barrier limit the delivery of drugs into the brain. Strategies to overcome ABCB1/ABCG2 have been largely unsuccessful, which poses a tremendous clinical problem to successfully treat CNS diseases. Understanding basic transporter biology, including intracellular regulation mechanisms that control these transporters, is critical to solve this clinical problem. In this comprehensive review, we summarize current knowledge on signaling pathways that regulate ABCB1/ABCG2 at the blood-brain barrier. In part I, we give a historical overview on blood-brain barrier research and introduce the role ABCB1 and ABCG2 play in this context. In part II, we summarize the most important strategies that have been tested to overcome the ABCB1/ABCG2 efflux system at the blood-brain barrier. In part III, the main component of this review, we provide detailed information on the signaling pathways that have been identified to control ABCB1/ABCG2 at the blood-brain barrier and their potential clinical relevance. This is followed by part IV, where we explain the clinical implications of ABCB1/ABCG2 regulation in the context of CNS disease. Lastly, in part V, we conclude by highlighting examples of how transporter regulation could be targeted for therapeutic purposes in the clinic. Significance Statement The ABCB1/ABCG2 drug efflux system at the blood-brain barrier poses a significant problem to successful drug delivery to the brain. Here, we review signaling pathways that regulate blood-brain barrier ABCB1/ABCG2 and could potentially be targeted for therapeutic purposes.
... In addition to the potential role of astrocyte dysfunction in the excessive permeability of the blood-brain barrier during seizures, pathological changes in perivascular astrocytes may adversely affect penetrance of anti-seizure drugs into the brain by overexpressing multidrug transporters and multidrug resistance proteins (Aronica et al., 2012b;Lazarowski et al., 2007;Tishler et al., 1995;van Vliet et al., 2005). These changes have been documented in the resected tissue of human epilepsy patients (Tishler et al., 1995). ...
The epilepsies are a diverse spectrum of disease states characterized by spontaneous seizures and associated comorbidities. Neuron-focused perspectives have yielded an array of widely used anti-seizure medications and are able to explain some, but not all, of the imbalance of excitation and inhibition which manifests itself as spontaneous seizures. Furthermore, the rate of pharmacoresistant epilepsy remain high despite the regular approval of novel anti-seizure medications. Gaining a more complete understanding of the processes that turn a healthy brain into an epileptic brain (epileptogenesis) as well as the processes which generate individual seizures (ictogenesis) may necessitate broadening our focus to other cell types. As will be detailed in this review, astrocytes augment neuronal activity at the level of individual neurons in the form of gliotransmission and the tripartite synapse. Under normal conditions, astrocytes are essential to the maintenance of blood-brain barrier integrity and remediation of inflammation and oxidative stress, but in epilepsy these functions are impaired. Epilepsy results in disruptions in the way astrocytes relate to each other by gap junctions which has important implications for ion and water homeostasis. In their activated state, astrocytes contribute to imbalances in neuronal excitability due their decreased capacity to take up and metabolize glutamate and an increased capacity to metabolize adenosine. Furthermore, due to their increased adenosine metabolism, activated astrocytes may contribute to DNA hypermethylation and other epigenetic changes that underly epileptogenesis. Lastly, we will explore the potential explanatory power of these changes in astrocyte function in detail in the specific context of the comorbid occurrence of epilepsy and Alzheimer's disease and the disruption in sleep-wake regulation associated with both conditions.
... High transporter levels in surgically resected brain tissues of PWE associated with a known structural brain abnormality such as cortical dysplasia or brain tumors, suggest disease etiology as the underlying cause (Leandro et al., 2019). An alternative explanation comes from the data obtained from animal models with experimentally-induced seizures, showing recurrent seizure activity and the associated neuronal stress as the responsible factor (Bankstahl and Loscher, 2008;van Vliet et al., 2005;van Vliet et al., 2007). However, these studies are few in number and the focus of these experiments has been ABCB1 with very little knowledge on other MDTs. ...
Despite the progress made in the development of new antiepileptic drugs (AEDs), poor response to them is a rising concern in epilepsy treatment. Of several hypotheses explaining AED treatment failure, the most promising theory is the overexpression of multidrug transporters belonging to ATP-binding cassette (ABC) transporter family at blood-brain barrier. Previous data show that AEDs themselves can induce these transporters, in turn affecting their own brain bioavailability. Presently, this induction and the underlying regulatory mechanism involved at human blood-brain barrier is not well elucidated. Herein, we sought to explore the effect of most prescribed first and second-line AEDs on multidrug transporters in human cerebral microvascular endothelial cells, hCMEC/D3. Our work demonstrated that exposure of these cells to valproic acid (VPA) induced mRNA, protein and functional activity of breast cancer resistance protein (BCRP/ABCG2). On examining the substrate interaction status of AEDs with BCRP, VPA, phenytoin and lamotrigine were found to be potential BCRP substrates. Furthermore, we observed that siRNA-mediated knockdown of peroxisome proliferator-activated receptor alpha (PPARα) or use of PPARα antagonist, resulted in attenuation of VPA-induced BCRP expression and transporter activity. VPA was found to increase PPARα expression and trigger its translocation from cytoplasm to nucleus. Findings from chromatin immunoprecipitation and luciferase assay showed that VPA enhances the binding of PPARα to its response element in the ABCG2 promoter, resulting in elevated ABCG2 transcriptional activity. Taken together, these in vitro findings highlight PPARα as the potential molecular target to prevent VPA-mediated BCRP induction, which may have important implications in VPA pharmacoresistance. Significance Statement Induction of multidrug transporters at blood-brain barrier can largely affect the bioavailability of the substrate antiepileptic drugs in the brains of patients with epilepsy, thus affecting their therapeutic efficacy. The present study reports a mechanistic pathway of breast cancer resistance protein (BCRP/ABCG2) upregulation by valproic acid in human brain endothelial cells via peroxisome proliferator-activated receptor alpha involvement, thereby providing a potential strategy to prevent valproic acid pharmacoresistance in epilepsy.
... In the field of brain tumors, MRP1 promotes the brain efflux of etoposide and vincristine (gliomas) [30]; MRP3 promotes the brain efflux of etoposide and cisplatin (gliomas) [30]; P-glycoprotein promotes the brain efflux of paclitaxel (intracerebral melanoma testing model) [31], ceritinib [32], temozolomide (glioblastoma) [33,34], and doxorubicin (glioblastoma) [35]; BCRP promotes the brain efflux of temozolomide (glioblastoma) [34,36] and ceritinib [32]. In epilepsy, P-glycoprotein is responsible for the brain efflux of phenytoin [37,38], topiramate [38,39], phenobarbital [40], and levetiracetam [38], while MRP1, MRP2, and BCRP efflux phenytoin [41]. Moreover, neuroinflammation associated with the course of epilepsy induces BBB alterations, such as an upregulation of the P-glycoprotein expression, hence contributing even more for the development of drug-resistant epilepsy [42]. ...
Brain diseases represent a substantial social and economic burden, currently affecting one in six individuals worldwide. Brain research has been focus of great attention in order to unravel the pathogenesis and complexity of brain diseases at the cellular, molecular, and microenvironmental levels. Due to the intrinsic nature of the brain, the presence of the highly restrictive blood-brain barrier (BBB), and the pathophysiology of most diseases, therapies can hardly be considered successful purely by the administration of one drug to a patient. Apart from improving pharmacokinetic parameters, tailoring biodistribution, and reducing the number of side effects, nanomedicines are able to actively co-target the therapeutics to the brain parenchyma and brain lesions, as well as to achieve the delivery of multiple cargos with therapeutic, diagnostic, and theranostic properties. Among other multivalent effects that can be personalized according to the disease needs, this represents a promising class of novel nanosystems, termed multifunctional nanomedicines. Herein, we review the principal mechanisms of therapeutic resistance of the most prevalent brain diseases, how to overcome this therapeutic resistance through the use of multifunctional nanomedicines that tackle multiple fronts of the disease microenvironment, and the promising therapeutic responses achieved by some of the most cutting-edge multifunctional nanomedicines reported in literature.
... In the normal brain, the expression of MRP-1 in the neurons and glial cells has not been constantly stated in the literature (Ashraf, Kao, & Bendayan, 2014). However, in the present study, MRP-1 IR was detected in Edelbroek , & Gorter, 2005). Contrary to this in the present study MRP-1 immuno-positivity was not seen in endothelial cells of capillaries in either control or diseased tissues (Fig.3 a-d). ...
Introduction
About 30% of patients with epilepsy do not respond to anti-epileptic drugs, leading to refractory seizures. The pathogenesis of drug-resistance in mesial temporal lobe epilepsy (MTLE) is not completely understood. Increased activity of drug-efflux transporters might be involved, resulting in subclinical concentrations of the drug at the target site. The major drug-efflux transporters are permeability glycoprotein (P-gp) and multidrug-resistance associated protein-1 (MRP-1). The major drawback so far is the expressional analysis of transporters in equal numbers of drug-resistant epileptic tissue and age-matched non-epileptic tissue.
Methods
We have studied P-gp and MRP-1 drug-efflux transporters in the sclerotic hippocampal tissues resected from the epilepsy surgery patients (n=15) and compared their expression profile with the tissues resected from non-epileptic autopsy cases (n=15).
Results
Statistically significant over expression of both P-gp (P<0.0001) and MRP-1 (P=0.01) at gene and protein levels were found in the MTLE cases. The fold change of P-gp was more pronounced than MRP-1. Immunohistochemistry of the patient group showed increased immunoreactivity of P-gp at blood-brain barrier and increased reactivity of MRP-1 in the parenchyma. The results were confirmed by confocal immunofluorescence microscopy.
Conclusion
Our results suggested that P-gp in association with MRP-1 might be responsible for the multi-drug resistance in epilepsy. P-gp and MRP-1 could be important determinants of bio availability and tissue distribution of anti-epileptic drugs in the brain which can pharmacologically inhibited to achieve optimal drug penetration to target site.
... Experimental data mostly from animal studies support that P-gp upregulation in epileptic regions of the brain occurs mainly as a result of seizure activity. Van Vliet et al. [50] also reported increased MRP1, MRP2, and BCRP protein expression levels in rat astrocytes and cerebral blood vessels after acute status epilepticus and in chronic epilepsy. Similar to the finding with P-gp, overexpression of these transporters was greater in chronic epileptic rats that demonstrated progression of epilepsy. ...
Background: Septic purulent nosocomial infections (SPNI) are one of the most significant healthcare challenges of post-surgical procedures. SPNI are associated with increased morbidity, mortality and admission costs. It is a priority to determine the level of nosocomial infections (NI). This study aims to evaluate the bacterial contaminations after cardiac surgery within the Department of Acquired Heart Defects (DAHD). Material and methods: A cross-sectional study was designed and the medical records of 1189 patients who underwent cardiac surgery within the DAHD of a multiprofile hospital were retrospectively analyzed. The data were collected and stored in a Microsoft Excel spreadsheet. Results: The incidence rate of SPNI following cardiac surgery was 317.57‰ compared to 15.02‰ officially reported (p <0.001). Of the most common infections among the total of 418 cases of SPNI studied, 32.06% were surgical site infections, 23.18% were associations of infections, 19.14% – respiratory tract infections. A patient with SPNI has an average of 22.25 days/bed spent in hospital, compared with the average for a patient without SPNI of 12.27 days/bed. The etiological structure includes 28 species of microorganisms including gram-positive (61.92%) and gram-negative (38.08%). Conclusions: Given the relatively high incidence of the SPNI and its impact, it is imperative to take more serious measures to prevent and control these infections
... Experimental data mostly from animal studies support that P-gp upregulation in epileptic regions of the brain occurs mainly as a result of seizure activity. Van Vliet et al. [50] also reported increased MRP1, MRP2, and BCRP protein expression levels in rat astrocytes and cerebral blood vessels after acute status epilepticus and in chronic epilepsy. Similar to the finding with P-gp, overexpression of these transporters was greater in chronic epileptic rats that demonstrated progression of epilepsy. ...
Background: Drug-resistant epilepsy is the cause of severe disability. Multiple questions remain unanswered both in terms of pathogenesis and therapeutic management. For this narrative review, PubMed database and Infomedica library were searched by using “drug-resistance in epilepsy” and “treatment of drug-resistant epilepsy” as key words. The following filters were applied: “Clinical Trial”, “Meta-analysis”, “Multicenter Study”, and “Randomized Controlled Trial”, covering the period of 01.01.2005–06.01.2021.Several hypotheses have been proposed, i.e., pharmacokinetic, intrinsic severity, gene, target, transporter, and neural network hypotheses. Many controlled trials showed different results in terms of seizure control after combined methods of therapies. Immunotherapy, palliative epilepsy surgery alone or associated with neurostimulation procedures including vagus nerve, trigeminal nerve, or deep brain stimulation may be efficient, however, seizure freedom is not always achieved. Genetic epilepsies might benefit from gene and exosome therapy; however, further studies are needed to verify their safety.
Conclusions: Neuroscience of drug-resistant epilepsy faces many challenges. Inflammatory mediators, biomarkers, and genes might allow the identification of new treatment targets, contribute to an earlier diagnosis, and assess the clinical outcomes.
... 43 Besides, highly expressed MRP1 has been verified in astrocytes and blood vessels of the brain following acute status epilepticus and during chronic epilepsy. 44 Therefore, the role of miR-139-5p in augmenting sensitivity to antiepileptic drugs was achieved by suppressing expression of MRP1. ...
Aim
Drug resistance is an intractable issue urgently needed to be overcome for improving efficiency of antiepileptic drugs in treating refractory epilepsy. microRNAs (miRNAs) have been proved as key regulators and therapeutic targets in epilepsy. Accordingly, the aim of the present study was to identify a novel differentially expressed miRNA which could improve the efficiency of antiepileptic drugs during the treatment of refractory epilepsy.
Methods and Results
Serum samples were collected from children with refractory epilepsy. An in vivo refractory epilepsy model was developed in SD rats by electrical amygdala kindling. We identified that miR‐139‐5p was decreased and multidrug resistance‐associated protein 1 (MRP1) was remarkably upregulated in the serum samples from children with refractory epilepsy and the brain tissues from rat models of refractory epilepsy. After phenobarbitone injection in rat models of refractory epilepsy, the after discharging threshold in kindled amygdala was detected to screen out drug‐resistant rats. Dual‐luciferase reporter gene assay demonstrated that MRP1 was a target of miR‐139‐5p. In order to evaluate the effect of miR‐139‐5p/MRP1 axis on drug resistance of refractory epilepsy, we transfected plasmids into the hippocampus of drug‐resistant rats to alter the expression of miR‐139‐5p and MRP1. TUNEL staining and Nissl staining showed that miR‐139‐5p overexpression or MRP1 downregulation could reduce the apoptosis and promote survival of neurons, accompanied by alleviated neuronal damage.
Conclusion
Collectively, these results suggest an important role of miR‐139‐5p/MRP1 axis in reducing the resistance of refractory epilepsy to antiepileptic drugs.
... In drug-resistant epilepsy, the role of specific transporters is emphasized: P-glycoprotein [96,99], the multidrug resistance-associated protein (MRP) [96], and the breast cancer resistance protein (BCRP) [100]. The blood-brain barrier overexpression of these transporters in patients and drug-resistant epilepsy experimental models supports this hypothesis [96,97,[100][101][102][103][104][105]. Also, this hypothesis is supported by the fact that the administration of drug-transporter antagonists reverts the low parenchymal concentration of antiepileptic drugs [98,103,104,[106][107][108][109][110][111]. ...
Drug-resistant epilepsy affects approximately one-third of the patients with epilepsy. The pharmacoresistant condition in epilepsy is mainly explained by six hypotheses. In addition, several experimental models have been used to understand the mechanisms involved in pharmacoresistant epilepsy and to identify novel therapies to control this condition. However, the global prevalence of this disease persists without changes. Several factors can explain this situation. First of all, the pharmacoresistant epilepsy is explained by different and independent hypotheses. Each hypothesis indicates specific mechanisms to explain the drug-resistant condition in epilepsy. However, there are different findings suggesting common mechanisms between the different hypotheses. Other important situation is that the experimental models designed for the screening of drugs with potential anticonvulsant effect do not consider factors such as age, gender, type of epilepsy, and comorbid disorders. The present review focuses on indicating the limitations for each hypothesis and the relationships among them. The relevance to consider central and peripheral phenomena associated with the drug-resistant condition in different types of epilepsy is also indicated. The necessity to establish a global hypothesis that integrates all the phenomena associated with the pharmacoresistant epilepsy is proposed.
This article is part of the Special Issue “NEWroscience 2018".
... MRP1 (ABCC1) has also been implicated but levels of this transporter are thought to be low in brain endothelial cells in situ and only increase in cultured brain endothelial cells once they are removed from the brain microenvironment [180,184,218,[222][223][224][225][226]. MRP1 and MRP2 are apparently upregulated and clearly expressed in epilepsy [227][228][229]. ...
This review considers efflux of substances from brain parenchyma quantified as values of clearances (CL, stated in µL g⁻¹ min⁻¹). Total clearance of a substance is the sum of clearance values for all available routes including perivascular pathways and the blood–brain barrier. Perivascular efflux contributes to the clearance of all water-soluble substances. Substances leaving via the perivascular routes may enter cerebrospinal fluid (CSF) or lymph. These routes are also involved in entry to the parenchyma from CSF. However, evidence demonstrating net fluid flow inwards along arteries and then outwards along veins (the glymphatic hypothesis) is still lacking. CLperivascular, that via perivascular routes, has been measured by following the fate of exogenously applied labelled tracer amounts of sucrose, inulin or serum albumin, which are not metabolized or eliminated across the blood–brain barrier. With these substances values of total CL ≅ 1 have been measured. Substances that are eliminated at least partly by other routes, i.e. across the blood–brain barrier, have higher total CL values. Substances crossing the blood–brain barrier may do so by passive, non-specific means with CLblood-brain barrier values ranging from < 0.01 for inulin to > 1000 for water and CO2. CLblood-brain barrier values for many small solutes are predictable from their oil/water partition and molecular weight. Transporters specific for glucose, lactate and many polar substrates facilitate efflux across the blood–brain barrier producing CLblood-brain barrier values > 50. The principal route for movement of Na⁺ and Cl⁻ ions across the blood–brain barrier is probably paracellular through tight junctions between the brain endothelial cells producing CLblood-brain barrier values ~ 1. There are large fluxes of amino acids into and out of the brain across the blood–brain barrier but only small net fluxes have been observed suggesting substantial reuse of essential amino acids and α-ketoacids within the brain. Amyloid-β efflux, which is measurably faster than efflux of inulin, is primarily across the blood–brain barrier. Amyloid-β also leaves the brain parenchyma via perivascular efflux and this may be important as the route by which amyloid-β reaches arterial walls resulting in cerebral amyloid angiopathy.
Electronic supplementary material
The online version of this article (10.1186/s12987-018-0113-6) contains supplementary material, which is available to authorized users.
... 36 ABCG2 and ABCCs proteins expressions also increased in epileptic patients' brains, but in contrast to ABCB1, both transporters seem to have a low affinity for AEDs. 37 Early evidence obtained from epileptic patients undergoing cortical resection, demonstrated abnormal expression of the glucose transporter SLC2A1 (GLUT1). Immunogold imaging of BBB GLUT1 performed on tissue resected from patients with seizures provided evidence that GLUT1 is downregulated in the endothelial cells in regions within and around the seizure focus. ...
Membrane transport proteins have central physiological function in maintaining cerebral homeostasis. These transporters are expressed in almost all cerebral cells where they regulate the movement of wide range of solutes including endogenous substrates, xenobiotic and therapeutic drugs. Altered activity/expression of CNS transporters has been implicated in the onset and progression of multiple neurological diseases. Neurological diseases are heterogeneous diseases that involve complex pathological alterations with only a few treatment options; therefore there is a great need for the development of novel therapeutic treatments. To that end, transporters have emerged recently to be promising therapeutic targets to halt or slow the course of neurological diseases. The objective of this review is to discuss implications of transporters in neurological diseases and summarize available evidence for targeting transporters as decent therapeutic approach in the treatment of neurological diseases. This article is protected by copyright. All rights reserved.
... There is evidence that genetic variation in P-gp influences treatment response to antipsychotic and antiepileptic drugs such as olanzapine, risperidone, paliperidone (9- hydroxy risperidone, the active metabolite of risperidone), phenobarbital and phenytoin (French, 2013; Wolking et al., 2015). Furthermore, P-gp and Bcrp are upregulated at the BBB in epilepsy and schizophrenia and contribute to pharmacoresistance by limiting the brain uptake and efficacy of anticonvulsant and antipsychotic drugs (Aronica et al., 2005; Bauer et al., 2014; De Klerk et al., 2010; Lazarowski et al., 2007; Van Vliet et al., 2005). Drugs that are not substrates of ABC transporters will then make better therapeutics, as they will be immune to drug resistance mediated by these proteins. ...
Cannabidiol (CBD) is currently being investigated as a novel therapeutic for the treatment of CNS disorders like schizophrenia and epilepsy. ABC transporters such as P-glycoprotein (P-gp) and breast cancer resistance protein (Bcrp) mediate pharmacoresistance in these disorders. P-gp and Bcrp are expressed at the blood brain barrier (BBB) and reduce the brain uptake of substrate drugs including various antipsychotics and anticonvulsants. It is therefore important to assess whether CBD is prone to treatment resistance mediated by P-gp and Bcrp. Moreover, it has become common practice in the drug development of CNS agents to screen against ABC transporters to help isolate lead compounds with optimal pharmacokinetic properties. The current study aimed to assess whether P-gp and Bcrp impacts the brain transport of CBD by comparing CBD tissue concentrations in wild-type (WT) mice versus mice devoid of ABC transporter genes. P-gp knockout (
Abcb1a/b
−∕−
), Bcrp knockout (
Abcg2
−∕−
), combined P-gp/Bcrp knockout (
Abcb1a/b
−∕−
Abcg2
−∕−
) and WT mice were injected with CBD, before brain and plasma samples were collected at various time-points. CBD results were compared with the positive control risperidone and 9-hydroxy risperidone, antipsychotic drugs that are established ABC transporter substrates. Brain and plasma concentrations of CBD were not greater in P-gp, Bcrp or P-gp/Bcrp knockout mice than WT mice. In comparison, the brain/plasma concentration ratios of risperidone and 9-hydroxy risperidone were profoundly higher in P-gp knockout mice than WT mice. These results suggest that CBD is not a substrate of P-gp or Bcrp and may be free from the complication of reduced brain uptake by these transporters. Such findings provide favorable evidence for the therapeutic development of CBD in the treatment of various CNS disorders.
... Altered expression of other ABC transporters has also been reported in the literature. For example, enhanced ABCG2 protein expression has been observed in a rat model of status epilepticus [202]. Among MRP isoforms, MRP1 expression has been detected in several brain abnormalities characteristic of refractory epilepsy including dysplastic neurons and reactive astrocytes [203]. ...
Pharmacotherapy of central nervous system (CNS) disease remains difficult due to limited drug permeation across the blood-brain barrier (BBB) and blood-cerebrospinal fluid barrier (BCSFB). In addition, brain parenchymal cellular compartments play an important role in regulating CNS drug distribution. This chapter reviews the expression, localization, and functional activity of drug transporters at brain barriers and in brain cellular compartments in normal physiological and pathological conditions. It discusses organic anion-transporting polypeptides (OATPs), organic anion transporters (OATs), organic cation transporters (OCTs), nucleoside transporters (NTs), peptide transporters (PTs), and monocarboxylate transporters (MCTs). The chapter describes relevance of drug transporters in CNS disorders, including brain tumors, epilepsy, neurodegenerative diseases and pain. Nuclear receptors have also been implicated in the regulation of other ATP-binding cassette (ABC) transporters. The dynamic and highly controlled environment of the brain is regulated, in part, by the brain barrier.
... Repeated abnormal discharges of the neurons in the brain and epileptic seizures are considered the major induction factor of a high MRP expression. For example, studies on rat in the status epilepticus have shown that the expression of MRP1 and MRP2 in neurons in the hippocampus and surrounding cortex (29), vascular endothelial cells as well as astrocyte increased significantly. The long-term intervention treatment of certain anti-epileptic drugs, such as oxcarbazepine, may also induce the expression of MRP1 in rat (30). ...
Intactable epilepsy (IE) is relatively common in pediatric epilepsy. The resistance mechanism of IE has been previously investigated. Multidrug‑resistant associated protein 1 (MRP1) and MRP2 are associated with drug transport. The aim of the present study was to investigate the expression of MRP1 and MRP2 in peripheral blood mononuclear cells of children with IE. Fifty outpatient or inpatient children were included in the study as the experimental group. Additionally, 50 children with epilepsy controlled by anti‑epileptic drugs (AEDs) and 50 healthy children without epilepsy, who served as the control group, were included in the present study. Expression of MRP1 and MRP2 in the peripheral blood mononuclear cells of children in all the groups was detected using RT-PCR and western blot analysis. The results showed that the relative expression of MRP1 and MRP2 mRNA in the peripheral blood mononuclear cells of children with IE (MRP1, 0.795±0.042; MRP2, 0.804±0.023) was higher than that in epilepsy controlled by AEDs (MRP1, 0.682±0.030; MRP2, 0.675±0.021) and healthy children without epilepsy (MRP1, 0.665±0.031; MRP2, 0.654±0.029) (P<0.01). The mean relative expression of MRP1 and MRP2 protein in the peripheral blood mononuclear cells of children with IE (MRP1, 2.027±0.034; MRP2, 1.902±0.021) was higher than that in children with epilepsy controlled by AEDs (MRP1, 1.131±0.042; MRP2, 1.086±0.027) and healthy children without epilepsy (MRP1, 1.093±0.023; MRP2, 1.045±0.018) (P<0.01). The difference in the MRP1 and MRP2 mRNA and protein expression between the children with epilepsy controlled by AEDs and healthy children without epilepsy was not statistically significant (P>0.05). In conclusion, a higher expression of MRP1 and MRP2 in the peripheral blood mononuclear cells of children with IE may be relevant to the drug-resistant mechanism of IE.
... The energetics driving drug uptake or effl ux mediated by these transporters will be discussed below, as it is relevant to the role these transporters play in drug disposition. (van Vliet et al., 2005 ;Kubota et al., 2006 ). The upregulation of MRP2 in the blood -brain barrier in patients with epilepsy may have implications toward treatment of epilepsy with pharmacotherapy. ...
IntroductionABC TransportersSLC TransportersIn vitro Assays in Drug DevelopmentConclusions and PerspectivesReferences
... (Dallas et al. 2006). There is also evidence that supports the idea that a number of MRPs are expressed at such low levels under basal conditions that they are virtually undetectable and it is only upon pathogenic stimulus that they can be detected by current molecular techniques (Aronica et al. 2005;Hoffmann and Loscher 2007). ...
ATP-binding cassette (ABC) transporters play a crucial role for the efflux of a wide range of substrates across different cellular membranes. In the central nervous system (CNS), ABC transporters have recently gathered significant attention due to their pivotal involvement in brain physiology and neurodegenerative disorders, such as Alzheimer’s disease (AD). Glial cells are fundamental for normal CNS function and engage with several ABC transporters in different ways. Here, we specifically highlight ABC transporters involved in the maintenance of brain homeostasis and their implications in its metabolic regulation. We also show new aspects related to ABC transporter function found in less recognized diseases, such as Huntington’s disease (HD) and experimental autoimmune encephalomyelitis (EAE), as a model for multiple sclerosis (MS). Understanding both their impact on the physiological regulation of the CNS and their roles in brain diseases holds promise for uncovering new therapeutic options. Further investigations and preclinical studies are warranted to elucidate the complex interplay between glial ABC transporters and physiological brain functions, potentially leading to effective therapeutic interventions also for rare CNS disorders.
This chapter is devoted to drug-resistant epilepsy and its genetic mechanisms. There are currently six hypotheses proposed for pharmacoresistant epilepsy. The genetic aspects of five of the six hypotheses are addressed in this chapter except from the intrinsic hypothesis as it does not comprise genetic mechanisms.In the end, we propose two assertions in the definition of genetic “drug-resistant” epilepsies: (1) when the antiseizure medication (ASM) has a proven effect on the molecular lesion, but seizures persist in spite of ASM treatment; this latter, we believe is true genetic pharmacorresistant epilepsy and (2) epilepsies are drug resistant because the antiseizure drug ASM does not have an effect on the specific molecular lesion of the epilepsy syndrome. The epilepsy is supposedly “drug resistant,” but seizures do not stop because “the key does not fit the lock in the door”.In diagnosis poor response to treatment, it is also important to consider that incorrect diagnosis in some epilepsies can also lead to “pseudo-drug resistance,” where mistreatment can lead to poor response or aggravation of seizures, this happens more frequently in genetic epilepsies.KeywordsGenesDrug-resistant epilepsyEpilepsyHypothesis, PharmacogenomicsGenetic Epilepsies
The blood-brain barrier is essential for maintaining the stability of the central nervous system and is also crucial for regulating drug metabolism, changes of blood-brain barrier's structure and function can influence how drugs are delivered to the brain. In high-altitude hypoxia, the central nervous system's function is drastically altered, which can cause disease and modify the metabolism of drugs in vivo. Changes in the structure and function of the blood-brain barrier and the transport of the drug across the blood-brain barrier under high-altitude hypoxia, are regulated by changes in brain microvascular endothelial cells, astrocytes, and pericytes, either regulated by drug metabolism factors such as drug transporters and drug-metabolizing enzymes. This article aims to review the effects of high-altitude hypoxia on the structure and function of the blood-brain barrier as well as the effects of changes in the blood-brain barrier on drug metabolism. We also hypothesized and explore the regulation and potential mechanisms of the blood-brain barrier and associated pathways, such as transcription factors, inflammatory factors, and nuclear receptors, in regulating drug transport under high-altitude hypoxia.
Adenosine-triphosphate-(ATP)-binding cassette (ABC) transport proteins are ubiquitously present membrane-bound efflux pumps that distribute endo- and xenobiotics across intra- and intercellular barriers. Discovered over 40 years ago, ABC transporters have been identified as key players in various human diseases, such as multidrug-resistant cancer and atherosclerosis, but also neurodegenerative diseases, such as Alzheimer’s disease (AD). Most prominent and well-studied are ABCB1, ABCC1, and ABCG2, not only due to their contribution to the multidrug resistance (MDR) phenotype in cancer, but also due to their contribution to AD. However, our understanding of other ABC transporters is limited, and most of the 49 human ABC transporters have been largely neglected as potential targets for novel small-molecule drugs. This is especially true for the ABCA subfamily, which contains several members known to play a role in AD initiation and progression. This review provides up-to-date information on the proposed functional background and pathological role of ABCA transporters in AD. We also provide an overview of small-molecules shown to interact with ABCA transporters as well as potential in silico, in vitro, and in vivo methodologies to gain novel templates for the development of innovative ABC transporter-targeting diagnostics and therapeutics.
The function of ATP-binding cassette (ABC) transporters at the blood-brain barrier (BBB) is to protect the brain from toxic compounds. Additionally, they play a crucial role in the onset and progression of several central nervous system (CNS) diseases as well as in drug resistance. Many compounds were identified as substrates, inhibitors, inducers, or activators for ABC transporters, causing important drug-drug interactions. PET imaging represents an excellent tool for assessing the function and expression of ABC transporters. Over the last years, many PET tracers with different characteristics have been developed, mainly for measuring P-glycoprotein (P-gp) function at the BBB. Although (R)-[11C]verapamil or [11C]N-desmethyl-loperamide are considered as the “gold standard” P-gp tracers, they have several drawbacks such as its high affinity to P-gp which limits its use for assessing P-gp increased function. Therefore, PET tracers with lower affinity to the transporter have been developed and studied in different species. The assessment of ABC transporters by PET imaging can provide new insight into the physiology and pathophysiology of different CNS diseases and may open new avenues for therapies. Moreover, PET can be used for screening the affinity of new entities toward various ABC transporters and thus enhance the development of CNS drugs.
Blood‐brain barrier dysfunction in epilepsy contributes to seizures and resistance to antiseizure drugs. Reports show that seizures increase brain glutamate levels, leading to barrier dysfunction. One component of barrier dysfunction is overexpression of the drug efflux transporters P‐glycoprotein (P‐gp) and breast cancer resistance protein (BCRP). Based on our previous studies, we hypothesized that glutamate released during seizures activates cytosolic phospholipase A2 (cPLA2), resulting in P‐gp and BCRP overexpression. We exposed isolated rat brain capillaries to glutamate ex vivo and used an in vivo‐ex vivo approach of isolating brain capillaries from rats after status epilepticus (SE) and in chronic epileptic (CE) rats. Glutamate increased cPLA2, P‐gp, and BCRP protein and activity levels in isolated brain capillaries. We confirmed the role of cPLA2 in the signaling pathway in brain capillaries from male and female mice lacking cPLA2. We also demonstrated, in vivo, that cPLA2 inhibition prevents overexpression of P‐gp and BCRP at the blood‐brain barrier in rats after status epilepticus and in CE rats. Our data support the hypothesis that glutamate signals cPLA2 activation, resulting in overexpression of blood‐brain barrier P‐gp and BCRP.—Hartz, A. M. S., Rempe, R. G., Soldner, E. L. B., Pekcec, A., Schlichtiger, J., Kryscio, R., Bauer, B. Cytosolic phospholipase A2 is a key regulator of blood‐brain barrier function in epilepsy. FASEB J. 33, 14281‐14295 (2019). www.fasebj.org
Up-regulation of efflux transporters in brain capillaries may lead to the decreased therapeutic efficacy of antiepileptic drugs in patients with Drug Resistant Epilepsy. Adenosine receptor activation in brain capillaries can modulate blood-brain barrier permeability by decreasing the protein levels and function of efflux transporters. Therefore, we aimed to investigate whether the activation of adenosine receptors improves convulsions outcome in carbamazepine (CBZ) resistant animals and modulates the protein levels of efflux transporters (P-GP, MRP1, MRP2) in brain capillaries. We employed the window-pentylenetetrazol (PTZ) kindling model to develop CBZ resistant rats by CBZ administration during the post-kindling phase, and tested if these animals displayed subsequent resistance to other antiepileptic drugs. Crucially, we investigated if the administration of a broad-spectrum adenosine agonist (NECA) improves convulsions control in CBZ resistant rats. Of potential therapeutic relevance, in CBZ resistant rats NECA restored the anticonvulsant effect of CBZ. We also evaluated how the resistance to CBZ and the activation of adenosine receptors with NECA affect protein levels of efflux transporters in brain capillaries, as quantified by western blot. While CBZ resistance was associated with the up-regulation of both P-GP/MRP2 in brain capillaries, with the administration of NECA in CBZ resistant rats, we observed a decrease of P-GP and an increase of MRP2 levels, in brain capillaries. Since the activation of adenosine receptors improves the outcome of convulsions probably through the modulation of the efflux transporters protein levels in brain capillaries, adenosine agonists could be useful as an adjunct therapy for the control of Drug Resistant Epilepsy.
Epilepsy is a neurological disorder affecting around 1%-2% of population worldwide and its treatment includes use of antiepileptic drugs to control seizures. Failure to respond to antiepileptic drug therapy is a major clinical problem and over expression of ATP-binding cassette transporters is considered one of the major reasons for pharmacoresistance. In this review, we have summarized the regulation of ABC transporters in response to oxidative stress due to disease and antiepileptic drugs. Further, ketogenic diet and antioxidants were examined for their role in pharmacoresistance. The understanding of signalling pathways and mechanism involved may help in identifying potential therapeutic targets and improving drug response.
In the first part of the study, the authors gave an overview of the concept of therapy-resistance and of the mechanism of bacterial antibiotic resistance, and describe the function and structure of "efflux-pumps". In the beginning of the second part, they discuss the role of P glycoprotein (one of the most important efflux-pumps) in the therapy-resistance of canine neoplastic diseases. They give an overview of some diseases (epilepsy, autoimmune diseases), in which the role of efflux-pumps in therapy resistance had already been proven. After summarising other factors of therapy-resistance, they describe some methods for in vivo and in vitro detection of efflux-pumps, and in connection with this, they mention different pump function inhibitors. The clinical use of these drugs in the future may lead to a breakthrough in the treatment of cancer and some non-neoplastic diseases.
Membrane transporters play a pivotal role in many organs to maintain their normal physiological functions and contribute significantly to drug absorption, distribution and elimination. Knowledge gained from gene modified animal models or human genetic disorders has demonstrated that interruption of the transporter activity can lead to debilitating diseases or organ toxicities. Herein we describe transporter associated diseases and organ toxicities resulting from transporter gene deficiency or functional inhibition in the liver, kidney, gastrointestinal tract (GIT) and central nervous system (CNS). While proposing additional transporters as targets for drug-induced organ toxicity, strategies and future perspectives are discussed for transporter risk assessment in drug discovery and development.
Despite the launch of a series of new antiepileptic drugs, epilepsy in about one third of the patient population is considered to be pharmacoresistant. Thereby, pharmacoresistance can exist with onset of the disease, but can also develop with progression of epilepsy. The analysis of the underlying mechanisms promises findings that can be used for the development of new antiepileptic drugs, and can be used for the development of new therapeutic strategies to overcome pharmacoresistance. Research during the last years indicates that multidrug transporters at the blood-brain barrier are overexpressed in pharmacoresistant patients and can mediate efflux of antiepileptic drugs, thereby restricting their efficacy. Furthermore epilepsy-associated changes have been detected in antiepileptic drug target structures, which can limit the drug effect. Future investigations have to clarify, whether the findings can be used for the optimization of the therapeutic situation.
This chapter is devoted to resistance to antiepileptic drugs (AEDs) and its genetic mechanisms. There are three general hypothesis proposed for pharmacoresistant epilepsy: (1) Target hypothesis, (2) Drug transporter hypothesis, and the (3) Intrinsic Severity Hypothesis (Gorter and Potschka, Jasper’s basic mechanisms of the epilepsies, 4th ed. National Center for Biotechnology Information (USA), Bethesda, MD, 2012).
In diagnosing poor response to treatment, it is also important to separate drug resistance from incorrect diagnosis of epilepsy syndrome for example: (a) Epilepsy caused by mutations in Glucose transporter gene 1 (GLUT1) being treated with valproate (VPA) worsens the seizures in this disease whereas replacement of glucose with ketogenic diet alleviates seizures and the glucose deficit in the central nervous system. (Klepper, Epilepsia 49(Suppl 8):46–49, 2008; Klepper et al., Neuropediatrics 40(5):207–210, 2009) (b) Genetic or idiopathic epilepsies such as Childhood Absence Epilepsy (CAE), Juvenile Myoclonic Epilepsy (JME) and Dravet’s Syndrome can be aggravated when treated with Na+ channel blockers (Genton, Brain Dev 22(2):75–80, 2000; Guerrini et al., Epilepsia 39(5):508–512, 1998; Thomas et al., Brain 129(Pt 5):1281–1292, 2006; Martínez-Juárez et al., Brain 129(Pt 5):1269–1280, 2006) and (c) Mitochondrial disorders can also be aggravated by VPA (Finsterer and Zarrouk Mahjoub, Expert Opin Drug Metab Toxicol 8(1):71–79, 2012).
Herein, we describe the three general hypothesis; we also summarize the “difficult to treat” genetic epilepsies.
This chapter focuses on two factors possibly contributing to the poor translatability of the animal models of central nervous system (CNS) disorders, namely the species variability in CNS drug disposition and the effect of disease states. The blood–brain barrier (BBB) is one of the most important and most studied blood-CNS interfaces. One might add species difference in BBB transporter activity as a potential factor. Species differences in BBB properties are not the only hurdle affecting the translatability of the animal models for CNS disorders. The modulation of BBB transporter activities in disease states is expected to impact on the brain disposition of actively transported drugs. A change in a single BBB parameter might overall have a rather limited influence on CNS drug disposition, as suggested for brain P-gp activity. Efforts should be made to incorporate all these variables into more predictive physiologically based pharmacokinetic (PBPK) modeling.
Breast cancer resistance protein (BCRP) functions as a major molecular gatekeeper at the blood-brain barrier. Considering its impact on access to the brain by therapeutic drugs and harmful xenobiotics, it is of particular interest to elucidate the mechanisms of its regulation. Excessive glutamate concentrations have been reported during epileptic seizures or as a consequence of different brain insults including brain ischemia. Previously, we have demonstrated that glutamate can trigger an induction of the transporter P-glycoprotein. These findings raised the question whether other efflux transporters are affected in a comparable manner. Glutamate exposure proved to down-regulate BCRP transport function and expression in isolated porcine capillaries. The reduction was efficaciously prevented by coincubation with N-methyl-d-aspartate (NMDA) receptor antagonist MK-801. The involvement of the NMDA receptor in the down-regulation of BCRP was further confirmed by experiments showing an effect of NMDA exposure on brain capillary BCRP transport function and expression. Pharmacological targeting of cyclooxygenase-1 and -2 (COX-1 and -2) using the nonselective inhibitor indomethacin, COX-1 inhibitor SC-560, and COX-2 inhibitor celecoxib revealed a contribution of COX-2 activity to the NMDA receptor's downstream signaling events affecting BCRP. Translational studies were performed using human capillaries isolated from surgical specimens of epilepsy patients. The findings confirmed a glutamate-induced down-regulation of BCRP transport activity in human capillaries, which argued against major species differences. In conclusion, our data reveal a novel mechanism of BCRP down-regulation in porcine and human brain capillaries. Moreover, together with previous data sets for P-glycoprotein, the findings point to a contrasting impact of the signaling pathway on the regulation of BCRP and P-glycoprotein. The effect of glutamate and arachidonic acid signaling on BCRP function might have implications for brain drug delivery and for radiotracer brain access in epilepsy patients and patients with other brain insults.
Overexpression of ATP-binding cassette (ABC) transporters may contribute to intractable epilepsy (IE) by reducing brain accumulation of antiepileptic drugs (AEDs). We conducted a meta-analysis of studies on expression and cellular distribution of multidrug resistance-associated protein 1 (MRP1) in IE patients to evaluate the contribution of this protein to AED resistance. In addition, we summarize experiments examining MRP1 expression and substrates in animal models of IE.
The literature search, based on pre-established inclusion and exclusion criteria, as well as quality assessment, data extraction, and statistical analyses were conducted concurrently by two independent researchers. We identified 9 high-quality studies (Jadad score ≥3) published between 2000 to 2014 on the expression and cellular distribution of MRP1 in IE patients. A fixed effect model was used to calculate pooled odds ratios (ORs) and corresponding 95% confidence intervals (95%CIs). Forest and funnel plots were constructed to assess study heterogeneity and publication bias, respectively.
MRP1 expression was significantly higher in both astrocytes (OR = 17.04, 95%CI: 7.69-37.76, P<0.00001) and neurons (OR = 22.13, 95%CI: 8.52-57.46, P<0.00001) of IE patients compared to controls, while there was no significant difference in endothelial cell MRP1 expression (OR: 1.47, 95%CI: 0.09-1.79, P = 0.48). Funnel plot symmetry indicated no substantial publication bias. Most relevant preclinical studies from 2000-2014 found higher MRP1 expression in IE model rodents. Furthermore, MRP1 overexpression reduced the extracellular concentration of AEDs in brain, while MRP1 inhibitors enhanced brain AED concentrations.
Pooled results strongly suggest that MRP1 is overexpressed in both neurons and astrocytes of IE patients. Inhibition of MRP1 may enhance AED efficacy by increasing local drug availability.
As a member of the multidrug-resistance associated protein family MRP2 affects the brain entry of different endogenous and exogenous compounds. Considering the role of this transporter at the blood-brain barrier the regulation is of particular interest. However, there is limited knowledge regarding the factors that regulate MRP2 in neurological disease states. Thus, we addressed the hypothesis that MRP2 might be affected by a glutamate-induced signaling pathway that we previously identified as one key mechanism in the regulation of P-glycoprotein. Studies in isolated porcine brain capillaries confirmed that glutamate and NMDA exposure up-regulates expression and function of MPR2. The involvement of the NMDA receptor was further suggested by the fact that the NMDA receptor antagonist MK-801 as well as the NMDA receptor glycine binding site antagonist L-701,324 prevented the impact of glutamate. A role of cyclooxygenase-2 was indicated by co-incubation with the cyclooxygenase-2 inhibitor celecoxib and the cyclooxygenase-1/-2 inhibitor indomethacin, which both efficaciously abolished a glutamate-induced up-regulation of MRP2. Translational studies in human capillaries from surgical specimen demonstrated a relevant MRP2 efflux function, and indicated an effect of glutamate exposure as well as its prevention by cyclooxygenase-2 inhibition. Taken together the findings provide first evidence for a role of a glutamate-induced NMDA receptor/cyclooxygenase-2 signaling pathway in the regulation of MRP2 expression and function. The response to excessive glutamate concentrations might contribute to overexpression of MRP2, which has been reported in neurological diseases including epilepsy. The overexpression might have implications for brain access of various compounds including therapeutic drugs.
The American Society for Pharmacology and Experimental Therapeutics.
Drug permeability in the central nervous system (CNS) across blood-brain and blood-cerebrospinal fluid barriers is an important determinant of neurological disorders therapeutic efficacy and is highly regulated by the expression of membrane-associated transporters belonging to the ATP-binding cassette (ABC) and solute carrier (SLC) superfamilies. Functional expression of ABC efflux transporters exists not only at the brain barriers (primary biochemical barrier) but also in astrocytes, microglia, neurons, and oligodendrocytes can significantly restrict drug penetration into these cells, thus creating a secondary biochemical barrier to permeability in brain parenchyma. In contrast, SLC members primarily contribute to the uptake of endogenous substrates (i.e., hormones, neurotransmitters) and pharmacological agents and can play a critical role in maintaining CNS homeostasis and drug response. In this chapter, we review the functional expression and localization of drug transporters in the brain, their role in CNS drug delivery, and their regulation in neuropathological conditions.
Multidrug resistance-associated protein (MRP) is a recently identified drug efflux transport system that actively transports organic acids and selected glucuronide or glutathione conjugates out of the cell. The current study presents, for the first time, both functional and biochemical data demonstrating the presence of MRP in the brain microvessel endothelial cells that form the blood-brain barrier (BBB). Using known MRP inhibitors, such as indomethacin and probenecid, fluorescein accumulation in primary cultured bovine brain microvessel endothelial cell (BBMEC) monolayers was significantly enhanced compared to control. The specificity of the MRP inhibitors on cellular fluorescein accumulation was confirmed using both MRP positive (Panc-1) and MRP negative (KBv) cell lines. Furthermore, western blot analysis using a specific antibody for MRP (MRPm6) and RT-PCR studies using a complementary sequence probe for human MRP demonstrate the expression of MRP in BBMEC. Previous studies have demonstrated the significance of the P-glycoprotein drug efflux transporter in the BBB. Given its function as a drug efflux transport system, it is anticipated that MRP in the BBB will also have an important role in limiting the exposure of the brain to many endogenous and exogenous compounds, including both toxic and therapeutic agents.
The multidrug resistance-associated protein (MRP) mediates the cellular excretion of many drugs, glutathione S-conjugates (GS-X) of lipophilic xenobiotics and endogenous cysteinyl leukotrienes. Increased MRP levels in tumor cells can cause multidrug resistance (MDR) by decreasing the intracellular drug concentration. The physiological role or roles of MRP remain ill-defined, however. We have generated MRP-deficient mice by using embryonic stem cell technology. Mice homozygous for the mrp mutant allele, mrp-/-, are viable and fertile, but their response to an inflammatory stimulus is impaired. We attribute this defect to a decreased secretion of leukotriene C4 (LTC4) from leukotriene-synthesizing cells. Moreover, the mrp-/- mice are hypersensitive to the anticancer drug etoposide. The phenotype of mrp-/- mice is consistent with a role for MRP as the main LTC4-exporter in leukotriene-synthesizing cells, and as an important drug exporter in drug-sensitive cells. Our results suggest that this ubiquitous GS-X pump is dispensable in mice, making treatment of MDR with MRP-specific reversal agents potentially feasible.
The mrp (multidrug resistance protein) gene has been associated with the multidrug resistance of cancer cells in vitro and in vivo. To gain information on its physiological role, embryonic stem cells were used to generate mice homozygous for a disruption of the mrp gene, resulting in complete abrogation of mrp expression. No physiological abnormalities were observed, at least up to 4 months of age. Viability, fertility, and a range of histological, hematological, and serum-chemical parameters were similar in mrp(+/+) and mrp(-/-) mice. mrp(-/-) mice displayed an increased sensitivity to etoposide phosphate (2-fold) accompanied by greater bone marrow toxicity, whereas the acute toxicity of sodium arsenite was equivalent in mrp(+/+) and mrp(-/-) mice. Tissue levels of glutathione (GSH) were elevated in breast, lung, heart, kidney, muscle, colon, testes, bone marrow cells, blood mononuclear leukocytes, and blood erythrocytes of mrp(-/-) mice and were unchanged in organs known to express little if any mrp, such as the liver and small intestine. The increase in GSH was not due to an increase in the activity of gamma-glutamylcysteine synthetase, the rate-limiting enzyme for GSH synthesis. The findings demonstrate that mrp is dispensable for development and growth but exerts a role in drug detoxification and GSH metabolism.
Different sampling techniques for collecting nasal mucus have been used. The eosinophil and its granule toxin proteins, especially the eosinophil cationic protein (ECP), is an important inflammatory cell.
In the healthy subjects (with no acute or chronic disease of the respiratory tract), no detailed quantitative data on mucus nasal proteins have been reported, so the aim of the study is to demonstrate a simple and reliable method for the collection of nasal mucus and for measuring nasal ECP.
Our method consists in collecting two specimens of mucus from each nasal fossa, using a square of sterile pre-humidified gauze, centrifuged at 2000g for 20 min and stored at -20 degrees C until tested. To evaluate the reproducibility of the technique, 30 healthy subjects were retested after 24 h.
The amounts of secretion gathered in the 89 collections did not show any statistical difference between the sexes. In the pooled subjects (n=59), the mean levels of ECP were 108 ng/mL and 325 ng/g and total protein (TP) levels were 701 mg/dL and 2.5 mg/g (expressed in absolute concentration and concentration related to the weight in grams). In the classes, according to age, no statistical differences were found on analysis of the groups by: sex, recovery rate, and ECP and TP concentrations. The Reproducibility Index was very good for the recovery rate (0.78), ECP (ng/mL=0.95, ng/g=0.83) and TP (mg/dL=0.89, mg/g=0.76).
With our method, the sample collections were taken with minimal stimulation of the mucosa and it was well tolerated by the subjects; the low known dilution factor is also adequate for analysing small quantities of mucus recovered, and the protein concentration can be measured as an absolute value.
Mechanisms of drug resistance other than P-glycoprotein are of increasing interest as the list of newly identified members of the ABC transport family has grown. We sought to characterize the phenotype of the newly discovered ABC transporter encoded by the mitoxantrone resistance gene, MXR, also known as ABCP1 or BCRP. The pharmacodynamics of mitoxantrone and 12 other fluorescent drugs were evaluated by confocal microscopy in four multidrug-resistant human colon (S1) and breast (MCF-7) cancer cell lines. We utilized two sublines, MCF-7 AdVp3000 and S1-M1-80, and detected overexpression of MXR by PCR, immunoblot assay and immunohistochemistry. These MXR overexpressing sublines were compared to cell lines with P-glycoprotein- and MRP-mediated resistance. High levels of cross-resistance were observed for mitoxantrone, the anthracyclines, bisantrene and topotecan. Reduced levels of mitoxantrone, daunorubicin, bisantrene, topotecan, rhodamine 123 and prazosin were observed in the two sublines with high MXR expression. Neither the P-glycoprotein substrates vinblastine, paclitaxel, verapamil and calcein-AM, nor the MRP substrate calcein, were extruded from MCF-7 AdVp3000 and S1-M1-80 cells. Thus, the multidrug-resistant phenotype due to MXR expression is overlapping with, but distinct from, that due to P-glycoprotein. Further, cells that overexpress the MXR protein seem to be more resistant to mitoxantrone and topotecan than cells with P-glycoprotein-mediated multidrug resistance. Our studies suggest that the ABC half-transporter, MXR, is a potent, new mechanism for conferring multiple drug resistance. Definition of its mechanism of transport and its role in clinical oncology is required.
Tumor cells may display a multidrug resistant phenotype by overexpression of ATP-binding cassette transporters such as multidrug resistance (MDRI) P-glycoprotein, multidrug resistance protein 1 (MRP1), and breast cancer resistance protein (BCRP). The presence of BCRP has thus far been reported solely using mRNA data. In this study, we describe a BCRP-specific monoclonal antibody, BXP-34, obtained from mice, immunized with mitoxantrone-resistant, BCRP mRNA-positive MCF-7 MR human breast cancer cells. BCRP was detected in BCRP-transfected cells and in several mitoxantrone- and topotecan-selected tumor cell sublines. Pronounced staining of the cell membranes showed that the transporter is mainly present at the plasma membrane. In a panel of human tumors, including primary tumors as well as drug-treated breast cancer and acute myeloid leukemia samples, BCRP was low or undetectable. Extended studies will be required to analyze the possible contribution of BCRP to clinical multidrug resistance.
4-Hydroxynonenal (4HNE) is the most prevalent toxic lipid peroxidation product formed during oxidative stress. It exerts its cytotoxicity mainly by the modification of intracellular proteins. The detection of 4HNE-modified proteins in several degenerative disorders suggests a role for 4HNE in the onset of these diseases. Efficient protection mechanisms are required to prevent the intracellular accumulation of 4HNE. The toxicity of 4HNE was tested with the small cell lung cancer cell lines GLC(4) and the multidrug-resistance-protein (MRP1)-overexpressing counterpart GLC(4)/Adr. In the presence of the MRP1 inhibitor MK571 or the GSH-depleting agent buthionine sulphoximine, both cell lines became more sensitive and showed decreased survival. Transport experiments were performed with the (3)H-labelled glutathione S-conjugate of 4HNE ([(3)H]GS-4HNE) with membrane vesicles from GLC(4)-derived cell lines with different expression levels of MRP1. [(3)H]GS-4HNE was taken up in an ATP-dependent manner and the transport rate was dependent on the amount of MRP1. The MRP1 inhibitor MK571 decreased [(3)H]GS-4HNE uptake. MRP1-specific [(3)H]GS-4HNE transport was demonstrated with membrane vesicles from High Five insect cells overexpressing recombinant MRP1. Kinetic experiments showed an apparent K(m) of 1.6+/-0.21 microM (mean+/-S.D.) for MRP1-mediated [(3)H]GS-4HNE transport. In conclusion, MRP1 has a role in the protection against 4HNE toxicity and GS-4HNE is a novel MRP1 substrate. MRP1, together with GSH, is hypothesized to have a role in the defence against oxidative stress.
Tumor cells may display a multidrug resistance phenotype by overexpression of ATP binding cassette transporter genes such as multidrug resistance (MDR) 1 P-glycoprotein (P-gp) or the multidrug resistance protein 1 (MRP1). MDR3 P-gp is a close homologue of MDR1 P-gp, but its role in MDR is probably minor and remains to be established. The MRP1 protein belongs to a family of at least six members. Three of these, i.e., MRP1, MRP2, and MRP3, can transport MDR drugs and could be involved in MDR. The substrate specificity of the other family members remains to be defined. Specific monoclonal antibodies are required for wide-scale studies on the putative contribution of these closely related transporter proteins to MDR. In this report, we describe the extensive characterization of a panel of monoclonal antibodies (Mabs) detecting several MDR-related transporter proteins in both human and animal tissues. The panel consists of P3II-1 and P3II-26 for MDR3 P-gp; MRPr1, MRPm6, MRPm5, and MIB6 for MRP1; M2I-4, M2II-12, M2III-5 and M2III-6 for MRP2; M3II-9 and M3II-21 for MRP3; and M5I-1 and M5II-54 for MRP5. All Mabs in the panel appeared to be fully specific for their cognate antigens, both in Western blots and cytospin preparations, as revealed by lack of cross-reactivity with any of the other family members. Indeed, all Mabs were very effective in detecting their respective antigens in cytospins of transfected cell lines, whereas in flow cytometric and immunohistochemical analyses, distinct differences in reactivity and suitability were noted. These Mabs should become valuable tools in studying the physiological functions of these transporter proteins, in screening procedures for the absence of these proteins in hereditary metabolic (liver) diseases, and in studying the possible contributions of these molecules to MDR in cancer patients.
To identify specific transporters that drive xenobiotics from central nervous system to blood, the accumulation of fluorescent drugs was studied in isolated capillaries from rat and pig brain using confocal microscopy and quantitative image analysis. Luminal accumulation of daunomycin and of fluorescent derivatives of cyclosporine A (CSA) and ivermectin was concentrative, specific, and energy-dependent (inhibition by NaCN). Transport was reduced by PSC 833, ivermectin, verapamil, CSA, and vanadate, but not by leukotriene C(4) (LTC(4)), indicating the involvement of P-glycoprotein. Luminal accumulation of the fluorescent organic anions sulforhodamine 101 and fluorescein methotrexate was also concentrative, specific, and energy-dependent. LTC(4), chlorodinitrobenzene, and vanadate reduced transport of these compounds, but PSC 833 and verapamil did not, indicating the involvement of a multidrug resistance-associated protein (Mrp). Immunostaining localized P-glycoprotein and Mrp2 to the luminal surface of the capillary endothelium and quantitative polymerase chain reaction showed Mrp1 and Mrp2 expression. Finally, the HIV protease inhibitors saquinavir and ritonavir were potent inhibitors of transport mediated by both P-glycoprotein and Mrp. These results validate a new method for studying drug transport in isolated brain capillaries and implicate both P-glycoprotein and one or more members of the Mrp family in drug transport from central nervous system to blood.
High expression of the Breast Cancer Resistance Protein (BCRP) gene has been shown to be involved in resistance to chemotherapeutic drugs. Knowledge of the localization of BCRP protein in normal tissues may help unravel the normal function of this protein. Therefore, we characterized the tissue distribution and cellular localization of BCRP in frozen sections of normal human tissues. For this purpose, we used the recently described monoclonal antibody BXP-34 and another independently developed monoclonal antibody directed against BCRP, BXP-21. Both monoclonal antibodies show specific BCRP plasma membrane staining on cytospins obtained from topotecan- or mitoxantrone-selected cell lines, as well as from BCRP-transfected cell lines. Immunoprecipitation experiments using either BXP-21 or BXP-34 yielded a clear M(r) 72,000 BCRP band from BCRP-overexpressing tumor cells. In the topotecan-selected T8 and mitoxantrone-selected MX3 tumor cell lines, BCRP turned out to be differentially glycosylated. In contrast to BXP-34, BXP-21 is able to detect the M(r) 72,000 BCRP protein on immunoblots and is reactive with BCRP in formalin-fixed, paraffin-embedded tissues. Using BXP-21 and BXP-34, prominent staining of BCRP was observed in placental syncytiotrophoblasts, in the epithelium of the small intestine and colon, in the liver canalicular membrane, and in ducts and lobules of the breast. Furthermore, BCRP was present in veinous and capillary endothelium, but not in arterial endothelium in all of the tissues investigated. In the tissues studied, the mRNA levels of BCRP were assessed using reverse transcription-PCR, and these corresponded with the levels of BCRP protein estimated from immunohistochemical staining. The presence of BCRP at the placental syncytiotrophoblasts is consistent with the hypothesis of a protective role of BCRP for the fetus. The apical localization in the epithelium of the small intestine and colon indicates a possible role of BCRP in the regulation of the uptake of p.o. administered BCRP substrates by back-transport of substrate drugs entering from the gut lumen. Therefore, it may be useful to attempt to modulate the uptake of p.o. delivered BCRP substrates, e.g., topotecan or irinotecan, by using a BCRP inhibitor. Clinical trials testing this hypothesis have been initiated in our institute.
The causes and mechanisms underlying multidrug resistance (MDR) in epilepsy are still elusive and may depend on inadequate drug concentration in crucial brain areas. We studied whether limbic seizures or anticonvulsant drug treatments in rodents enhance the brain expression of the MDR gene (mdr) encoding a permeability glycoprotein (P-gp) involved in MDR to various cancer chemotherapeutic agents. We also investigated whether changes in P-gp levels affect anticonvulsant drug concentrations in the brain. Mdr mRNA measured by RT-PCR increased by 85% on average in the mouse hippocampus 3-24 hr after kainic acid-induced limbic seizures, returning to control levels by 72 hr. Treatment with therapeutic doses of phenytoin or carbamazepine for 7 d did not change mdr mRNA expression in the mouse hippocampus 1-72 hr after the last drug administration. Six hours after seizures, the brain/plasma ratio of phenytoin was reduced by 30% and its extracellular concentration estimated by microdialysis was increased by twofold compared with control mice. Knock-out mice (mdr1a/b -/-) lacking P-gp protein showed a 46% increase in phenytoin concentrations in the hippocampus 1 and 4 hr after injection compared with wild-type mice. A significant 23% increase was found in the cerebellum at 1 hr and in the cortex at 4 hr. Carbamazepine concentrations were measurable in the hippocampus at 3 hr in mdr1a/b -/- mice, whereas they were undetectable at the same time interval in wild-type mice. In rats having spontaneous seizures 3 months after electrically induced status epilepticus, mdr1 mRNA levels were enhanced by 1.8-fold and fivefold on average in the hippocampus and entorhinal cortex, respectively. Thus, changes in P-gp mRNA levels occur in limbic areas after both acute and chronic epileptic activity. P-gp alterations significantly affect antiepileptic drugs concentrations in the brain, suggesting that seizure-induced mdr mRNA expression contributes to MDR in epilepsy.
Delivery of drugs to the brain is impeded by the activity of efflux pumps expressed by endothelial cells of brain vasculature. The ATP binding cassette (ABC) transporters, among which ABCB1/MDR1 P-glycoprotein and ABCC1/multidrug resistance-associated protein 1 are expressed in brain endothelial cells, participate in drug efflux properties of the blood-brain barrier (BBB). Searches of the EST (expressed sequence tags) database with the conserved ABC domain, conducted to identify other ABC transporters expressed in the BBB, recovered 15 ABC transporter sequences expressed in human brain cDNA libraries. One of these sequences, identical to ABCG2, was highly expressed in cultured human cerebromicrovascular endothelial cells and human brain tissue at both mRNA and protein levels. Overexpression of human ABCG2 in immortalized rat brain endothelial cells resulted in enhanced polarized abluminal to luminal transport of various substrates tested in the in vitro BBB model. Brain vessels extracted from tissue sections of nonmalignant human brain and glioblastoma tumors by laser capture microdissection microscopy and analyzed by real-time polymerase chain reaction showed higher expression of ABCG2 relative to ABCB1/MDR1 and ABCC1/MRP1. ABCG2 was up-regulated in both glioblastoma vessels and parenchymal tissue. These studies suggest a role for brain endothelial ABCG2 transporter in modulating drug delivery to the brain and in conferring drug resistance to glioblastomas.
Active drug efflux transporters of the ATP binding cassette (ABC)-containing family of proteins have a major impact on the pharmacological behavior of most of the drugs in use today. Pharmacological properties affected by ABC transporters include the oral bioavailability, hepatobiliary, direct intestinal, and urinary excretion of drugs and drug-metabolites and -conjugates. Moreover, the penetration of drugs into a range of important pharmacological sanctuaries, such as brain, testis, and fetus, and the penetration into specific cell- and tissue compartments can be extensively limited by ABC transporters. These interactions with ABC transporters determine to a large extent the clinical usefulness, side effects and toxicity risks of drugs. Many other xenotoxins, (pre-)carcinogens and endogenous compounds are also influenced by the ABC transporters, with corresponding consequences for the well-being of the individual. We aim to provide an overview of properties of the mammalian ABC transporters known to mediate significant transport of clinically relevant drugs.
DRUG resistance in epilepsy due to malformations of cortical development (MCD) is unexplained. P-glycoprotein is a mediator of drug resistance, and we propose that MCD lesions over-express P-glycoprotein. Because P-glycoprotein expression may be induced by some antiepileptic drugs (AEDs), we studied brain samples from MCD cases before the onset of seizures or treatment with AEDs. Sixteen MCD cases and 16 age-matched controls were examined using immunohistochemistry. Glial labelling, representing over-expression, was seen in 10 of 16 MCD samples and in two of 16 control samples,(p=0.003). Semiquantitative assessment showed many immunoreactive glia in-five of 16 MCD and one of 16 controls. We conclude that there is constitutive over-expression of P-glycoprotein in many MCD. NeuroReport 10:3437-3441 (C) 1999 Lippincott Williams & Wilkins.
Limbic status epilepticus was induced in rats by unilateral 60-min electrical stimulation of the CA3 region of the ventral hippocampus. As assessed by RT-PCR followed by Southern blot analysis, transcripts of interleukin-1β, interleukin-6, interleukin-1 receptor antagonist and inducible nitric oxide synthase were significantly increased 2 h after status epilepticus in the stimulated hippocampus. Induction was maximal at 6 h for interleukin-1β (445%), interleukin-6 (405%) and tumour necrosis factor-α (264%) and at 24 h for interleukin-1 receptor antagonist (494%) and inducible nitric oxide synthase (432%). In rats with spontaneous seizures (60 days after status epilepticus), interleukin-1β mRNA was still higher than controls (241%). Immunocytochemical staining of interleukin-1β, interleukin-6 and tumour necrosis factor-α was enhanced in glia with a time-course similar to that of the respective transcripts. Sixty days after status epilepticus, interleukin-1β immunoreactivity was increased exclusively in neurons in one third of the animals. Multiple intracerebroventricular injections of interleukin-1 receptor antagonist (0.5 μg/3 μL) significantly decreased the severity of behavioural convulsions during electrical stimulation and selectively reduced tumour necrosis factor-α content in the hippocampus measured 18 h after status epilepticus. Thus, the induction of spontaneously recurring seizures in rats involves the activation of inflammatory cytokines and related pro- and anti-inflammatory genes in the hippocampus. These changes may play an active role in hyperexcitability of the epileptic tissue.
Why some patients with seizures are successfully treated with antiepileptic drugs (AEDs) and others prove medically intractable is not known. Inadequate intraparenchymal drug concentration is a possible mechanism of resistance to AEDs. The multiple drug resistance gene (MDRI) encodes P-glycoprotein, an energy-dependent efflux pump that exports planar hydrophobic molecules from the cell. If P-glycoprotein is expressed in brain of some patients with intractable epilepsy and AEDs are exported by P-glycoprotein, lower intraparenchymal drug concentrations could contribute to lack of drug response in such patients. Eleven of 19 brain specimens removed from patients during operation for intractable epilepsy had MDR1 mRNA levels > 10 times greater than those in normal brain, as determined by quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) method. Immunohistochemistry for P-glycoprotein from 14 of the patients showed increased staining in capillary endothelium in samples from epileptic patients as compared with staining in normal brain samples. In epileptic brain specimens with high MDR1 mRNA levels, expression of P-glycoprotein in astrocytes also was identified. Last, steady-state intracellular phenytoin (PHT) concentrations in MDR1 expressing neuroectodermal cells was one fourth that in MDR1-negative cells. MDR1 expression is increased in brain of some patients with medically intractable epilepsy, suggesting that the patients’ lack of response to medication may be caused by inadequate accumulation of AED in brain.
Reactive gliosis is a prominent morphological feature of mesial temporal lobe epilepsy. Because astrocytes express glutamate receptors, we examined changes in metabotropic glutamate receptor (mGluR) 2/3, mGluR5 and transforming growth factor (TGF)-β in glial cells of the hippocampal regions in an experimental rat model of spontaneous seizures. Rats that exhibited behavioural status epilepticus (SE) directly after 1 h of electrical angular bundle stimulation, displayed chronic spontaneous seizures after a latent period of 1–2 weeks as observed using continuous electrographic monitoring. SE resulted in hypertrophy of astrocytes and microglia activation throughout the hippocampus as revealed by immunolabelling studies. A dramatic, seizure intensity-dependent increase in vimentin immunoreactivity (a marker for reactive astrocytes) was revealed in CA3 and hilar regions where prominent neuronal loss occurs. Increased vimentin labelling was first apparent 24 h after onset of SE and persisted up to 3 months. mGluR2/3 and mGluR5 protein expression increased markedly in glial cells of CA3 and hilus by 1 week after SE, and persisted up to 3 months after SE. Double immunolabelling of brain sections with vimentin confirmed co-localization with glial fibrillary acidic protein (GFAP), mGluR2/3 and mGluR5 in reactive astrocytes. TGF-β, a cytokine implicated in mGluR3-mediated neuroprotection, was also upregulated during the first 3 weeks after SE throughout the hippocampus. This study demonstrates seizure-induced upregulation of two mGluR subtypes in reactive astrocytes, which − together with the increased production of TGF-β − may represent a novel mechanism for modulation of glial function and for changes in glial-neuronal communication in the course of epileptogenesis.
Purpose: It has been suggested that altered drug permeability across the blood–brain barrier (BBB) may be involved in pharmacoresistance to antiepileptic drugs (AEDs). To test this hypothesis further, we measured multiple drug resistance (MDR) gene expression in endothelial cells (ECs) isolated from temporal lobe blood vessels of patients with refractory epilepsy. ECs from umbilical cord or temporal lobe vessels obtained from aneurysm surgeries were used as comparison tissue.
Methods: cDNA arrays were used to determine MDR expression. MDR protein (MRP1) immunocytochemistry and Western blot analysis were used to confirm cDNA array data.
Results: We found overexpression of selected MDR and significantly higher P-glycoprotein levels in “epileptic” versus “control” ECs. Specifically, MDR1, cMRP/MRP2, and MRP5 were upregulated in epileptic tissue, whereas Pgp3/MDR3 levels were comparable to those measured in comparison tissue. The gene encoding cisplatin resistance–associated protein (hCRA-α) also was overexpressed in epileptic tissue. Immunocytochemical analysis revealed that MDR1 immunoreactivity was localized primarily in ECs; MRP1 protein levels also were significantly higher in epileptic tissue.
Conclusions: Complex MDR expression changes may play a role in AEDs pharmacoresistance by altering the permeability of AEDs across the BBB.
4-Hydroxynonenal (4HNE) is the most prevalent toxic lipid peroxidation product formed during oxidative stress. It exerts its cytotoxicity mainly by the modification of intracellular proteins. The detection of 4HNE-modified proteins in several degenerative disorders suggests a role for 4HNE in the onset of these diseases. Efficient protection mechanisms are required to prevent the intracellular accumulation of 4HNE. The toxicity of 4HNE was tested with the small cell lung cancer cell lines GLC4 and the multidrug-resistance-protein (MRP1)-overexpressing counterpart GLC4/Adr. In the presence of the MRP1 inhibitor MK571 or the GSH-depleting agent buthionine sulphoximine, both cell lines became more sensitive and showed decreased survival. Transport experiments were performed with the 3H-labelled glutathione S-conjugate of 4HNE ([3H]GS-4HNE) with membrane vesicles from GLC4-derived cell lines with different expression levels of MRP1. [3H]GS-4HNE was taken up in an ATP-dependent manner and the transport rate was dependent on the amount of MRP1. The MRP1 inhibitor MK571 decreased [3H]GS-4HNE uptake. MRP1-specific [3H]GS-4HNE transport was demonstrated with membrane vesicles from High Five insect cells overexpressing recombinant MRP1. Kinetic experiments showed an apparent K m of 1.6±0.21 µ M (mean±S.D.) for MRP1-mediated [3H]GS-4HNE transport. In conclusion, MRP1 has a role in the protection against 4HNE toxicity and GS-4HNE is a novel MRP1 substrate. MRP1, together with GSH, is hypothesized to have a role in the defence against oxidative stress.
The expression of glial and neuronal glutamate transporter proteins was investigated in the hippocampal region at different time points after electrically induced status epilepticus (SE) in the rat. This experimental rat model for mesial temporal lobe epilepsy is characterized by cell loss, gliosis, synaptic reorganization, and chronic seizures after a latent period. Despite extensive gliosis, immunocytochemistry revealed only an up-regulation of both glial transporters localized at the outer aspect of the inner molecular layer (iml) in chronic epileptic rats. The neuronal EAAC1 transporter was increased in many somata of individual CA1-3 neurons and granule cells that had survived after SE; this up-regulation was still present in the chronic epileptic phase. In contrast, a permanent decrease of EAAC1 immunoreactivity was observed in the iml of the dentate gyrus. This permanent decrease in EAAC1 expression, which was only observed in rats that experienced progressive spontaneous seizure activity, could lead to abnormal glutamate levels in the iml once new abnormal glutamatergic synaptic contacts are formed by means of sprouted mossy fibers. Considering the steady growth of reorganizing mossy fibers in the iml, the absence of a glutamate reuptake mechanism in this region could contribute to progression of spontaneous seizure activity, which occurs with a similar time course.
Bicinchoninic acid, sodium salt, is a stable, water-soluble compound capable of forming an intense purple complex with cuprous ion (Cu1+) in an alkaline environment. This reagent forms the basis of an analytical method capable of monitoring cuprous ion produced in the reaction of protein with alkaline Cu2+ (biuret reaction). The color produced from this reaction is stable and increases in a proportional fashion over a broad range of increasing protein concentrations. When compared to the method of Lowry et al., the results reported here demonstrate a greater tolerance of the bicinchoninate reagent toward such commonly encountered interferences as nonionic detergents and simple buffer salts. The stability of the reagent and resulting chromophore also allows for a simplified, one-step analysis and an enhanced flexibility in protocol selection. This new method maintains the high sensitivity and low protein-to-protein variation associated with the Lowry technique.
Why some patients with seizures are successfully treated with antiepileptic drugs (AEDs) and others prove medically intractable is not known. Inadequate intraparenchymal drug concentration is a possible mechanism of resistance to AEDs. The multiple drug resistance gene (MDR1) encodes P-glycoprotein, an energy-dependent efflux pump that exports planar hydrophobic molecules from the cell. If P-glycoprotein is expressed in brain of some patients with intractable epilepsy and AEDs are exported by P-glycoprotein, lower intraparenchymal drug concentrations could contribute to lack of drug response in such patients. Eleven of 19 brain specimens removed from patients during operation for intractable epilepsy had MDR1 mRNA levels > 10 times greater than those in normal brain, as determined by quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) method. Immunohistochemistry for P-glycoprotein from 14 of the patients showed increased staining in capillary endothelium in samples from epileptic patients as compared with staining in normal brain samples. In epileptic brain specimens with high MDR1 mRNA levels, expression of P-glycoprotein in astrocytes also was identified. Last, steady-state intracellular phenytoin (PHT) concentrations in MDR1 expressing neuroectodermal cells was one fourth that in MDR1-negative cells. MDR1 expression is increased in brain of some patients with medically intractable epilepsy, suggesting that the patients' lack of response to medication may be caused by inadequate accumulation of AED in brain.
Resistance to multiple natural product drugs associated with reduced drug accumulation in human tumor cells may be conferred by either the 170 kDa P-glycoprotein or the 190 kDa multidrug resistance protein, MRP. Both MRP and P-glycoprotein belong to the large and ancient ATP-binding cassette (ABC) superfamily of transport proteins but share only 15% amino acid identify. Unlike P-glycoprotein, MRP actively transports conjugated organic anions such as the cysteinyl leukotriene C4 and glutathione-conjugated aflatoxin B1. Transport of unconjugated chemotherapeutic agents appears to require cotransport of glutathione. MRP and several more recently discovered ABC proteins contain an additional NH2-proximal membrane-spanning domain not found in previously characterized ABC transporters. This domain, whose NH2-terminus is extracytosolic, is essential for MRP-mediated transport activity. This review summarizes current knowledge of the structural and transport characteristics of MRP which suggest that the physiologic functions of this protein could range from a protective role in chemical toxicity and oxidative stress to mediation of inflammatory responses involving cysteinyl leukotrienes.
The membrane proteins mediating the ATP-dependent transport of lipophilic substances conjugated to glutathione, glucuronate, or sulfate have been identified as members of the multidrug resistance protein (MRP) family. Several isoforms of these conjugate export pumps with different kinetic properties and domain-specific localization in polarized human cells have been cloned and characterized. Orthologs of the human MRP isoforms have been detected in many different organisms. Studies in mutant rats lacking the apical isoform MRP2 (symbol ABCC2) indicate that anionic conjugates of endogenous and exogenous substances cannot exit from cells at a sufficient rate unless an export pump of the MRP family is present in the plasma membrane. Several mutations in the human MRP2 gene have been identified which lead to the absence of the MRP2 protein from the hepatocyte canalicular membrane and to the conjugated hyperbilirubinemia of Dubin-Johnson syndrome. Overexpression of recombinant MRP2 confers resistance to multiple chemotherapeutic agents. Because of its function in the terminal excretion of cytotoxic and carcinogenic substances, MRP2 as well as other members of the MRP family, play an important role in detoxification and chemoprevention.
Drug resistance in epilepsy due to malformations of cortical development (MCD) is unexplained. P-glycoprotein is a mediator of drug resistance, and we propose that MCD lesions over-express P-glycoprotein. Because P-glycoprotein expression may be induced by some antiepileptic drugs (AEDs), we studied brain samples from MCD cases before the onset of seizures or treatment with AEDs. Sixteen MCD cases and 16 age-matched controls were examined using immunohistochemistry. Glial labelling, representing over-expression, was seen in 10 of 16 MCD samples and in two of 16 control samples (p = 0.003). Semiquantitative assessment showed many immunoreactive glia in five of 16 MCD and one of 16 controls. We conclude that there is constitutive over-expression of P-glycoprotein in many MCD.
Reactive gliosis is a prominent morphological feature of mesial temporal lobe epilepsy. Because astrocytes express glutamate receptors, we examined changes in metabotropic glutamate receptor (mGluR) 2/3, mGluR5 and transforming growth factor (TGF)-beta in glial cells of the hippocampal regions in an experimental rat model of spontaneous seizures. Rats that exhibited behavioural status epilepticus (SE) directly after 1 h of electrical angular bundle stimulation, displayed chronic spontaneous seizures after a latent period of 1-2 weeks as observed using continuous electrographic monitoring. SE resulted in hypertrophy of astrocytes and microglia activation throughout the hippocampus as revealed by immunolabelling studies. A dramatic, seizure intensity-dependent increase in vimentin immunoreactivity (a marker for reactive astrocytes) was revealed in CA3 and hilar regions where prominent neuronal loss occurs. Increased vimentin labelling was first apparent 24 h after onset of SE and persisted up to 3 months. mGluR2/3 and mGluR5 protein expression increased markedly in glial cells of CA3 and hilus by 1 week after SE, and persisted up to 3 months after SE. Double immunolabelling of brain sections with vimentin confirmed co-localization with glial fibrillary acidic protein (GFAP), mGluR2/3 and mGluR5 in reactive astrocytes. TGF-beta, a cytokine implicated in mGluR3-mediated neuroprotection, was also upregulated during the first 3 weeks after SE throughout the hippocampus. This study demonstrates seizure-induced upregulation of two mGluR subtypes in reactive astrocytes, which - together with the increased production of TGF-beta - may represent a novel mechanism for modulation of glial function and for changes in glial-neuronal communication in the course of epileptogenesis.
Despite accumulating evidence that multidrug resistance transporter proteins play a part in drug resistance in some clinical cancers, it remains unclear whether the relatively low levels of multidrug resistance transporter expression found in most untreated tumors could substantially affect their basal sensitivity to antineoplastic drugs. To shed light on this problem, the drug sensitivities of wild-type mouse cell lines were compared with those of lines in which the Mdr1a and Mdr1b genes encoding P-glycoprotein (P-gp) were inactivated and lines in which the Mrp1 gene was inactivated in addition to Mdr1a and Mdr1b. These models permit a clean dissection of the contribution of each transporter to drug resistance at expression levels similar to those in normal tissues and avoid complications that might arise from previous exposure of cell lines to drug selection. For substrate drugs, we found that these contributions can indeed be very substantial. Lines lacking functional P-gp were, on average, markedly more sensitive to paclitaxel (16-fold), anthracyclines (4-fold) and Vinca alkaloids (3-fold). Lines lacking both P-gp and Mrp1 were (compared with wild-type lines) hypersensitive to an even broader array of drugs, including epipodophyllotoxins (4-7-fold), anthracyclines (6-7-fold), camptothecins (3-fold), arsenite (4-fold) and Vinca alkaloids, especially vincristine (28-fold). Thus, even very low levels of P-gp and Mrp1 expression that may be difficult to detect in tumors could significantly affect their innate sensitivity to a wide range of clinically important substrate drugs. An implication is that the use of resistance reversal agents to sensitize drug-naive tumors may be appropriate in more cases than is presently appreciated.
Drug resistance in epilepsy is poorly understood. We used routine immunohistochemistry to assess overexpression of a multidrug-resistance protein in dysplastic neurons, glia, and around vessels in surgically resected epileptogenic human brain tissue. We showed non-tumoral overexpression of this multidrug-resistance protein, which might contribute to drug resistance in epilepsy caused by focal cortical dysplasia.
The development of spontaneous limbic seizures was investigated in a rat model in which electrical tetanic stimulation of the angular bundle was applied for up to 90 min. This stimulation produced behavioural and electrographic seizures that led to a status epilepticus (SE) in most rats (71%). Long-term EEG monitoring showed that the majority of the rats (67%) that underwent SE, displayed a progressive increase of seizure activity once the first seizure was recorded after a latent period of about 1 week. The other SE rats (33%) did not show this progression of seizure activity. We investigated whether these different patterns of evolution of spontaneous seizures could be related to differences in cellular or structural changes in the hippocampus. This was the case regarding the following changes. (i) Cell loss in the hilar region: in progressive SE rats this was extensive and bilateral whereas in nonprogressive SE rats it was mainly unilateral. (ii) Parvalbumin and somatostatin-immunoreactive neurons: in the hilar region these were almost completely eliminated in progressive SE rats but were still largely present unilaterally in nonprogressive SE rats. (iii) Mossy fibre sprouting: in progressive SE rats, extensive mossy fibre sprouting was prominent in the inner molecular layer. In nonprogressive SE rats, mossy fibre sprouting was also present but less prominent than in progressive SE rats. Although mossy fibre sprouting has been proposed to be a prerequisite for chronic seizure activity in experimental temporal lobe epilepsy, the extent of hilar cell death also appears to be an important factor that differentiates between whether or not seizure progression will occur.
The 190 kDa multidrug resistance protein 1 (MRP1/ABCC1) is a founding member of a subfamily of the ATP binding cassette (ABC) superfamily of transport proteins and was originally identified on the basis of its elevated expression in multidrug resistant lung cancer cells. In addition to its ability to confer resistance in tumour cells, MRP1 is ubiquitously expressed in normal tissues and is a primary active transporter of GSH, glucuronate and sulfate conjugated and unconjugated organic anions of toxicological relevance. Substrates include lipid peroxidation products, herbicides, tobacco specific nitrosamines, mycotoxins, heavy metals, and natural product and antifolate anti-cancer agents. MRP1 also transports unmodified xenobiotics but often requires GSH to do so. Active efflux is generally an important aspect of cellular detoxification since it prevents the accumulation of conjugated and unconjugated compounds that have the potential to be directly toxic. The related transporters MRP2 and MRP3 have overlapping substrate specificities with MRP1 but different tissue distributions, and evidence that they also have chemoprotective functions are discussed. Finally, MRP homologues have been described in other species including yeast and nematodes. Those isolated from the vascular plant Arabidopsis thaliana (AtMRPs) decrease the cytoplasmic concentration of conjugated toxins through sequestration in vacuoles and are implicated in providing herbicide resistance to plants.
Despite considerable advances in the pharmacotherapy of epilepsy, about 30% of epileptic patients are refractory to antiepileptic drugs (AEDs). In most cases, a patient who is resistant to one major AED is also refractory to other AEDs, although these drugs act by different mechanisms. The mechanisms that lead to drug resistance in epilepsy are not known. Recently, over-expression of multidrug transporters, such as P-glycoprotein (PGP) and multidrug resistance-associated protein (MRP), has been reported in surgically resected epileptogenic human brain tissue and suggested to contribute to the drug resistance of epilepsy. However, it is not known to what extent multidrug transporters such as PGP or MRP are involved in transport of AEDs. In the present study, we used in vivo microdialysis in rats to study whether the concentration of carbamazepine in the extracellular fluid of the cerebral cortex can be enhanced by inhibition of PGP or MRP, using the PGP inhibitor verapamil and the MRP inhibitor probenecid. Local perfusion with verapamil or probenecid via the microdialysis probe increased the extracellular concentration of carbamazepine. The data indicate that both PGP and MRP participate in the regulation of extracellular brain concentrations of the major AED carbamazepine.
The present study was designed to address the question of whether recurrent spontaneous seizures cause progressive neuronal damage in the brain. Epileptogenesis was triggered by status epilepticus (SE) induced by electrically stimulating the amygdala in rat. Spontaneous seizures were continuously monitored by video-EEG for up to 6 months. The progression of damage in individual rats was assessed with serial magnetic resonance imaging (MRI) by quantifying the markers of neuronal damage (T2, T1 rho, and Dav) in the amygdala and hippocampus. The data indicate that SE induces structural alterations in the amygdala and the septal hippocampus that progressively increased for approximately 3 weeks after SE. T2, T1 rho, and Dav did not normalize during the 50 days of follow-up after SE, suggesting ongoing neuronal death due to spontaneous seizures. Consistent with these observations, Fluoro-Jade B-stained preparations revealed damaged neurons in the hippocampus of spontaneously seizing animals that were sacrificed up to 62 days after SE. The presence of Fluoro-Jade B-positive neurons did not, however, correlate with the number of spontaneous seizures, but rather with the time interval from SE to perfusion. Further, there were no Fluoro-Jade B-positive neurons in frequently seizing rats that were perfused for histology 6 months after SE. Also, the number of lifetime seizures did not correlate with the severity of neuronal loss in the hilus of the dentate gyrus assessed by stereologic cell counting. The methodology used in the present experiments did not demonstrate a clear association between the number or occurrence of spontaneous seizures and the severity of hilar cell death. The ongoing hippocampal damage in these epileptic animals detected even 2 month after SE was associated with epileptogenic insult, that is, SE rather than spontaneous seizures.
Breast cancer resistance protein (BCRP/MXR/ABCP/ABCG2; hereafter ABCG2) is a member of the ATP-binding-cassette family of transporters that causes multi-drug resistance to various anticancer drugs. The expression of ABCG2 in human tumours and its potential involvement in clinical drug resistance are unknown. Recently, two monoclonal antibodies against human ABCG2 were produced, BXP-34 and BXP-21. This study describes an immunohistochemical method using BXP-21 to study ABCG2 expression in formalin-fixed, paraffin-embedded tissues. No staining was seen using BXP-34 with the same protocols. The expression of ABCG2 was then investigated in a panel of 150 untreated human solid tumours comprising 21 tumour types. Overall, ABCG2 expression was frequent. Specificity of immunohistochemistry was confirmed by the detection of a 72 kD band in western blotting. ABCG2 expression was seen in all tumour types, but it seemed more frequent in adenocarcinomas of the digestive tract, endometrium, and lung, and melanoma. Positive tumours showed membranous and cytoplasmic staining. In certain adenocarcinomas, prominent membranous staining was seen. Endothelial cells frequently displayed moderate to strong staining. ABCG2 is widely present in untreated human solid tumours and may represent a clinically relevant mechanism of drug resistance. Future studies in specific tumour types are needed to ascertain its clinical relevance. BXP-21 and the immunohistochemical protocol described here will be of value in these investigations.
Rat brain astrocyte and microglia cultures express different members of ATP-binding-cassette (ABC) proteins. RT-PCR analysis showed that astrocytes are equipped with P-glycoprotein (mdr1a, mdr1b), multidrug resistance-associated-protein (mrp1, mrp4, mrp5) and cystic fibrosis transmembrane conductance regulator (CFTR). No transcripts for mrp5 and CFTR were detected in microglia. The ABC protein functional activities are shown by the following results: (i) cyclosporin A (50 microM), verapamil (50 microM), probenecid (1 mM) or sulfinpyrazone (2 mM) enhanced [3H]vincristine accumulation; (ii) cyclosporin A or verapamil but not probenecid or sulfinpyrazone enhanced [3H]digoxin accumulation; (iii) glibenclamide (100 microM) inhibited 36Cl efflux from astrocytes. ATP release from glial cells was inhibited by the pretreatment with ABC protein inhibitors indicating that ABC proteins are involved in nucleotide efflux from glial cells which represent the main source of cerebral extracellular purines.
Movement of substrates between blood and brain is known to be influenced by P-glycoprotein (P-gp) at the luminal surface of the endothelium lining brain microvessels and by multidrug resistance associated protein 1 (MRP1) at the basolateral surface of the choroid plexus epithelium. Here, using RT-PCR and Western blotting, we investigate other ABC transporters in both normal and tumour human brain tissue and demonstrate the presence of breast cancer resistance protein (BCRP). Immunofluorescence confocal microscopy demonstrates that BCRP is located at the blood-brain barrier, mainly at the luminal surface of microvessel endothelium. This localization closely resembles that of P-gp. BCRP has several substrates in common with P-gp and may pose an additional barrier to drug access to the brain.
Active drug efflux transporters of the ATP binding cassette (ABC)-containing family of proteins have a major impact on the pharmacological behavior of most of the drugs in use today. Pharmacological properties affected by ABC transporters include the oral bioavailability, hepatobiliary, direct intestinal, and urinary excretion of drugs and drug-metabolites and -conjugates. Moreover, the penetration of drugs into a range of important pharmacological sanctuaries, such as brain, testis, and fetus, and the penetration into specific cell- and tissue compartments can be extensively limited by ABC transporters. These interactions with ABC transporters determine to a large extent the clinical usefulness, side effects and toxicity risks of drugs. Many other xenotoxins, (pre-)carcinogens and endogenous compounds are also influenced by the ABC transporters, with corresponding consequences for the well-being of the individual. We aim to provide an overview of properties of the mammalian ABC transporters known to mediate significant transport of clinically relevant drugs.
Accumulating evidence implicates drug-transporter proteins ABCB1 and ABCC1 in resistance to multiple antiepileptic drugs (AEDs) in refractory epilepsy. These proteins are upregulated in surgically resected human brain tissue containing epileptogenic pathologies, including cortical dysplasia. In surgically resected cases, no upregulation is seen in normal adjacent tissue, suggesting that neither seizures nor prolonged exposure to AEDs need induce ABCB1 or ABCC1 expression. We wished to determine if status epilepticus might cause upregulation of these proteins.
Immunohistochemistry was performed for ABCB1 and ABCC1 in postmortem human brain tissue from a patient who died in status epilepticus and was found to have unihemispheric cortical dysplasia.
There was upregulation of both proteins, as expected, in the hemisphere containing dysplasia. There also was widespread upregulation of both proteins in glia from the normal hemisphere. Previous work shows that drug treatment does not cause such upregulation.
Upregulation of these proteins in histologically normal brain tissue is most likely the result of seizures in status, as seen in animal models. The findings provide a possible explanation for the appearance of AED resistance in prolonged status and emphasise the importance of prompt treatment of status epilepticus.
The cell-specific distribution of multidrug resistance extrusion pumps was studied in developmental glioneuronal lesions, including focal cortical dysplasia (15 cases) and ganglioglioma (15 cases) from patients with medically intractable epilepsy. Lesional, perilesional, as well as normal brain regions were examined for the expression of the multidrug resistance gene 1 encoded P-glycoprotein (P-gp) and the multidrug resistance-associated protein 1 (MRP1) by immunocytochemistry. In normal brain MRP1 expression was below detection, whereas P-gp staining was present only in blood vessels. MRP1 and P-gp immunoreactivity was observed in dysplastic neurons of 11/15 cases of focal cortical dysplasia, as well as in the neuronal component of 14/15 ganglioglioma. Glial cells with astrocytic morphology within the lesion showed multidrug-resistant protein immunoreactivity (P-gp>MRP1). Moderate to strong MRP1 and P-gp immunoreactivity was observed in a population of large ballooned neuroglial cells. P-gp appeared to be most frequently expressed in glial fibrillary acidic protein-positive balloon cells (glial type), whereas MRP1 was more frequently expressed in microtubule-associated protein 2-positive balloon cells (neuronal type). In both types of lesions strong P-gp immunoreactivity was found in lesional vessels. Perilesional regions did not show increased staining in vessels or in neuronal cells compared with normal cortex. The predominant intralesional cell-specific distribution of multidrug transporter proteins supports the hypothesis of a constitutive overexpression as common mechanism underlying the intrinsic pharmaco-resistance to antiepileptic drugs of both malformative and neoplastic glioneuronal developmental lesions.
The blood-brain barrier (BBB) plays the predominant role in controlling the passage of endogenous and xenobiotic substances between the circulating blood and the extracellular fluid environment of the brain. The transfer of compounds is strictly regulated by brain capillary endothelial cells (BCEC), which are interconnected to each other by well developed tight junctions, without fenestrations. Although hydrophobic molecules such as nicotine and ethanol readily cross the BBB by diffusion, the brain microvasculature shows a highly restrictive permeability to hydrophobic antitumor agents. So far, this multidrug resistance has been almost exclusively attributed to the most prominent member of the ATP-binding cassette (ABC) transporter family, P-glycoprotein located in the luminal membrane of brain capillary endothelial cells and to a minor extent to the multidrug resistance-associated proteins (MRPs). The brain multidrug resistance protein (BMDP) has recently been discovered at the porcine BBB and was shown to be highly homologous to the human breast cancer resistance protein (BCRP/ABCG2). Here, we demonstrate by northern blot and RT-PCR analysis that BMDP mRNA is more highly expressed in the capillary endothelial cells compared to other cell types of the brain. Immunocytochemistry of porcine BCEC showed a clear plasma membrane localisation of BMDP. Analysis of the total mRNA pool revealed that BMDP is more strongly expressed than P-glycoprotein and MRP1. Consistently, first transport studies indicate that active exclusion of the chemotherapeutic drug daunorubicin from the central nervous system is mediated mainly by this new transporter compared to P-glycoprotein or MRP1. Thus, we hypothesise that BMDP might play an important role in the exclusion of xenobiotics from the porcine brain.
To determine whether repeated seizures contribute to hippocampal sclerosis, we investigated whether cell loss in the (para) hippocampal region was related to the severity of chronic seizure activity in a rat model for temporal lobe epilepsy (TLE).
Chronic epilepsy developed after status epilepticus (SE) that was electrically induced 3-5 months before. The presence of neuronal damage was assessed by using Fluoro-Jade and dUTP nick end-labeling (TUNEL) of brain sections counterstained with Nissl.
We found a negative correlation between the numbers of surviving hilar cells and the duration of the SE (r = -0.66; p < 0.01). In the chronic phase, we could discriminate between rats with occasional seizures (0.15 +/- 0.05 seizures per day) without progression and rats with progressive seizure activity (8.9 +/- 2.8 seizures/day). In both groups, the number of TUNEL-positive cells in parahippocampal regions was similar and higher than in controls. In the hippocampal formation, this was not significantly different from controls. Fluoro-Jade staining showed essentially the same pattern at 1 week and no positive neurons in chronic epileptic rats.
Cell death in this rat model is related to the initial SE rather than to the frequency of spontaneous seizures. These results emphasize that it is of crucial importance to stop the SE as soon as possible to prevent extended cell loss and further progression of the disease. They also suggest that neuroprotectants can be useful during the first week after SE, but will not be very useful in the chronic epileptic phase.
The molecular basis of drug resistance in epilepsy is being explored. Two proteins associated with drug resistance in cancer, P-glycoprotein and multidrug resistance-associated protein 1, are upregulated in human epileptogenic pathologies. Other proteins associated with resistance in cancer include major vault protein (MVP) and breast cancer resistance protein (BCRP). We hypothesized that these proteins would also be upregulated in human epileptogenic pathologies.
Hippocampal sclerosis (HS), focal cortical dysplasia (FCD), and dysembryoplastic neuroepithelial tumor (DNT) were studied by using immunohistochemistry for MVP and BCRP. Nonepileptogenic control and histologically normal brain adjacent to epileptogenic tissue were used for comparison.
MVP and BCRP were expressed ubiquitously in brain capillary endothelium. Ectopic upregulation of MVP was seen in hilar neurons in HS, dysplastic neurons in FCD, and lesional neurons in DNT. Only in HS cases were rare extralesional neurons immunoreactive. Glial upregulation was not seen. There was no qualitative upregulation of BCRP.
These results show that more than one resistance protein may be upregulated in a given epileptogenic pathology and may contribute to drug resistance. Determination of the types, amounts, and distribution of such proteins will be necessary for rational treatment for drug resistance in epilepsy.
In the brain, the efflux transporter P-glycoprotein (Pgp) is predominantly located on the luminal membrane of endothelial cells lining brain microvessels and forming the blood-brain barrier. Many lipophilic drugs, including antiepileptic drugs, are potential substrates for Pgp. Overexpression of Pgp in endothelial cells of the blood-brain barrier has been determined in patients with drug resistant forms of epilepsy such as temporal lobe epilepsy and rodent models of temporal lobe epilepsy and suggested to lead to reduced penetration of antiepileptic drugs into the brain. Expression of Pgp after seizures has also been described in astrocytes, whereas it is not clear whether neurons can express Pgp. In the present study, Pgp expression was studied by immunohistochemistry in rats 24 h after a status epilepticus induced by either pilocarpine or kainate, widely used models of temporal lobe epilepsy. Unexpectedly, in addition to endothelial Pgp staining, intense Pgp staining was found in neurons in the CA3c/CA4 sectors and hilus of the hippocampus formation, but not in other brain regions examined. The neuronal Pgp staining was confirmed by two different Pgp antibodies. Double immunolabeling and confocal microscopy showed that Pgp was colocalized with the neuronal marker neuronal nuclear antigen, but not with the glial marker glial fibrillary acidic protein. No neuronal Pgp staining was seen in control rats. The expression of Pgp in neurons after limbic seizures was substantiated by determining Pgp encoding genes (mdr1a, mdr1b) in neurons by real time quantitative RT-PCR. Increased Pgp expression in hippocampal neurons is likely to affect the action of drugs with intraneuronal targets and, in view of recent evidence from other cell types, could be associated with prevention of apoptosis which is involved in neuronal damage developing after seizures such as produced by pilocarpine.
ABCG2, also termed BCRP/MXR/ABCP, is a half ATP-binding cassette (ABC) transporter expressed on plasma membranes. ABCG2 was independently cloned from placenta as well as cell lines selected for resistance to mitoxantrone or anthracyclines. ABCG2 consists of a nucleotide-binding domain (NBD) at the amino terminus and a transmembrane domain (TMD) at the carboxyl terminus and it is postulated to form a homodimer to perform its biological functions. Over-expression of ABCG2 in cell lines confers resistance on a wide variety of anticancer drugs including mitoxantrone, daunorubicin, doxorubicin, topotecan and epirubicin. The expression of ABCG2 has been implicated in multidrug resistance (MDR) of acute myeloid leukemia and some solid tumors. In addition, ABCG2 can transport several fluorescent dyes or toxins. ABCG2 is found to be expressed in epithelial cells of intestine and colon, liver canaliculi, and renal tubules, where it serves to eliminate the plasma level of orally administered anticancer drugs as well as ingested toxins. ABCG2 is found to be highly expressed in placenta and the luminal surface of microvessel endothelium blood-brain barrier where it may play a role in limiting the penetration of drugs, such as topotecan from the maternal plasma into the fetus and from blood to brain. A variety of inhibitors for ABCG2 including GF120918 may prove useful for sensitizing cancer cells to chemotherapy or altering the distribution of orally administered drug substrates of ABCG2. Interestingly, ABCG2 is also expressed highly in hematopoietic stem cells. However, the function of ABCG2 in stem cells is currently unknown, although it may provide protection to stem cells from a variety of xenobiotics.
Increased expression of the multidrug transporter P-glycoprotein (Pgp; ABCB1) has previously been found in epileptogenic brain tissue from patients with pharmacoresistant temporal lobe epilepsy (TLE) as well as in the hippocampus and other limbic brain regions in the rat kainate model of TLE. Approaches to the quantification of Pgp expression have mainly been based on subjective visual estimation of the level of Pgp immunoreactivity in brain sections. In the present study, computer-assisted image analysis based on optical density (OD) measurements was used to examine immunohistochemical expression of Pgp in the kindling model of TLE. Sections from kainate-treated rats were used for comparison. Using diaminobenzidine as chromogen, Pgp was exclusively located in brain capillary endothelial cells, which was confirmed by double-labeling with an antibody against the endothelial glucose transporter (GLUT-1). After kainate-induced seizures, the intensity of endothelial Pgp staining significantly increased by 70-80% in the dentate gyrus. A significant, albeit less marked increase in Pgp expression in this area was also seen after amygdala-kindled seizures. Furthermore, Pgp was upregulated after kindling in the hilus of the dentate gyrus, the CA1 and CA3 sectors of the hippocampus, and the piriform and cerebral cortex. In kindled rats, most Pgp alterations occurred ipsilateral to the electrode in the basolateral amygdala. The data demonstrate that computer-assisted image analysis using OD is an accurate and rapid method to determine the relative amount of Pgp protein in brain sections and the effects of seizures on this multidrug transporter. The fact that Pgp overexpression in brain capillary endothelial cells occurs in two established models of difficult-to-treat TLE substantiates the notion that seizure-induced upregulation of Pgp contributes to multidrug resistance (MDR) in epilepsy.
The basis for low brain permeability of valproic acid (VPA) appears to be the result of efflux transport at the blood-brain barrier (BBB); however, the identity of the putative efflux transporter has not been investigated. The objective of our studies was to determine whether the multidrug resistance-associated protein (MRP) might be involved in efflux transport of VPA. Brain microvessel endothelial cells (BMEC) were isolated from cow brains and grown to confluence. MRP messenger RNA (mRNA) in BMEC were verified by reverse transcriptase-polymerase chain reaction (RT-PCR). Functional activity was demonstrated using the steady-state retention of calcein and MRP inhibitors, indomethacin (IND) and probenecid (PRB). Probenecid (0.50 mM) and indomethacin (10 microM) produced a 26 and 13% ( P<0.05 ) elevation in steady-state cellular VPA uptake following a 30-min-incubation with tracer 3H-VPA and 30 microM cold VPA. In contrast, at higher concentrations of probenecid (2 mM) and indomethacin (500 microM), an 11 and 31% reduction in VPA uptake was observed. The biphasic pattern of VPA uptake suggested concurrent inhibition of uptake and efflux transporters by the inhibitor with differing sensitivities, i.e. the efflux transporter being more susceptible to inhibition than the influx transporter. Similar results were obtained in the MRP overexpressing cell line A549. Overall, the results suggest that MRP(s) is(are) involved in the efflux transport of VPA, but do not preclude the possible contribution(s) of other organic anion transporters. The findings also adds to the growing evidence that up-regulation of active drug efflux transporters at the BBB may contribute to the development of drug resistance to antiepileptic drug therapy.