Address correspondence and reprint requests to Dr. W. Löscher, Department of Pharmacology, Toxicology and Pharmacy, University of Veterinary Medicine, Bunteweg 17, D-30559 Hannover, Germany, E-mail: email@example.com
Summary: Current estimates indicate that up to one-third of all individuals with epilepsy are refractory to antiepileptic drug (AED) therapy. Moreover, most of these people are resistant to multiple drugs with a wide range of mechanistic actions. These observations suggest that the development of multidrug resistance involves nonspecific, global changes in the brain. The multidrug transporter hypothesis of pharmacoresistant epilepsy proposes that regional-specific overexpression of drug efflux transporters in the blood–brain barrier limits the brain penetration of AEDs. Consequently, drug concentrations are too low to induce antiepileptic effects at target brain sites. Cumulative clinical and experimental data support this hypothesis and offer novel therapeutic approaches for the treatment of drug-resistant epilepsy.
Victor Ling identified the first multidrug transporter, also referred to as a multidrug resistance (MDR) protein, in chemotherapy-resistant cancer cells in the 1970s (1). He coined the name, P-glycoprotein (P-gp); P for permeability because he found that this protein was responsible for decreasing the concentration of chemotherapeutic agents in target cancer cells. Since this discovery, various additional multidrug transporters, including the family of multidrug-resistant proteins (MRPs) and breast cancer resistant protein (BCRP), have been identified in both chemotherapy-resistant cancer cells and in normal cells of the intestine, liver, kidney, and endothelial cells lining the blood–brain barrier (BBB) (2). The primary function of these proteins is to pump lipophilic drugs and other xenobiotics out of cells and thereby prevent the accumulation of potentially toxic substances. In doing so, however, these proteins may also decrease the efficacy of pharmacological agents by limiting their access to target tissues in the brain. The fact that multidrug transporters are highly expressed in capillary endothelial cells of the BBB and overexpressed in human epileptic tissue is highly significant (3). This article reviews the clinical and experimental evidence that supports the multidrug transporter hypothesis, focusing on the role of drug efflux transporter in the genesis of drug-resistant epilepsy.
Fig. 1 contains an illustration of a brain microvessel showing the tight junctions within the endothelial cell layer, in addition to the pericytes, basal membrane, and the astrocytic foot processes that together form the physical barrier between blood and brain. To demonstrate the presence of P-gp in brain microvessels, we used monoclonal antibodies to double stain for both glucose transporter 1 (GLUT1), a marker for brain capillary endothelial cells (green), and the P-gp drug transporter (red) in normal rat brain (3). In the confocal laser micrograph included in Fig. 1, one can see that P-gp is highly expressed and colocalized with GLUT1 only on the luminal surface of the vessel. This would be the expected location of a protein that functions to transport drugs back into the blood before they reach the BBB. Although there are a number of other drug efflux transporters located on the luminal side of endothelial cells, P-gp is most relevant to drug-resistant epilepsy because it actively transports a large number of frequently prescribed AEDs, including carbamazepine, felbamate, gabapentin, lamotrigine, phenobarbital, phenytoin, and topiramate (3). For this reason, P-gp has been a target for intense experimental study.
MULTIDRUG TRANSPORTERS IN EPILEPTIC AND NONEPILEPTIC BRAIN
A number of drug transporter genes and their proteins are overexpressed in the BBB of individuals with refractory epilepsy. This has been demonstrated in tissue taken from epileptic foci at the time of resective surgery. Specific data indicate that there is a 130% increase in the expression of MDR1, the gene encoding for P-gp, a 180% increase in MRP5, and a 225% increase in MRP2 in brain capillary endothelial cells isolated from epileptic individuals in comparison with nonepileptic controls (4). Similarly, there is a marked overexpression of P-gp (≈200%) and MRP2 (≈100%) transporter proteins in human epileptogenic tissue (5). Additional data indicate that P-gp, MRP1, and MRP2 are also overexpressed in glial and/or neuronal cells of patients with intractable epilepsy (4–7). Experimental models of temporal lobe epilepsy (TLE), a diagnosis that is most frequently associated with multidrug resistance in man, have yielded similar results—an overexpression of P-gp, MRP1, MRP2, and BCRP in endothelial cells of the BBB and/or glial and neuronal cells in the brain parenchyma (3,8,9). Using monoclonal antibody staining, we have observed regional-specific overexpression of P-gp in the hilus of the dentate gyrus and CA3 region of the hippocampus in rats 1 week after pilocarpine-induced status epilepticus. Most importantly, there was no comparable staining of hippocampal neurons in nonseizing controls, indicating that the upregulation of P-gp was a consequence of the experimentally induced seizure activity (9).
There are at least two reasons why multidrug efflux transporters are overexpressed in epileptogenic brain tissue. First, there may be a constitutive or inherited overexpression that occurs as a result of a genetic polymorphism. We know, for example, that certain polymorphisms in the MDR1 gene that encodes for P-gp in humans are associated with an increased expression and functionality of P-gp, which may be associated with refractory epilepsy (10). A second possibility is that the overexpression is acquired or induced by some epilepsy-related factor, such as uncontrolled seizures. As mentioned before, animal models have shown that there is a transient and regionally restricted increase in the expression of P-gp and other drug efflux transporters following intense seizure activity (11,12). The upregulation persists for a couple of days, and then levels return to normal. At this time we do not fully understand why the upregulation of transporters occurs following seizure activity. It is possible that the acquired overexpression is a second-line defense mechanism to protect the brain during transient opening of the BBB, which typically occurs in response to prolonged seizure activity. Although it is also conceivable that AEDs directly induce overexpression of multidrug transporters to prevent toxic build-up of these substances in the brain, this has not been demonstrated in animal models.
EXPERIMENTAL VALIDATION OF THE DRUG TRANSPORTER HYPOTHESIS
According to the drug transporter hypothesis, the expression of drug efflux transporters in the brain capillaries of normal brain should not restrict the penetration of AEDs to any significant extent. However, in epileptic brain, where multidrug transporters are overexpressed in brain capillaries as well as in the astrocytic foot processes surrounding these capillaries, brain penetration of AEDs should be reduced in a regional-specific fashion. Moreover, overexpression of drug efflux transporters in neurons and glia of the brain parenchyma would further limit the effectiveness of AEDs by restricting access to intracellular target sites. Hence, AED concentrations would be insufficient to cause antiepileptic activity. To verify this hypothesis, we first need to determine if AEDs are substrates for multidrug transporters in the BBB. Recent data from multiple laboratories support this claim. Animal models in which P-gp activity is reduced, through transport inhibition or genetic manipulation, for instance, have all demonstrated a concomitant increase in phenytoin brain penetration (Fig. 2). Using microdialysis in freely moving rats, we found that brain penetration of systemically administered phenytoin nearly doubled 0.5–1.5 h after a unilateral intracerebral injection of the P-gp transport inhibitor, verapamil, in comparison with the contralateral, noninjected hemisphere (Panel A) (13). Similarly, phenytoin brain concentrations are significantly increased in mdr1 knockout mice that lack the gene encoding for P-gp in comparison with wild-type controls (Panel B) (14). Conversely, overexpression of P-gp, produced as a result of kainate-induced status epilepticus, significantly decreases the BBB permeability of phenytoin in the hippocampus in comparison with nonepileptic control rats (Panel C) (14). Taken together, these results demonstrate that the multidrug transporter, P-gp, regulates phenytoin entry into the brain. Additional data have shown that a number of other AEDs, including carbamazepine, phenobarbital, lamotrigine, gabapentin, and topiramate, are also substrates for P-gp, MRPs, or both (3). Hence, it appears that an overexpression of P-gp could potentially affect the efficacy of a variety of different AEDs.
Overexpression of multidrug transporters is an attractive and plausible hypothesis to explain multidrug resistance in epilepsy. However, further studies are needed to firmly establish proof of concept. First and foremost, we need to determine if the expression of multidrug transporters, such as P-gp, differ in drug-resistant and drug-responsive individuals. This, of course, is much easier to do in animals than in humans because we cannot readily obtain tissue samples from people with drug-responsive epilepsy. However, using two different models of experimentally induced TLE, we have been able to correlate P-gp expression with subsets of epileptic rats that vary in their response to AED therapy. In our first model, repeated administration of phenytoin in amygdala-kindled Wistar rats produced three subgroups: responders that always responded to the antiepileptic effects of phenytoin with enhanced seizure thresholds, nonresponders that never responded to phenytoin with enhanced seizure thresholds, and variable responders that showed a response 1 week and not the next (15). This is illustrated in Fig. 3. Panels A and B show the mean seizure thresholds that were recorded from responders and nonresponders following phenytoin injection on three separate trials in comparison with those of vehicle control recordings. In Panel C, one can see that there was also more than a two-fold increase in the area of the amygdala that stained positive for P-gp in phenytoin resistant rats indicating that P-gp expression was significantly elevated in comparison with that of phenytoin responders. Overexpression of P-gp and pharmacoresistance to phenobarbital have also been observed in a subgroup of rats that developed intractable spontaneous seizures following status epilepticus induced by sustained electrical stimulation of the basolateral amygdala (12). Most importantly, phenobarbital-resistant rats displayed P-gp overexpression only in areas of the limbic system, a region of the brain thought to be intimately involved in epileptogenesis. Specifically, significant increases in P-gp were noted in the hilus of the dentate gyrus, CA1 layer of the hippocampus, and the piriform cortex.
CLINICAL SUPPORT FOR THE DRUG TRANSPORTER HYPOTHESIS
Positron emission tomography (PET) is a very promising technique that may help elucidate the role of multidrug transporters in the development of pharmacoresistance in epilepsy. Although not yet validated in patients with epilepsy, results from animal studies have shown that PET can be used to evaluate P-gp substrate kinetics in the BBB of rodents. Through the use of high-resolution micro-PET scans, scientists have been able to visualize the movement of C11-labeled P-gp substrates, such as verapamil, in the BBB of both wild-type and MDR1 knockout mice (16). In wild-type mice, verapamil does not penetrate into the brain to any significant extent because it binds to P-gp and is immediately transported out of the capillary endothelial cells of the BBB. However, verapamil passes freely through the BBB in mdr1 knockout mice because these mutants lack the P-gp transporter. These results suggest that PET may provide a means to study BBB function in patients with epilepsy and other neurological disorders. Indeed, recent observations indicate that C11-labeled verapamil penetrates the BBB only in the midbrain of patients with Parkinson's disease, suggesting that there is a local disturbance in BBB function (17).
Verapamil is a calcium channel blocker that binds to P-gp and competitively blocks the transport of other substrates by P-gp. Using PET, Bart and colleagues (18) have demonstrated that there is a dose–response relationship between the brain uptake of 11C-labeled verapamil and P-gp expression. These findings suggest that it may be possible to quantify the brain penetration of an AED by coadministrating a labeled drug such as 11C-phenytoin with increasing doses of a nonlabeled P-gp inhibitor, such as verapamil or cyclosporin A.
Researchers have also attempted to derive clinical support for the multidrug transporter hypothesis of pharmacoresistant epilepsy by studying the effects of polymorphisms in the genes that encode for drug efflux transporters. The most well-known study demonstrated a significant correlation between the C3435T polymorphism in exon 26 of the MDR1 gene and pharmacoresistance in patients with epilepsy (11). However, due to the failure of independent laboratories to consistently replicate these findings, the link between this polymorphism and drug-resistant epilepsy remains tenuous, at least in man (19,20). In rats, however, AED-resistance and AED-responsiveness appear to be associated with a different frequency of several polymorphisms in the mdr1a gene that encodes P-gp in the rodent BBB (21).
There is an overexpression of P-gp in the BBB of individuals with drug resistant epilepsy (5). Only been recently, however, have we been able to link this overexpression with reduced brain penetration of systemically administered AEDs, as it has been in animals (13,14). In a very eloquent study, Rambeck and colleagues used microdialysis probes to measure AED concentrations in the extracellular space of epileptogenic tissue, cerebrospinal fluid, and blood plasma of patients who were undergoing resective surgery (22). Analysis of perfusates, collected over 1 h prior to tissue excision, revealed that AED concentrations were significantly decreased in the epileptogenic zone of these individuals when compared with CSF concentrations (Fig. 4). These data are the first indication that decreased drug levels in the epileptogenic zone may be involved in drug resistance of patients with epilepsy, which substantiates the multidrug transporter hypothesis. In order to firmly establish a role for drug transporters in the development of refractory epilepsy, one must demonstrate that pharmacoresistance can be reversed or prevented via functional or transcriptional modulation, direct inhibition, or the bypassing of multidrug transporters in the BBB. Anecdotal clinical reports have shown that coadministration of the P-gp inhibitor, verapamil, counteracts drug resistancy in single patients (23,24). A large clinical trial is currently planned in Germany and Austria to confirm these observations. In addition to verapamil, there are a number of more selective P-gp inhibitors that have been developed for use in patients with chemotherapy-resistant cancers. Very recent experimental data indicate that coadministration of these inhibitors reverses drug resistance in animal models of TLE:
• Cyclosporin A reverses resistance to phenytoin in a rat model of AED resistant status epilepticus (25).
• Verapamil reverses resistance to oxcarbazepine in rats with pilocarpine-induced seizures (26).
• Tariquidar potentiates the effect of phenytoin in a rat model of TLE (27).
• Tariquidar counteracts resistance to phenobarbital in a rat model of TLE (28).
Convergent experimental and clinical findings support the hypothesis that overexpression of drug efflux transporters in the BBB can produce multidrug resistance in epilepsy. For ultimate proof of principle, however, we must demonstrate that pharmacoresistance can be reversed or prevented by inhibiting multidrug transporters in man. Initial studies indicate that AED resistance can be reversed through an inhibition of P-gp. Hence, coadministration of an AED and P-gp inhibitor may provide a novel treatment strategy for patients with intractable (drug-resistant) epilepsy. Clinical trials are currently underway to evaluate the effectiveness of this treatment regimen.