Jasper's Basic Mechanisms of the Epilepsies Jasper's Basic Mechanisms of the Epilepsies Antiepileptogenesis, Plasticity of AED Targets, Drug resistance, and Targeting the Immature Brain

Heinz Beck1 Yoel Yaari2,3 1 Department of Epileptology, Laboratory of Experimental Epileptology and Cognition Research, University of Bonn Medical Center, Sigmund-Freud Str. 25, 53105 Bonn, Germany 2 Department of Medical Neurobiology, Institute of Medical Research Israel-Canada, Hebrew University- Hadassah School of Medicine, Jerusalem, Israel 3 The Interdisciplinary Center for Neuronal Computation, the Hebrew University, Jerusalem 91904, Israel

Summary Resistance to currently available antiepileptic drugs is a major problem in the treatment of temporal lobe epilepsy that affects ~30% of patients. The antiepileptic drug targets are mostly voltage-gated ion channels or neurotransmitter receptors. Manifold changes in the molecular composition and pharmacology of common antiepileptic drug targets, such as sodium channels, hyperpolarization- activated cationic ion channels and GABA receptors have been described. Available evidence suggests that plasticity of antiepileptic drug targets, with a concomitantly decreased sensitivity to antiepileptic drugs, coexists with other cellular mechanisms to cause pharmacoresistance. A better understanding of the mechanisms of pharmacoresistance is crucial for the development of new antiepileptic therapies.

Introduction The cellular basis of epileptic seizures consists of high frequency, synchronized discharges of neuronal ensembles. The ultimate goal of all antiepileptic therapies is to prevent the occurrence of such episodes or to substantially attenuate their severity. To this end, a multitude of antiepileptic compounds have been developed that are currently in clinical use. However, seizures remain uncontrolled by carefully monitored drug treatment in a substantial portion (~30 %) of epilepsy patients. Therefore, a better understanding of the mode of action of different antiepileptic drugs is mandatory, along with an improved understanding of why these compounds fail in some epilepsy patients, with the ultimate goal of developing new therapeutic avenues.

So far, two hypotheses have been advanced to account for the cellular basis of pharmacoresistance in chronic epilepsy. The first hypothesis proposes that pharmacoresistance involves an up-regulation of multidrug-transporters at the blood-brain barrier. This up- regulation limits the access of antiepileptic drugs to the brain parenchyma, and therefore leads to a reduced drug concentration at the respective drug target. Because multidrug transporter proteins are of central importance to this hypothesis, it has been termed the ‘transporter hypothesis’. The second hypothesis contends that the molecular targets of antiepileptic drugs are modified in chronic epilepsy. Consequently, they are less sensitive to these compounds. This hypothesis has been coined the ‘target hypothesis’.1,2 Clearly, these two hypotheses are not mutually exclusive. Rather, the underlying mechanisms may co-exist and perhaps even act in synergy. The subject of the following review article is the target hypothesis. We describe the molecular mechanisms that alter the targets of antiepileptic drugs and how these mechanisms may interact with altered drug transporter function to cause pharmacoresistance. Page 2

Defining drug targets In general, antiepileptic drug targets are proteins involved in neuronal function, which mediate a reduction in excitability after binding the antiepileptic drug at clinically relevant Jasper's Basic Mechanisms of the Epilepsies Jasper's Basic Mechanisms of the Epilepsies concentrations. A plethora of drug targets has been identified in central neurons (see Table 1). By far most of these targets are neurotransmitter receptors or voltage-gated ion channels. A comprehensive review of the different ion channel types that are affected by antiepileptic drugs is beyond the scope of this review. However, a number of classes of ion channels are particularly prominent targets.

Table 1 Drug targets for common antiepileptic drugs

Voltage-gated ion channels Neurotransmission

INaT INaP ICa IK IH GABA Glu Presynaptic.

Primarily targeting voltage-gated ion channels

Phenytoin + + + +

Carbamazepine + +

Oxcarbazepine + +

Lamotrigine + + + + +

Valproic acid + +/− + +

Losigamone +

Retigabine +

Zonisamide + +

Ethosuximide − + + +

Mixed mechanism

Felbamate + + + +

Topiramate + + + + + +

Primarily affecting neurotransmitter receptors, release or metabolism

Levetiracetam − − + + + (SV2A)

Phenobarbital + +

Benzodiazepine +

Vigabatrin +

Tiagabin +

Other

Gabapentin, primary target alpha2delta-1 subunit − − − − + − − −

INaP: persistent sodium current, INaT: transient sodium current, ICa: calcium currents, IK: voltage-gated potasium currents, IH: H-current, GABA: activity on the GABAergic system, Glu: activity on the glutamatergic system.

Sodium channels Voltage-gated sodium channels are implicated in the mode of action of a large number of common antiepileptic drugs, such as phenytoin, carbamazepine, valproate, lamotrigine, lacosamide and eslicarbazepine.3–5 These channels are found in all excitable cells and are

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capable of extremely rapid channel gating that mediates millisecond scale physiological processes.6 They open rapidly upon membrane depolarization, causing the upstroke of the action potential, and subsequently undergo fast inactivation, thus contributing to its

Jasper's Basic Mechanisms of the Epilepsies Jasper's Basic Mechanisms of the Epilepsies downstroke. Upon repolarization of the cell membrane, sodium channels recover from inactivation with a complex time course. In many cells, however, a non-inactivating, persistent component of the sodium current is also observed in addition to the rapidly inactivating component. This current component activates even during subthreshold depolarizations, and therefore is important in controlling subthreshold activity. Both the transient and the persistent components of the sodium current are potently inhibited by antiepileptic drugs, such as phenytoin, carbamazepine and lamotrigine, in addition to other drugs. A characteristic effect of most of these sodium channel blockers is that they preferentially bind to channels that have entered an inactivated state following depolarization of the cell. In addition, a second major mechanism is that the recovery from depolarization-induced inactivation is prolonged by many antiepileptic drugs.5,4 These two effects together explain why these drugs preferentially block repetitive high-frequency neuronal activity and long-lasting depolarizations, both of which typically occur during epileptic seizures.

Calcium channels Voltage-gated calcium channels are also targets for numerous antiepileptic drugs. CNS neurons express multiple types of calcium channels, which can be separated into two classes based on their biophysical properties, namely, high-threshold and low-threshold calcium channels.7 A number of antiepileptic drugs has been shown to inhibit high threshold calcium channels in native neurons at high therapeutic concentrations.8–10 and this has been proposed to inhibit neurotransmitter release via a reduction of presynaptic action potential-induced calcium rises.11 Some antiepileptic drugs potently inhibit low-threshold (also known as T-type) calcium channels, which are expressed in postsynaptic compartments,12–14 and powerfully control postsynaptic excitability and the propensity to generate burst discharges.15 Intriguingly, the antiepileptic drug gabapentin has been shown to exhibit strong and specific binding to the accessory calcium channel alpha2delta subunit,16 but this binding seems to have no effect on the functional properties of the channel complex, suggesting that gabapentin binding to these subunits exerts effects that are not dependent on calcium channel modulation.17

HCN channels HCN channels, responsible for generating the so called H-current, are cation permeable channels that are activated by hyperpolarization and deactivate upon depolarization of the membrane potential.18,19 H-currents modulate membrane resistance and resting potential, and can mediate pacemaker activity in some types of neurons due to their particular biophysical properties.19 H-currents, and their corresponding HCN channel subunits, appear to be located mainly in dendrites.20–22 Accordingly, they potently modify dendritic integration of excitatory input.22,23 Interestingly, dendritic H-currents are potentiated by application of the antiepileptic drugs lamotrigine or Gabapentin.24,25 Thus, H-currents appear to be dendritic antiepileptic drug targets.

GABAA receptors A further group of antiepileptic drugs seems to exert its main actions via an increase in synaptic inhibition. Gamma-aminobutyric acid (GABA) is the predominent inhibitory neurotransmitter in the adult brain. The ionotropic GABAA receptor, which conducts chloride upon the binding of GABA, is an important target for antiepileptic drugs. GABAA receptor-active drugs include direct modulators such as benzodiazepines and barbiturates, which increase the GABAA receptor-mediated chloride currents via allosteric modulation.26 A number of additional antiepileptic drugs indirectly enhance GABAA receptor-mediated action by inhibiting the re-

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uptake of GABA or its catabolism. This class of compounds includes tiagabine and vigabatrin. Tiagabine inhibits the high-affinity GABA transporter GAT1 that normally terminates synaptic action of GABA via rapid uptake into astrocytes, whereas vigabatrin is a GABA analogue that

Jasper's Basic Mechanisms of the Epilepsies Jasper's Basic Mechanisms of the Epilepsies inhibits GABA transaminase, one of the main GABA degrading enzymes in the brain. Both compounds are capable of causing large elevations in brain GABA levels.

Glutamate receptors A number of newer antiepileptic drugs exert their action primarily or in part by reducing the activity of excitatory neurotransmitter receptors, namely, glutamate receptors. Felbamate exerts complex effects on the NMDA receptor, perhaps by interacting with the modulatory glycine binding site of this receptor.27–30 Some of the effects of felbamate have been shown to be dependent upon NMDA receptor subunit composition.31,32 Topiramate,33 as well as a number of compounds currently in clinical trials,34 have been shown to reduce excitatory synaptic transmission via an inhibition of AMPA receptors.

Presynaptic proteins Recently, the presynaptic vesicle protein SV2A has been identified as a target for the antiepileptic levetiracetam. More intriguingly, SV2A seems to be the only high-affinity receptor for levetiracetam in the rodent brain.35 The SV2A protein is a vesicular component of the complex presynaptic release machinery, and inhibits presynaptic neurotransmitter release in a use-dependent manner. Studies in knock-out mice have suggested that SV2A is involved in regulating the release probability in quiescent neurons, either via protein interactions that facilitate vesicle priming or inhibit depriming, or via transporting a protein involved in priming.36–39 However, the precise effects of levetiracetam binding to SV2A, and how this in turn affects neurotransmitter release are still unknown.

Unconventional drug targets It should be emphasized that many of the antiepileptic drugs discussed so far show multiple modes of action. It is also very probable that the list of drug targets will have to be expanded. Research on drug targets has hitherto focused on voltage-gated or neurotransmitter gated receptor ion channels, which are the most easily investigated drug targets in a quantitative manner. Studies of alternative drug targets that control processes on a slower time scale (e.g., neuroendocrine processes or neuronal structural and functional plasticity) have been conducted more rarely, likely because they are more technically demanding and labour intensive. One intriguing study has identified the calcium channel subunit alpha2delta-1 as a receptor for thrombospondin.17 Thrombospondin is an astrocyte-secreted protein that promotes synaptogenesis via an alpha2delta-1 interaction. Intriguingly, gabapentin antagonizes thrombospondin binding to alpha2delta-1 and powerfully inhibits excitatory synapse formation in vitro and in vivo.17 These findings were the first to identify a specific drug target that may act by inhibiting pathological structural plasticity. It should also be noted that studies concerning mechanisms of antiepileptic drug action and refractoriness have so far focused mostly upon drug targets in neurons, while neglecting many putative targets in glia. Indeed, glial function may also be affected by common antiepileptic drugs.40

Altered pharmacology and molecular plasticity of drug targets in chronic epilepsy In a number of cases, our knowledge of the cellular mechanisms of action of antiepileptic drugs has fuelled studies aimed at finding whether these drug actions are altered in chronically epileptic tissue. Indeed, many drug targets undergo plastic changes on the molecular level in epilepsy models, and probably also in human epilepsy. In the section, we will summarize what is known about epilepsy-associated modifications of drug targets.

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Altered pharmacology of voltage-gated sodium channels A reduced activity of the antiepileptic drug carbamazepine on the transient sodium current in dentate granule cells of the hippocampus has been shown in vitro, both in patients with Jasper's Basic Mechanisms of the Epilepsies Jasper's Basic Mechanisms of the Epilepsies pharmacoresistant epilepsy, as well as in the pilocarpine model of temporal lobe epilepsy. This loss of activity was selective for the use-dependent or frequency-dependent sodium channel blocking effects of carbamazepine, whereas other effects of this drug where not altered.41 A similar but weaker effect was seen in pyramidal cells in the CA1 subregion of the hippocampus.42 Intriguingly, in the pilocarpine model, phenytoin showed a similar but much less pronounced loss of its use- and voltage-dependent sodium channel blocking activity. In contrast, the use- and voltage-dependent blocking effects of lamotrigine and valproate were unchanged.43 Likewise, data from both epilepsy patients and the kindling models of epilepsy, suggest that valproate effects on Na+ channels are unaltered in epileptic tissue.44,45

These data indicate that the molecular mechanisms of pharmacoresistance likely are drug specific, so that changes in sodium channel properties affecting the efficacy of one antiepileptic drug, e.g., carbamazepine, are less relevant for other drugs interacting with the same channel.

The molecular mechanisms leading to the selective loss of use-dependent sodium channel block by carbamazepine, while other actions of carbamazepine on these channels remain untouched, are completely unclear. One potential molecular mechanism is down-regulation of accessory beta-subunits. shown to occur in epilepsy models.46,47 However, this mechanism is unlikely to be relevant, because mice lacking either β1 or β2 subunits display undiminished use- and frequency-dependent block of sodium channels by Carbamazepine.48 A number of additional changes in the subunit composition of sodium channels have been discovered in epilepsy, which may account for the reduced efficacy of Carbamazepine.49 Further possibilities are alternative splicing of sodium channels or the re-expression of neonatal isoforms of sodium channels.50 In addition, posttranscriptional changes in sodium channel properties also may alter the sensitivity of drug targets. For instance, there is evidence that phosphorylation of sodium channels alters the efficacy of some antiepileptic drugs in suppressing the persistent sodium current component.51

Loss of ion channel drug sensitivity occurs not only in the pilocarpine model of chronic epilepsy. A diminished effect of carbamazepine has also been observed on the steady-state inactivation properties of Na+ channels in CA1 neurons from kindled animals.45

Plasticity of calcium channels: Emergence of a novel drug target? The altered expression of calcium channels and their functional consequences have been studied extensively in chronic epilepsy. So far, these studies have yielded no clear evidence of altered Ca2+ channel pharmacology in chronic epilepsy, or of the formation of pharmacoinsensitive channels. However, in the pilocarpine model of epilepsy, the T-type calcium current was shown to be upregulated in CA1 pyramidal cells, at least during the early stage of epileptogenesis, causing the conversion of these ordinarily regularly firing neurons into aberrantly burst-firing neurons.52,53 An up-regulation of T-type calcium current was shown to result from a transcriptional up-regulation of Cav3.2 calcium channel subunits, and 54 was critical for the development of the epileptic condition. This finding suggests that Cav3.2 may constitute a novel drug target that emerges during epileptogenesis. However, studies so far have yielded no clear evidence of altered calcium channel pharmacology or of the formation of pharmacoresistant calcium channels in chronic epilepsy.

Plasticity of H-current expression: Loss of a drug target? In chronic epilepsy, dramatic changes in the magnitude of dendritic H-currents, and expression of the underlying HCN subunits, particularly in pyramidal cells, have been reported in chronic

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epilepsy models.55–57 Given that HCN channels are likely a major target in the antiepileptic action of lamotrigine and gabapentin, it is tempting to speculate that their downregulation causes pharmacoresistance to these drugs. Jasper's Basic Mechanisms of the Epilepsies Jasper's Basic Mechanisms of the Epilepsies

Altered subunit composition of GABAA receptors

An enormous diversity of GABAA receptors has been reported in the CNS, reflecting the fact that in each receptor at least three different subunits are present, deriving from one of eight structurally distinct and genetically distinct families.58,59 An elegant study by Brooks-Kayal et al. has shown that GABAA receptor subunit composition is specifically altered in epilepsy 60 models, and that this is associated with a reduced activity of GABAA receptor agonists. Whether these changes are pertinent for pharmacoresistance to antiepileptic drugs acting on GABAergic inhibition remains to be explored. It is noteworthy that neither the efficacy of GABA uptake, nor the sensitivity of the GABA transporter GAT-1 to tiagabine, are altered in chronic experimental epilepsy.61

Increasing evidence suggests that the actions of GABA in epileptic brain tissue, as in immature 62 brain, are not strictly inhibitory. For instance, in the epileptic human subiculum, GABAA receptor activation in a subset of principal neurons causes depolarisation rather than the ordinary hyperpolarization of the membrane potential.63,64 This change in GABA action is caused by the altered expression of chloride transporters, leading to the intracellular accumulation of chloride.65 Depolarizing GABAergic action in subsets of neurons would be expected to strongly reduce the antiepileptic activity of drugs acting on synaptic inhibition and thus may contribute to pharmacoresistance. This consideration illustrates a more general point: changes in drug targets have to be considered within the context of other epilepsy related cellular changes.

Relationship between changes in antiepileptic drug targets and in-vivo pharmacoresistance What is the relationship of changes in specific drug targets to pharmacoresistance observed either in epilepsy patients or in animal models of chronic epilepsy? Addressing this question requires first to assess the antiepileptic efficacy of a drug in vivo in chronic epilepsy models, and then subsequently to compare it to its efficacy in modifying its putative drug target. This approach has been implemented in few studies only. One study has used kindled rats separated into two groups, one responsive and the other unresponsive to phenytoin.66 In these groups, gross differences in the sensitivity of transient sodium currents to suppression by phenytoin were not found.67 Further studies are needed to compare the two groups with respect to phenytoin’s use-dependent socium current block and its effect on the persistent sodium current. Notwithstanding, this experimental approach, i.e. comparing the pharmacosensitivity of putative antiepileptic drug targets in pharmacoresponsive versus pharmacoresistent epileptic animals, likely will lead to a better understanding of pharmacoresistance.68

A similar approach has been applied in studies of hippocampal tissue resected from carbamazepine-treated epileptic patients, separated into pharmacoresistant and pharmacoresponsive groups. Interestingly, the use-dependent block of sodium currents by carbamazepine was evident in CA1 pyramidal cells in the responsive group, but not in the resistant group.41 Furthermore, epileptiform activity induced acutely was strongly inhibited by carbamazepine in hippocampal slices from the responsive group, but not in slices from the resistant group.41,69 Because changes in blood-brain barrier cannot contribute to pharmacoresistance in these in vitro slice experiments, these findings support the presence of a target mechanism for pharmacoresistance.

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Relationship between the target and transporter hypotheses of pharmacoresistance Functionally relevant changes in antiepileptic drug targets, as well as in multi-drug transporters, occur in chronic epilepsy.2 To define the relative impact of these two mechanisms for each Jasper's Basic Mechanisms of the Epilepsies Jasper's Basic Mechanisms of the Epilepsies individual antiepileptic drug is of obvious therapeutic relevance. In the case of carbamazepine, for instance, a target mechanism is well established in human and experimental epilepsy.41,69 On the other hand, recent studies have revealed that carbamazepine is not transported by numerous human multi-drug transporters including P-glycoprotein, MRP1, 2 and 5.70–72 These results suggest that a target mechanism of pharmacoresistance may be dominant in the case of carbamazepine. On the other hand, a number of other antiepileptic drugs are excellent substrates for the human multi-drug transporter P-glycoprotein1.73,71 In the case of phenytoin or lamotrigine, which are transported efficiently by PGP1,71 target changes in drug sensitivity seem to be less pronounced than for Carbamazepine. 67,43,42 These data support the notion that potential pharmacoresistent mechanisms, and the predominance of either target or transporter mechanisms, have to be evaluated individually for each antiepileptic drug.

Table 2 Relative importance of target and transporter mechanisms MRP1, 2 and 5 and PGP denote types of multidrug transporters. The data given for drug transporters (left side of the table) are based on results described for human multidrug transporters.70–74

Transported by human multidrug transporters Target mechanism: reduction of use-or voltage-dependent block in epilepsy models

MRP1 MRP2 MRP5 PGP

Carbamazepine − − − − Dentate gyrus: +++ 41,69 CA1: +, smaller effects 42

Phenytoin − − − + Dentate gyrus: +, small effects 43 CA1: + small effects 42

Lamotrigine − − − + Dentate gyrus: no change in use-dependent block, only impaired tonic block 43

Valproic acid − − − − - 43,44,45

The therapeutic relevance of these considerations to pharmacoresistent epilepsy is underscored by the fact that a large number of increasingly selective inhibitors exist for multi-drug transporters. Co-medication of antiepileptic drugs with such compounds may overcome pharmacoresistance provided the antiepileptic drug is a substrate of the multi-drug transporter and that upregulation of the transporter contributes importantly to pharmacoresistance to this drug. If a dominant target mechanism exists, the efforts to overcome pharmacoresistance should be aimed at designing novel compounds that act on the modified drug targets in the epileptic brain. Thus, a comprehensive understanding of the dominant mechanisms underlying pharmacoresistance to each antiepileptic drug will be required to instruct strategies towards new therapeutic options.

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