doi:10.1093/brain/awh682Brain (2005) Page 1 of 18
Molecular and cellular mechanisms of
pharmacoresistance in epilepsy
Stefan Remy and Heinz Beck
Department of Epileptology, University of Bonn Medical Center, Bonn, Germany
Correspondence to: Heinz Beck, MD, Department of Epileptology, University of Bonn Medical Center,
Sigmund-Freud-Strasse 25, 53105 Bonn, Germany
Epilepsy is a common and devastating neurological disorder. In many patients with epilepsy, seizures are
well-controlled with currently available anti-epileptic drugs (AEDs), but a substantial (?30%) proportion of
patients continue to have seizures despite carefully optimized drug treatment. Two concepts have been put
forward to explain the development of pharmacoresistance. The transporter hypothesis contends that
the expression or function of multidrug transporters in the brain is augmented, leading to impaired access
of AEDs to CNS targets. The target hypothesis holds that epilepsy-related changes in the properties of the
drug targets themselves may result in reduced drug sensitivity. Recent studies have started to dissect the
molecular underpinnings of both transporter- and target-mediated mechanisms of pharmacoresistance in
human and experimental epilepsy. An emerging understanding of these underlying molecular and cellular
mechanisms is likely to provide important impetus for the development of new pharmacological treatment
Keywords: epilepsy; pharmacoresistance; anti-epileptic drugs; multidrug transporter; ion channel
Abbreviations: AED = anti-epileptic drug; ABC = adenosine triphosphate-binding cassette; PGP = P-glycoprotein
Received April 13, 2005. Revised July 22, 2005. Accepted October 13, 2005
Introduction to the clinical problem of
pharmacoresistance in epilepsy
Epilepsy is a common and devastating neurological disorder.
In many patients with epilepsy, seizures are well-controlled
with currently available anti-epileptic
However, seizures persist in a considerable proportion of
these patients. The exact fraction of epilepsy patients who
are considered refractory varies in the literature, mostly
because the criteria for classification as pharmacoresistant
have varied. Nevertheless, a substantial proportion (?30%)
line AEDs, despite administration in an optimally monitored
regimen (Regesta and Tanganelli, 1999). The fraction of
patients who are pharmacoresistant appears to correlate with
certain features of the epileptic condition, such as a high
seizure frequency or febrile seizures prior to treatment, early
onset of seizures or the presence of certain types of structural
brain lesions. In addition, pharmacoresistance occurs fre-
quently in patients with partial seizures (Aicardi and
Shorvon, 1997; Regesta and Tanganelli, 1999 for a more
complete discussion of clinical aspects of pharmacoresist-
ance). Despite the obvious clinical relevance of uncontrolled
seizures in a large fraction of epilepsy patients, the cellular
basis of pharmacoresistance has so far remained elusive. The
availability of tissue from epilepsy patients undergoing
surgery for focal epilepsies, primarily temporal lobe epilepsy,
has allowed to address some of the mechanisms underlying
pharmacoresistance of focal epilepsies. The mechanisms
underlying the development of resistance in certain forms
of generalized epilepsies are still enigmatic.
Introduction to the cellular candidate
mechanisms of pharmacoresistance
Which key mechanisms govern efficacy of CNS drugs? Firstly,
in the presence of adequate, carefully monitored serum AED
levels, drugs have to traverse the blood–brain barrier (BBB).
Subsequently, CNS activity of AEDs is determined by a
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multitude of factors, including physical properties, such as
lipophilicity, that affect their distribution in different
compartments within the CNS. Consequently, one scenario
to explain pharmacoresistance could be that sufficient intra-
presence of adequate AED serum levels. Such a phenomenon
could arise via an enhanced function of multidrug transport-
ers that control intraparenchymal AED concentrations
(transporter hypothesis of pharmacoresistance, Kwan and
Following permeation into the CNS parenchyma, drugs
have to bind to one or more target molecules to exert their
desired action. Thus, pharmacoresistance may also be caused
by a modification of one or more drug target molecules
(seeTable 1).Thesemodifications would thencause areduced
efficacy of a given AED at the target. This concept has been
collectively termed the target hypothesis of pharmacoresist-
ance (Fig. 1).
Modification in drug targets as basis
The cellular mechanisms of AEDs have been examined to
some extent in normal brain tissue, or ion channels and
receptors in expression systems. These data are summarized
qualitatively in Table 2. Many of these drug targets are altered
on a molecular level in epilepsy. In the following sections, we
will attempt to summarize briefly the known mechanisms of
AEDs on ion channels. We will then focus on emerging
experimental evidence supporting a loss of AED efficacy at
selected targets, and discuss the possible molecular basis of
Changes in molecular drug targets
Voltage-gated Na+currents are ubiquitously expressed in
excitable cells (Fig. 2A, Goldin, 1999; Goldin et al., 2002),
and appear to be targets for multiple first-line AEDs. Upon
depolarization of the membrane, the channels activate and
responsible for the rising phase of the action potential,
and—in some cells—a slowly-inactivating ‘persistent’ current
(INaP, see Fig. 2C). Both current components represent major
targets of several first-line AEDs including carbamazepine,
phenytoin (PHT), lamotrigine and valproate (Ragsdale and
Avoli, 1998; Catterall, 1999; Ko ¨hling, 2002, see also Table 2).
Most AEDs block Na+channels in their resting state (tonic
block) at hyperpolarized membrane potentials (Ragsdale and
Avoli, 1998), with a voltage-dependent enhancement of the
block towards more depolarizing potentials. This voltage-
dependent inhibition is associated with a shift of the steady-
Importantly, blocking effects are activity- or use-dependent,
i.e. blocking effects are enhanced when neurons are repetit-
ively depolarized at higher frequencies (Fig. 2E and F). This
fast Na+channel inactivation (Ragsdale and Avoli, 1998;
Catterall, 1999). It has been suggested that use-dependent
blocking effects are important because they result in a pref-
erential block of INaTduring prolonged high-frequency neur-
onal activity, such as that occurring during seizures.
Several lines of evidence so far have indicated that reduced
efficacy in inhibiting INaTmay be a candidate mechanism of
Table 1 Changes in known AED targets or drug efflux transporters in experimental epilepsy models and
human epileptic tissue
Target ModificationCell type Human
Voltage-gated sodium channels Downregulation of accessory subunits
Altered alpha subunit expression,
induction of neonatal isoforms
Dentate granule cells
CA1 pyramidal neurons
CA1 pyramidal neurons
CA3 pyramidal neurons
Dentate granule cells
CA1 pyramidal neurons
Entorhinal cortex layer 3 neurons
Voltage-gated calcium channels
Increased expression of T-type channels
Loss of dendritic IH
GABAAreceptors: decrease of a1
increase of a4subunits
Dentate granule cells Yes
Capillary endothelial cells
Capillary endothelial cells
MVP (major vault protein)Overexpression No
Page 2 of 18 Brain (2005) S. Remy and H. Beck
pharmacoresistance to some AEDs. Firstly, in CA1 neurons,
the effects of carbamazepine on the steady-state inactivation
properties of INaTwere transiently reduced in the kindling
model of epilepsy (Vreugdenhil and Wadman, 1999b). In
contrast to these comparatively modest and transient effects,
a complete and long-lasting loss of use-dependent blocking
effects of carbamazepine was found in the pilocarpine model
of epilepsy in hippocampal dentate granule cells, as well as in
epilepsy patients with carbamazepine-resistant temporal lobe
epilepsy (Remy et al., 2003a). This dramatic loss of a major
mechanism of action of carbamazepine did not extend to
other AEDs known to affect INaT. Following pilocarpine-
induced status epilepticus, the use-dependent effects of
PHT were reduced, but not completely lost, while the effects
of lamotrigine were completely unchanged (Remy et al.,
2003b). Although the mechanisms of INaTinhibition induced
by valproate are still controversial (Xie et al., 2001); but see
(Vreugdenhil et al., 1998; Vreugdenhil and Wadman, 1999b),
this substance exhibits potent voltage-dependent blocking
effects in various preparations (Fohlmeister et al., 1984;
Zona and Avoli, 1990; Vreugdenhil and Wadman, 1999b;
Ko ¨hling, 2002). Notably, in tissue obtained from pharma-
coresistant patients and in experimental epilepsy no differ-
(Vreugdenhil et al., 1998; Vreugdenhil and Wadman, 1999b;
Remy et al., 2003b). Collectively, these results suggest that
epileptogenesis causes changes in the properties of INaT
that may differ depending on the cell type examined
Fig. 1 In the pharmacoresistant patient the drug faces a modified target, with which it interacts less effectively (A, panel b). Putative
candidate mechanisms resulting in target modifications are seizure-induced changes in transcription or alternative splicing of ion channel
subunits, altered post-translational modification of the protein and/or phosphorylation by protein kinases. A second emerging concept to
explain pharmacoresistance contends that increased expression or function of multidrug transporter proteins decreases availability of the
AED at its target (B, panel b versus B, panel a). In the non-epileptic brain, drug transporter molecules are predominantly expressed in
endothelial cells, and in some cases astrocytes (see text), and appear to regulate intraparenchymal AED concentrations by extruding certain
AEDs over the blood–brain or blood–CSF barrier (indicated by arrows in B, panel a). An upregulation of various drug transporter
molecules has been described in human epilepsy as well as in experimental epilepsy models (B, panel b). This upregulation may decrease the
effective concentration of AEDs at their targets. In addition, in the setting of an epileptic brain, an ectopic expression of certain drug
transporter genes has been observed in astrocytes and in neurons. It remains unresolved whether such transporters control access of
AEDs to intracellular targets (indicated by the absence of an arrow in the neuron in B, panel b).
Pharmacoresistance in epilepsyBrain (2005)Page 3 of 18
Table 2 Summary of AED targets
Voltage-dependent ion channels
(Willow et al.,
1985; Schwarz and
Ragsdale et al.,
1991; Kuo, 1998;
Segal and Douglas,
1997; Lampl et al.,
(Stefani et al.,
Schumacher et al.,
(Todorovic et al.,
et al., 2001)
(Willow et al.,
1985; Schwarz and
Grigat, 1989; Kuo,
et al., 1999;
(Schumacher et al.,
(McLean et al.,
et al., 1994)
(Schmutz et al.,
1994; Stefani et al.,
(Xie et al., 2001;
Xie et al., 1995;
Zona and Avoli,
1997; Remy et al.,
2003b; Kuo, 1998)
(Spadoni et al.,
(Stefani et al.,
et al., 1996)
(Huang et al.,
2004; Zona et al.,
(Poolos et al.,
Zona and Avoli,
et al., 1998;
(Taverna et al.,
1998), but see
et al., 2004)
(Taverna et al.,
(Lo ¨scher, 1989)
(Gebhardt et al.,
(Main et al., 2000),
(Tatulian et al.,
2001; Yue and
(Suzuki et al.,
Page 4 of 18 Brain (2005)S. Remy and H. Beck
(Taglialatela et al.,
(Stefani et al.,
(Rho et al., 1997)
et al., 1995; White
et al., 1995; Kuo
et al., 2004)
(Zona et al., 1997;
Taverna et al.,
et al., 2000)
(Taverna et al.,
(Zhang et al.,
(Herrero et al.,
Gordey et al.,
(Gibbs, III et al.,
2000; Gryder and
(Leresche et al.,
(Coulter et al.,
and Lingle, 1998;
Gomora et al.,
2001), but see
(Leresche et al.,
(Leresche et al.,
No effect: (Zona
et al., 2001)
et al., 2004)
(Lukyanetz et al.,
effect: (Zona et al.,
(Madeja et al.,
(Alden and Garcia,
2001), but see
(Schumacher et al.,
(Freiman et al.,
(Surges et al.,
(McClelland et al.,
et al., 1993)
(Twyman et al.,
(Study and Barker,
1994; Eghbali et al.,
et al., 1999)
(Jolkkonen et al.,
1992; Lo ¨scher and
1994; Wu et al.,
(Fink-Jensen et al.,
and Ga ¨hwiler,
Pharmacoresistance in epilepsyBrain (2005) Page 5 of 18
(i.e. dentate granule cells versus CA1 neurons), and according
to the epilepsy model studied. Changes in INaTmay then
dramatically affect sensitivity to some, but not all, AEDs.
Block of INaPmay also be a crucially important mechanism
of AED action. Pharmacological augmentation of INaPcauses
an increased propensity of individual neurons to generate
burst discharges (Su et al., 2001), and several mutations
that give rise to increased INaPcause epilepsy in mice or
humans (Kearney et al., 2001; Lossin et al., 2002; Rhodes
et al., 2004; Spampanato et al., 2004). INaP is efficiently
Fig. 2 Expression, functional properties and AED pharmacology of voltage-gated sodium channels. The Na+channel alpha subunits
NaV1.1–NaV1.9 are widely distributed in excitable cells and exhibit a tissue-specific expression (A). Upon depolarization the channels
activate and give rise to a rapidly inactivating ‘transient’ inward Na+current (INaT, B), responsible for the rising phase of the action potential.
In addition, in some cells a slowly-inactivating ‘persistent’ current has been described (INaP, C). Slow voltage-ramp commands allow to
describe the voltage-dependent properties of this current component in isolation. The persistent Na+current contributes to spike after-
depolarizations in some neurons and plays a role in the generation and maintenance of subthreshold membrane oscillations. Na+channels
are the main targets for a subset of AEDs including carbamazepine, PHT and lamotrigine. The drugs acting on INaTchannels display two
predominant mechanisms of action: 1. Voltage-dependent inhibition, associated with a shift of the steady-state inactivation curve in a
hyperpolarized direction. This shift decreases the availability of the Na+channel during an action potential and, therefore, reduces the
excitability of the cell (D). 2. Activity- or use-dependent blocking effects, i.e. blocking effects are enhanced when neurons are repetitively
depolarized at higher frequencies (E and F). This results in a highly efficient block of INaTpreferentially during prolonged high-frequency
neuronal activity, such as that occurring during seizures.
Page 6 of 18Brain (2005) S. Remy and H. Beck
blocked by many AEDs, frequently at a concentration range
lower than that observed for INaT(Ko ¨hling, 2002). In native
well as losigamone (Chao and Alzheimer, 1995; Lampl et al.,
data). Despite the potential importance of INaPin the regu-
epileptogenesis have so far not been described.
What mechanisms can account for an altered sensitivity of
Na+channels in epileptic tissue? One possibility may be that
the subunit composition of these channels is altered, such
that the expression of AED-insensitive subunits or subunit
combinations is promoted. Indeed, numerous changes in Na+
channel subunit expression have been observed in both
human and experimental epilepsy (Bartolomei et al., 1997;
Aronica et al., 2001; Whitaker et al., 2001; Ellerkmann et al.,
2003). In this respect, the downregulation of accessory Na+
channel b1and b2subunits following experimentally induced
status epilepticus appears to be a consistent finding (Gastaldi
et al., 1998; Ellerkmann et al., 2003). In addition to changes in
mRNA levels, altered alternative splicing of pore-forming
subunit mRNAs has also been observed (Gastaldi et al.,
1997; Aronica et al., 2001). A recent, very interesting study
underscores the potential importance of the b1subunit in the
development of pharmacoresistance. In the paper by Lucas
et al. (2005), the pharmacology of Na+channels containing a
mutant b1subunit causing the epilepsy syndrome generalized
epilepsy with febrile seizures plus was examined. Surprisingly,
Na+channels containing mutant b1subunits displayed a dra-
matic and selective loss of use-dependent block by the AED
PHT, that was very similar to the effects observed in chronic
experimental epilepsy for PHT and carbamazepine (Remy
et al., 2003a, b). These results collectively suggest that changes
in accessory subunits might be promising candidates for
further investigation as a molecular correlate of the AED-
insensitive sodium channel. They are also intriguing because
they suggest that use-dependent effects on Na+channels
require some form of interaction with b1subunits, whereas
this may not be the case for tonic block of Na+channels.
It is at present unclear why use-dependent block by
carbamazepine and PHT is lost or reduced, whereas use-
dependent block by lamotrigine remains intact in experi-
mental epilepsy. This is an intriguing question because it
has been suggested that all three drugs bind to the same
site on Na+channels in CA1 neurons based on coapplication
experiments (Kuo et al., 1998). The mutual exclusivity among
the binding of drugs simultaneously applied to a channel may
however result from either an allosteric mechanism or from
direct competition of the drugs at a single binding site. Even
though a single binding site appears to provide the most
parsimonious explanation for these results, it is quite
conceivable that allosteric interactions between different
binding sites may also exist. This would provide a basis for
the AED-selective loss of sensitivity observed in chronic
Other types of voltage-gated channels
Other types of voltage-gated channels have also been screened
as potential drug targets. In many cases, effects of AEDs on
specific ion channel subunits or ion channels in native neur-
ons or expression systems have been described (see Table 2).
Ca2+channels can be subdivided into two groups: high-
threshold Ca2+currents, and a group of low-threshold
currents (also termed T-type Ca2+currents, Ertel et al.,
2000). A number of AEDs has been shown to inhibit high
threshold Ca2+channels in native neurons at high therapeutic
concentrations (Stefani et al., 1997b, 1998; Schumacher et al.,
1998; see Table 2). Additionally, the AED gabapentin has been
shown to exhibit strong and specific binding to the accessory
a2d subunit (Gee et al., 1996). It has been proposed that
this effect underlies inhibition of presynaptically expressed
high-threshold Ca2+channels by gabapentin, which causes
a reduction in neurotransmitter release (Fink et al., 2000;
see Table 2). Some AEDs potently inhibit low-threshold
T-typeCa2+channels, which are notexpressedpresynaptically
(Yaari et al., 1987; Coulter et al., 1989; Gomora et al., 2001;
see Table 2). The effects of AEDs on the three T-type Ca2+
channel subunits, as well as in native neurons, are diverse
(cf. Todorovic and Lingle, 1998; Todorovic et al., 2000;
Lacinova, 2004). T-type channels are critically important in
controlling the excitability of the postsynaptic compartment
of neurons (Huguenard, 1996), both in normal and epileptic
neurons. For instance, aberrant bursting is seen in CA1 hip-
pocampal neurons from epileptic animals (Sanabria et al.,
2001) that is mediated by increased expression of T-type
Ca2+channels (Su et al., 2002). Additionally, T-type Ca2+
channels in thalamic neurons have been implicated in the
generation of spike-wave discharges in absence epilepsy
(Huguenard, 2002 and references therein). Consequently,
inhibition of burst discharges in thalamic neurons is thought
to contribute tothe anti-epileptic effects of antiabsenceAEDs.
It is so far unknown if the sensitivity of either presynaptic or
post-synaptic Ca2+channels to AEDs changes during epilep-
togenesis. The same applies to other voltage-gated ion chan-
nels such as K+channels (see Table 2).
H-currents (IH) are mixed cationic currents that are activ-
ated by hyperpolarization and deactivated following repolar-
ization of the membrane. IHhas multiple functional roles; for
instance, it mediates some forms of pacemaker activity in
heart and brain, it regulates membrane resistance and dend-
ritic integration and stabilizes the level of the resting potential
(reviewed, Robinson and Siegelbaum, 2003). An interesting
predominantly located in dendrites, rather than the soma
of neurons (Poolos et al., 2002). Interestingly, dendritic
H-currents are potently enhanced by the AEDs lamotrigine
and gabapentin at clinically relevant concentrations (Poolos
et al., 2002; Surges et al., 2003, see Table 2), resulting in
IH-mediated inhibitory effects on action potential firing by
selectively reducing the excitability of the apical dendrites
(Poolos et al., 2002). Cell-type specific changes in IHhave
Pharmacoresistance in epilepsyBrain (2005)Page 7 of 18
been described in models of epilepsy (Chen et al., 2001),
including a dramatic loss of dendritic IHin entorhinal cortex
neurons (Shah et al., 2004). The importance of this change for
pharmacoresistance to lamotrigine has not been directly
addressed, but it is conceivable that a sufficiently large reduc-
tion of these channels could constitute a de facto loss of a
major drug target for lamotrigine in this subregion.
Neurotransmitter systems: GABA
GABA is the predominant inhibitory neurotransmitter in the
adult brain and plays a critical role in the regulation of excit-
ability of neuronal networks (Mody and Pearce, 2004). GABA
binding to ionotropic GABAAreceptors causes opening of the
receptor ionophore, which is permeable to Cl?and—to a
lesser extent—to HCO3. In the presence of a normal adult
transmembraneous Cl?gradient, this results in expression of
an inhibitory post-synaptic current that hyperpolarizes the
post-synaptic neuronal membrane. Direct modulators of
GABAAreceptors include benzodiazepines and barbiturates.
Benzodiazepines increase GABA affinity of the receptor com-
plex and may augment their Cl?conductance via allosteric
modulation (Twyman et al., 1989a; Rudolph et al., 1999,
2001). Substances that interact with the GABA system in a
more indirect way affect the handling and metabolism of
synaptically released GABA.
GABA) is a GABA analogue that inhibits one of the main
enzymes controlling GABA concentrations in the brain,
GABA transaminase. Consequently, application of vigabatrin
causes large elevations in brain GABA levels. The AED tiaga-
bine inhibits the high-affinity GABA transporter GAT1 that
normally terminates synaptic action of GABA via rapid
uptake. So far, available evidence indicates that neither the
efficacy of GABA uptake, nor its sensitivity to tiagabine is
altered in chronic experimental epilepsy (Frahm et al., 2003).
Regarding GABAAreceptor agonists, reduced activity of
such substances has been described in a chronic model of
epilepsy. In the pilocarpine model of epilepsy, GABAArecept-
ors of dentate granule cells show a reduced sensitivity to drugs
acting on the benzodiazepine receptor site 1. While augmen-
tation of GABA-evoked currents by the broad-spectrum
animals, augmentation by the benzodiazepine site 1-selective
agonist zolpidem was strongly decreased (Gibbs et al., 1997;
Brooks-Kayal et al., 1998; Cohen et al., 2003). In CA1 pyr-
amidal cells, the effects of clonazepam were dramatically
reduced in chronically epileptic animals (81% reduction rel-
ative to control, (Gibbs et al., 1997). This suggests that the
same might also apply to clinically employed benzo-
What is the molecular mechanism of this change in GABAA
receptor pharmacosensitivity? An enormous diversity of
GABAAreceptors has been reported in the CNS, reflecting
the fact that in each receptor at least three different sub-
units are present, which derive from one of eight structur-
ally distinct and genetically distinct families (Costa, 1998;
Sperk et al., 2004). Combined molecular and functional
studies indicate that a transcriptionally mediated switch in
the alpha subunit composition of GABAAreceptors occurs
in epileptic animals, in particular a decrease of a1subunits
and an increase of a4subunits (Brooks-Kayal et al., 1998).
These findings correlate well with the observed changes in
benzodiazepine receptor pharmacology.
Neurotransmitter systems: glutamate
Despite the undoubted importance of altered glutamate-
mediated excitatory neurotransmission in chronic experi-
mental (Mody and Heinemann, 1987; Martin et al., 1992;
Kohr et al., 1993) and human epilepsy (Isokawa and
Levesque, 1991), few substances acting on this system have
been developed to clinical use so far. Felbamate exerts com-
plex effects on the NMDA receptor (Kuo et al., 2004), some of
which may be mediated via the modulatory glycine binding
site (White et al., 1995). Some of the effects of felbamate have
been shown to be affected by NMDA receptor subunit com-
position (Kleckner et al., 1999; Harty and Rogawski, 2000).
AEDs acting on AMPA receptors are also scarce, some
drugs currently in clinical trials inhibit AMPA receptors
(talampanel, see Chappell et al., 2002). Likewise, topiramate
has been shown to reduce excitatory synaptictransmission via
an inhibition of AMPA receptors (Qian and Noebels, 2003).
Altered cellular expression of glutamate receptors in
epilepsy should be considered in future development of com-
pounds acting on these receptors. Given the paucity of estab-
lished AEDs acting on individual glutamate receptors,
however, we will abstain from an in depth discussion of
these changes here.
Role of changes in drug targets in the
setting of a chronically epileptic brain
The specific changes in drug targets described above are an
ant to realize, however, that not only changes in drug targets
themselves, but also changes in other molecules that affect
their function may have important consequences for AED
efficacy. This idea is exemplified by recent findings regarding
the role of GABA in epilepsy. GABA may on occasion act as
an excitatory neurotransmitter in the immature brain. A
depolarizing action of GABAA receptor activation arises
because of an altered chloride homeostasis, resulting in a
changed chloride gradient across the neuronal membrane.
The altered chloride reversal potential then results in a net
outward flux of Cl?through the GABAAreceptor ionophore,
causing depolarization of the neuron (Mody and Pearce,
2004). Interestingly, in addition to the developing brain,
depolarizing GABA responses appear to be a feature of
some neurons in the epileptic brain (Cohen et al.,
2002; Wozny et al., 2003). Augmenting such depolarizing
GABA-mediated potentials by application of GABA agonists
is likely to facilitate action potential generation to excitatory
Page 8 of 18Brain (2005) S. Remy and H. Beck
neuronal excitability instead of decreasing it. Whether depol-
arizing GABA responses really play a role in pharma-
coresistance to GABAmimetic drugs remains to be seen.
These considerations do, however, illustrate the need to con-
sider changes in drug targets within the more general setting
of a chronically epileptic brain.
Molecular mechanisms underlying altered
So far, most of the mechanisms implicated in altered AED
targets are changes in the transcription of ion channel sub-
units. Seizures appear to cause a highly coordinated change in
transcription of certain groups of ion channel subunits, both
in rat models of epilepsy (Brooks-Kayal et al., 1998) and in
human epilepsy patients (Brooks-Kayal et al., 1999; Bender
et al., 2003). This seizure-induced transcriptional plasticity
appears to be differentially regulated in different neuron types
(cf. Bender et al., 2003; Shah et al., 2004). These transcrip-
tional changes most probably affect both the density of ion
channels in the neuronal membrane, as well as the subunit
stochiometry of multisubunit channel complexes (Brooks-
Kayal et al., 1998). In addition to transcriptional mechanisms,
seizure activity may also evoke multiple post-translational
modifications of ion channel proteins, such as altered protein
transport and targeting, phosphorylation or glycosylation
(Bernard et al., 2004). Indeed, increased phosphorylation
of INaby protein kinase C has been shown to affect respons-
iveness to the AED topiramate in one study (Curia et al.,
2004). It is quite possible that other post-transcriptional
modifications of ion channel proteins induced by seizures
may profoundly affect their drug sensitivity. How seizures
may modify the pharmacosensitivity of AED targets is
summarized schematically in Fig. 3.
Relationship of molecular changes in AED
sensitivity to pharmacoresistance
observed in vivo
How is the loss of AED sensitivity on the level of an ion
channel such as INaTrelated to pharmacoresistance observed
in human epilepsy patients or intact animals? In epilepsy
patients, properties of INaTseemed to differ when patients
are separated into two groups, one resistant to carbamazepine
anda smaller one responsivetocarbamazepine.In theformer,
the use-dependent block of INaTproved to be abolished,
similar to the findings in the pilocarpine model of epilepsy.
use-dependent effects of carbamazepine on INaT. Thus, the
sensitivity to carbamazepine on a cellular level appeared to
correlate with the clinical responsiveness to the same drug.
Firstly, the number of patients for which both clinical and
in vitro data could be obtained is still limited, particularly
when considering the group of pharmacoresponsive epilepsy
patients (Remy et al., 2003a). Secondly, patients who are
resistant to carbamazepine very frequently are resistant also
to other AEDs (Kwan and Brodie, 2000). However, available
data indicate that altered sensitivity of Na+channels may not
be able to account for altered efficacy of other AEDs such as
valproic acid or lamotrigine (Remy et al., 2003b). This finding
may indicate that resistance to AEDs in epilepsy patients is a
complex phenomenon that possibly relies on multiple mech-
yield candidate gene polymorphisms that may be associated
with AED sensitivity (Tate et al., 2005).
The correlation of target pharmacosensitivity and sensitiv-
ity to AEDs in vivo in experimental models of epilepsy is quite
unclear. The pilocarpine model of epilepsy has been
frequently used to study changes in pharmacosensitivity of
drug targets. Leite and Cavalheiro (1995) have provided some
evidence that high doses of common AEDs such as car-
bamazepine, PHT and valproate reduce the spontaneous
seizure frequency in these animals. This is in apparent con-
tradiction to the finding that Na+channels in the same model
are resistant to carbamazepine. It is possible that this may be
due to the high doses of AEDs used, or alternatively to dif-
ferences in the rat strains and/or pilocarpine protocols used.
Furthermore, it is likely that some groups responsive and
resistant to AED may exist in chronic epilepsy models
(Lo ¨scher et al., 1998; Nissinen and Pitka ¨nen, 2000). Ideally,
these questions could, therefore, be resolved by first examin-
ing pharmacosensitivity in intact animals, and subsequently
comparing these in vivo results to the cellular effects of AEDs
in the same individuals. So far, this important approach has
only been implemented in few experiments (Jeub et al., 2002).
For these experiments, kindled rats were used that could be
separated into two groups based on their responsiveness to
compared to a group of rats that were not, no difference in
PHT sensitivity of INaTemerged. It should be noted, however,
that PHT effects on the recovery from inactivation and use-
dependent block, where the most dramatic effects were seen in
the pilocarpine model of epilepsy, were not examined in this
groups with differential pharmacosensitivity can be defined
represent a promising avenue to study mechanisms of phar-
macoresistance (Nissinen and Pitka ¨nen, 2000). It remains to
be seen how far such animal models mirror mechanisms of
pharmacoresistance in human epilepsy patients.
Modification in multidrug transporters
as basis for pharmacoresistance
Overview of multidrug transporter
molecules expressed in the brain
The second main emerging concept to explain pharma-
coresistance contends that increased expression or function
of multidrug transporter proteins decreases the effective
concentration of AEDs at their targets (see Fig. 1B). Intense
Pharmacoresistance in epilepsyBrain (2005)Page 9 of 18
interest has been focused on understanding the molecular
basis of multidrug transport in the brain in recent years,
primarily because of their potential importance in mediating
resistance to anticancer drugs. This effort has led to the dis-
function as drug efflux pumps. These genes are highly con-
served and the vast majority belongs to the superfamily of
adenosine triphosphate-binding cassette (ABC) proteins. A
large number of human genes belonging to this superfamily
have been identified, which have been systematically classified
into seven subfamilies [ABCA, ABCB, ABCC, ABCD, ABCE
ABCF and ABCG (Dean et al., 2001)]. Most of these genes
encode ATP-driven pumps that are able to transport a wide
range of substrates.
Studies addressing the role of multidrug transporters in
the development of pharmacoresistant epilepsy have hitherto
been focused mainly on a subset of these transporters. MDR1
(belonging to the ABCB subfamily, systematic nomenclature
ABCB1) encodes P-glycoprotein (PGP, Silverman et al.,
1991; Ueda et al., 1993), which transports a wide range of
lipophilic substances across cell membranes. A further family
of genes [multidrug-resistance associated proteins or MRPs,
transports a range of substances partially overlapping with
those transported by PGP. Most of the proteins encoded
by these genes (i.e. MRP1 to 6 and PGP) are expressed in
endothelial cells of the blood–brain or blood–CSF barrier
(Schinkel et al., 1996; Huai-Yun et al., 1998; Rao et al.,
1999; Zhang et al., 1999). In addition, MRP1 and one of
the two rodent isoforms of PGP are present in astrocytes
(Pardridge et al., 1997; Regina et al., 1998; Golden and
Pardridge, 1999; Decleves et al., 2000). The functional role
of this expression is currently a matter of debate (Golden and
Altered expression of multidrug
transporters in human and experimental
epilepsy, and consequences for
intraparenchymal AED concentrations
Several lines of evidence indicate a role of multidrug trans-
porters in the development of resistance to AEDs, which are
set out in more detail as follows. Firstly, drug transporters
transport some AEDsin isolated cell systems(Batrakova et al.,
1999; Marchi et al., 2004). Secondly, drug transporters appear
to regulate intraparenchymal drug concentrations in vivo in
many cases. Mice or rats lacking certain drug transporters
display increased accumulation of AEDs (Schinkel et al.,
1996, 1997; Rizzi et al., 2002; Potschka et al., 2003b, but see
Sills et al., 2002; see Table 3). It should be noted that inter-
pretation of data from such animal models is complicated by
two issues: firstly, there may be a compensatory regulation of
other drug transporter molecules and, secondly, the wide
Fig. 3 Epileptic seizures trigger an activity-dependent sequence of events resulting in alterations in neuronal firing and pharmacosensitivity.
Known changes triggered by seizures include coordinate changes in ion channel transcription, altered post-translational processing of ion
channel proteins, or altered modification of channels by second-messenger systems. Many of these changes result in defined
pharmacological and functional changes in ion channels. These changes may underlie altered responses to AEDs as well as altered
excitability. It should be noted that this model depicts acquired activity-dependent changes caused by seizures. Reduced responses to AEDs
may also be a pre-existing condition caused by genetic polymorphisms that affect specific AED targets (see discussion in text).
Page 10 of 18 Brain (2005) S. Remy and H. Beck
expression of drug transporters in other tissues may result in
complex pharmacokinetic effects of deleting drug transporter
genes. These issues do not apply when drug transporters are
inhibited pharmacologically in vivo. Indeed, pharmacological
inhibition of drug transporters alters brain distribution of
some AEDs (Potschka and Lo ¨scher, 2001; Potschka et al.,
2001, 2003b; Lo ¨scher and Potschka, 2002, see Table 3). It
employed thus far are not very specific, and that studies using
more specific novel inhibitors are currently being undertaken.
Interestingly, the results in the literature with regard to one
of the most frequently employed AEDs, carbamazepine, are
not uniform. This drug is not transported in PGP-containing
cell systems, and its brain concentration remains unaltered in
mice lacking PGP (Owen et al., 2001), or animals lacking
MRP2 (Potschka et al., 2003a). On the other hand, pharma-
cological (Potschka et al., 2001) or genetic (Rizzi et al., 2002)
intraparenchymal carbamazepine concentration under some
conditions. The reasons for this discrepancy are currently
under scrutiny. That not all AEDs may be transported equally
(Schinkel et al., 1996). As a further possibility, carbamazepine
has been shown to itself inhibit the activity of human
P-glycoprotein, albeit at high concentrations which may
not be clinically relevant (Weiss et al., 2003).
A large body of evidence suggests that different drug trans-
porter molecules are indeed upregulated in human epilepsy,
as well as in experimental models of epilepsy. For instance,
increased MDR1 expression on the mRNA and/or protein
level occurs in patients with different forms of epilepsy
(Tishler et al., 1995; Lazarowski et al., 1999; Sisodiya et al.,
1999, 2002; Aronica et al., 2003; Volk and Lo ¨scher, 2005) and
after chemically-induced status epilepticus or audiogenic
seizures (Zhang et al., 1999; Kwan et al., 2002; Rizzi et al.,
2002). Similar findings have been obtained for MRP1
[(Sisodiya et al., 2001, 2002), MRP2 (Dombrowski et al.,
2001)] and major vault protein (Van Vliet et al., 2004).
These studies have also shown that expression of drug
transporter genes in epileptic foci is observed in cell types
that do not usually express them. For instance, PGP and
MRP1 appear to be strongly upregulated on the protein
level in astrocytes, especially surrounding blood vessels
(Sisodiya et al., 2002). Likewise, there may be upregulation
of drug transporter expression in dysplastic neurons in focal
cortical dysplasia (Sisodiya et al., 2001), as well as in hippo-
Volk et al., 2004). It is yet unclear how this ectopic expression
might contribute to altered pharmacosensitivity. It is entirely
possible, however, that expression of multidrug transporters
in neuronal membranes inhibits access of AEDs to intracel-
lular sites of action.
Relationship of molecular changes in AED
sensitivity to pharmacoresistance
observed in vivo
If multidrug transporters play a significant role in phar-
macoresistance, then upregulation of transporters on the
molecular or functional level should correlate with the clin-
ically observed responsiveness to AEDs. Indeed, increased
drug transporter expression appeared to be correlated with
less efficient seizure control in one study (Tishler et al., 1995).
This correlation is also found inan animalmodelof resistance
to AEDs. Rats resistant to phenobarbital showed a dramatic
overexpression of PGP in limbic brain regions compared to
rats responsive to phenobarbital (Volk and Lo ¨scher, 2005).
Similar findings have been obtained in an elegant study using
MRP2-deficient rats. In this study, MRP2-deficient kindled
rats have higher PHT brain levels than wild-type rats, and are
more susceptible to PHT treatment (Potschka et al., 2003b).
These findings are interesting because they constitute the first
controlled experiment in which deficiency in a specific drug
transporter is associated with differential susceptibility to
Even though, collectively, these findings appear to support
a role for multidrug transporters in pharmacoresistant epi-
lepsy, there are a number of conceptual questions that remain
enigmatic. Firstly, epileptic seizures are known to result in a
disruption of the BBB, which would be expected to result in
better access of AEDs to brain parenchyma despite the
upregulation of multidrug transporters. Secondly, patients
are in many cases treated with AEDs until CNS side effects
develop. This seems to indicate that relevant CNS concentra-
tions of AEDs are reached despite transporter upregulation,
yet, these patients are resistant to treatment. This apparent
discrepancy could potentially arise via local upregulation of
drug transporters that only affects AED concentrations at the
The genetic basis of pharmacoresistance
How can the wide spectrum of pharmacoresistance observed
in human epilepsy patients and some animal models be
explained? A number of recent studies have suggested that
Table 3 Drug efflux transporters and their
anticonvulsant drug substrates (adapted from
Loescher and Potschka, 2005)
Drug efflux transporterSubstrate
PGP (MDR1, ABCB1)Phenytoin (Tishler et al., 1995;
Schinkel et al., 1996; Potschka and
Lo ¨scher, 2001; Rizzi et al., 2002)
Carbamazepine (Potschka et al., 2001)
Phenobarbital (Potschka et al., 2002)
Lamotrigine (Potschka et al., 2002)
Felbamate (Potschka et al., 2002)
Topiramate (Sills et al., 2002)
Phenytoin (Potschka et al., 2003b) MRP2 (ABCC2)
Pharmacoresistance in epilepsy Brain (2005)Page 11 of 18
sequence variants in drug transporter or ion channel genes
affect either function or expression of the corresponding
proteins. In the case of drug transporters, a number of func-
tionally relevant polymorphisms have been identified (Kerb
et al., 2001a, b). Furthermore, a polymorphism (C3435T) has
been identified in exon 25 of the gene encoding MDR1 that
is associated with increased expression of the protein (CC-
genotype). Based on these findings, Siddiqui et al. (2003)
conducted a population-based association study testing the
hypothesis that the C3435T polymorphism is associated with
resistance to AED treatment. They found that patients
with drug-resistant epilepsy were more likely to have the
CC-genotype than the TT-genotype [OR 2.66, 95% CI
suggested by the authors of this study, the C3435T poly-
morphism by itself is very unlikely to confer a biologically
relevant effect. Since this variant is localized in an extensive
block of linkage disequilibrium spanning the gene, the as yet
unidentified causal variant is supposed be in linkage disequi-
librium with the C-allele of the C3435T polymorphism. It
should be noted that the results of Siddiqui et al. (2003)
have not been confirmed in a subsequent study by Tan
et al. (2004). To further address how polymorphisms can
contribute to drug resistance, two major obstacles will have
to be overcome. Firstly, it will be necessary to address experi-
mentally whether polymorphisms found in association stud-
ies have biologically relevant effects. Secondly, it will be
patient cohorts to increase reproducibility of such results
(Soranzo et al., 2004; Cavalleri et al., 2005). Finally, it will
be interesting to extend current studies to include poly-
morphisms in other multidrug transporters. In this respect,
the development of single nucleotide polymorphism tagging
for classes of genes important in resistance is a very important
step that may enable screening of large numbers of patients
(Ahmadi et al., 2005).
It is important to note that gene polymorphisms
relevant for pharmacoresistance may occur both in promoter
regions as well as in introns and exons. Gene polymorphisms
within the coding regions of such genes would result in a
difference in ion channel or transporter proteins that
precedes the onset of epilepsy. Polymorphisms in promoter
regions, which affect the transcription of such genes, may
affect activity-dependent transcriptional regulation of these
genes by seizures. This provides a potential mechanism for
the acquisition of a pharmacoresistant phenotype during
et al., 2003)]. As
Interplay between target and
Collectively, the experimental results described in the
above sections indicate that functionally relevant alterations
in both AED targets and AED transporters exist. Clearly, these
mechanisms are not mutually exclusive. It is entirely possible
that decreased permeation of AEDs into brain tissue, in
synergy with changes in targets for these drugs, mediate phar-
macoresistance. This does not exclude that—for some
AEDs—predominant mechanisms underlying pharmacores-
istance to these drugs can be identified.
Carbamazepine, for instance, appears not to be a substrate
of some multidrug transporters (Owen et al., 2001); rather,
sodium channels display a potent loss of sensitivity to
carbamazepine (Remy et al., 2003a). These and other results
imply that expression of multidrug transporters at the BBB
is not the main factor in the development of resistance,
and imply a target mechanism for this drug. For other
AEDs, this may be different. For instance, intraparenchymal
PHT concentration is potently regulated by multidrug trans-
porters (Potschka and Lo ¨scher, 2001; Rizzi et al., 2002;
Potschka et al., 2003b), and epileptic animals lacking a mul-
tidrug transporter protein (MRP2) are more sensitive to PHT
treatment than wild-type animals (Potschka et al., 2003b). In
contrast, target mechanisms seem to be less important for
PHT compared to carbamazepine (Jeub et al., 2002;
Remy et al., 2003b). Although the evidence supporting this
view is far from conclusive, it is tempting to speculate that
predominant resistance mechanisms may exist for specific
Future directions of research
Whatare thekeypiecesofevidencethat weshould consider to
beprerequisites inorder tostate thatdrug transportersand/or
altered targets play a role in the development of resistance to a
given AED? We believe that the following sets of experimental
results should be available:
(i) Evidence that the multidrug transporter regulates intra-
parenchymal concentrations of the drug: this should
include work with specific drug transporter inhibitors,
and mice lacking specific drug transporter subtypes, as
well as a combination of pharmacological and genetic
inhibition of transporter function.
(ii) Evidence that multidrug transporter expression and/or
transporter function is upregulated in human and
experimental epilepsy. Regarding drug targets, evidence
should be available that drug targets are less sensitive to a
given AED in chronic epilepsy. In both cases, functional
and molecular changes should correlate with AED sens-
itivity of seizures in experimental animals or epilepsy
(iii) Evidence that genetic or pharmacological manipulation
of drug transporters/drug targets affects sensitivity of
spontaneous seizures to AEDs in vivo in chronic models
In addition, the following data on human epilepsy patients
should be obtained.
(iv) Association of polymorphisms in drug transporter/drug
target genes with clinical pharmacoresistance. This could
also include association of polymorphisms with the drug
Page 12 of 18Brain (2005) S. Remy and H. Beck
transporter function or drug target pharmacology
measured in vitro in tissue obtained from epilepsy
(v) Demonstration thatdrug
isms have functional effects resulting in a decreased
intraparenchymal AED concentration (i.e. effects on
either expression or function of drug transporter
molecules). Likewise, polymorphisms in drug target
genes should have demonstrable effects on expression
or pharmacology of these targets.
Fig. 4 Potential implications for treatment of pharmacoresistant epilepsy when taking into account different mechanisms underlying
pharmacoresistance. Responsive patients (A): In patients responsive to a given AED, the AED gains sufficient access to a target. In addition,
the AED has sufficient pharmacological effects on the target. Resistant patients, predominant transporter mechanism of resistance (B): In a
scenario of increased drug transport without any changes in the AED target, the use of AEDs that are not transporter substrates and may
additionally exhibit transporter-inhibiting properties would be advantageous in obtaining therapeutic drug levels at the site of action.
Alternatively, the use of transporter inhibitors may increase the intraparenchymal drug concentration to therapeutic levels and may help to
overcome the reduced efficacy of the drug. Resistant patients, predominant target mechanism of resistance (C): If resistance for a given
AED is due to a change in its target, increasing the intraparenchymal AED concentration by comedication with transporter inhibitors would
not be expected to be beneficial. In this case, the development of drugs that specifically act on the modified target would be the more
appropriate approach. Resistant patients, mixed resistance mechanism (D): If both transporter- and target-mediated mechanisms of
resistance apply, both strategies outlined in panel B and C would have to be combined.
Pharmacoresistance in epilepsyBrain (2005)Page 13 of 18
Potential future clinical implications
Once we have obtained a detailed picture of the mechanisms
underlying the development of pharmacoresistance to
individual AEDs, this knowledge may become increasingly
important both in drug development, as well as clinically.
First, detailed information on drug target changes can be
used to inform the development of new drugs for the treat-
ment of epilepsy. Currently, identification of AEDs is per-
formed mostlyin acute
experimental animals. These animals do not show any of
the chronic changes in ion channels or receptors discussed
here, and may not represent the best models to develop novel
compounds useful in human epilepsy. Targeting novel drugs
to ‘epileptic’ ion channels based on information from chronic
models of epilepsy or even tissue from epileptic patients
represents a promising avenue for rational drug development
in the future.
Information on specific resistance mechanisms might also
be used to guide potential treatment with drug transporter
inhibitors in conjunction with AEDs.The simplestscenario in
which such substances might be used would be as comedica-
tion with an AED that is ineffective predominantly due to
transporter-mediated mechanisms (depicted schematically in
Fig. 4B). On the other hand, comedication with transporter
inhibitors in a patient in whom resistance to an AED is
predominantly target mediated (see Fig. 4C) would not be
expected to be beneficial. In this case, the development of
drugs that specifically act on the modified target would be
the more appropriate approach. Ideally, these compounds
would not be substrates of drug transporter, but could also
be coadministered with transporter inhibitors. For some
AEDs, both resistance mechanisms may be relevant and syn-
ergistic (Fig. 4D); in this case, strategies to overcome resist-
ance would have to combine the approaches outlined in
Fig. 4B and C. A further clinical issue that may gain more
and more importance in the coming years is the identification
of predictors that identify clinically resistant or responsive
patient populations. If clear genetic polymorphisms in either
transporteror ion channelgenes can be identifiedthat reliably
predict the occurrence and the probable mechanism of drug
resistance(Soranzo et al., 2004; Tan et al., 2004; Ahmadi et al.,
2005), these data would obviously strongly influence initial
therapy, and perhaps increase the chances of its success.
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