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Keywords:

  • KCNQ;
  • M-current;
  • Antiepileptic drugs;
  • Anticonvulsant;
  • Antiepileptic

Summary

  1. Top of page
  2. Summary
  3. KCNQ Channels, a Family of K+ Channels That Control Cellular Excitability in Humans
  4. RTG/EZG Is a Positive Allosteric Modulator (Opener) of KCNQ2–5
  5. RTG/EZG Selectivity of Action: Primary MoA Is at KCNQ Channels
  6. Beyond EC50: Insights into RTG/EZG–KCNQ Pharmacology at the Receptor, Cellular, and Network Level
  7. Discussion
  8. Acknowledgments
  9. Disclosure
  10. References
  11. Supporting Information

The pharmacologic profile of retigabine [RTG (international nonproprietary name); ezogabine, EZG (U.S. adopted name)], is different from all currently approved antiepileptic drugs (AEDs). Its primary mechanism of action (MoA) as a positive allosteric modulator of KCNQ2–5 (Kv7.2–7.5) ion channels defines RTG/EZG as the first neuronal potassium (K+) channel opener for the treatment of epilepsy. KCNQ2–5 channels are predominantly expressed in neurons and are important determinants of cellular excitability, as indicated by the occurrence of human genetic mutations in KCNQ channels that underlie inheritable disorders including, in the case of KCNQ2/3, the syndrome of benign familial neonatal convulsions. In vitro pharmacologic studies demonstrate that the most potent action of RTG/EZG is at KCNQ2–5 channels, particularly heteromeric KCNQ2/3. Furthermore, mutagenesis and modeling studies have pinpointed the RTG/EZG binding site to a hydrophobic pocket near the channel gate, indicating how RTG/EZG can stabilize the open form of KCNQ2–5 channels; the absence of this site in KCNQ1 also provides a clear explanation for the inbuilt selectivity RTG/EZG has for potassium channels other than the KCNQ cardiac channel. KCNQ channels are active at the normal cell resting membrane potential (RMP) and contribute a continual hyperpolarizing influence that stabilizes cellular excitability. The MoA of RTG/EZG increases the number of KCNQ channels that are open at rest and also primes the cell to retort with a larger, more rapid, and more prolonged response to membrane depolarization or increased neuronal excitability. In this way, RTG/EZG amplifies this natural inhibitory force in the brain, acting like a brake to prevent the high levels of neuronal action potential burst firing (epileptiform activity) that may accompany sustained depolarizations associated with the initiation and propagation of seizures. This action to restore physiologic levels of neuronal activity is thought to underlie the efficacy of RTG/EZG as an anticonvulsant in a broad spectrum of preclinical seizure models and in placebo-controlled trials in patients with partial epilepsy. In this article, we consider the pharmacologic characteristics of RTG/EZG at the receptor, cellular, and network levels as a means of understanding the novel and efficacious MoA of this new AED as defined in both preclinical and clinical research.

The introduction of retigabine [RTG (international nonproprietary name), Marketing Authorization in Europe; ezogabine (EZG; U.S. adopted name)] as a novel antiepileptic drug (AED) for the adjunctive treatment of partial-onset seizures represents the culmination of >20 years of preclinical and clinical research. RTG/EZG, first named D-23129 (free base) or D-20443 (HCl salt version), was developed in the 1980s by the East German company Arzneimittelwerk Dresden, which became part of the ASTA Medica group after German reunification. Initial interest in its potential as a novel treatment for epilepsy stemmed from research efforts on flupirtine, a congener compound that was successfully developed as a nonopioid centrally acting analgesic in Europe (marketed as Katadolon). Flupirtine demonstrated weak evidence of anticonvulsant efficacy following its submission to the National Institutes of Health Anticonvulsant Drug Development program in the 1980s (Rostock et al., 1996) and a small exploratory clinical trial (Porter et al., 2007). These early efficacy findings were serendipitous and devoid of an appreciation of the mechanism of action (MoA) or pharmacologic properties that delivered them. Rather, similar to the majority of AEDs available today (Meldrum & Rogawski, 2007), in vivo efficacy from preclinical models was the key factor supporting development of both flupirtine and then retigabine as a novel epilepsy treatment; the understanding of the MoA would come later. In the case of RTG/EZG, quantitative structural optimization efforts led to an improved efficacy [10-fold based on characterization of ED50 (median effective dose) values in the rat maximal electroshock seizure (MES) test] and pharmaceutical profile relative to flupirtine that were key considerations in the selection of this agent for further development as a novel anticonvulsant (Rostock et al., 1996).

A large body of novel research has now yielded a detailed appreciation of the pharmacologic MoA of RTG/EZG. Early efforts demonstrated a lack of potent action at previously established AED targets such as sodium (Nav) and calcium channels (Cav) (Fig. 1A). In addition, action on the γ-aminobutyric acid (GABA)ergic system including GABAA receptors was restricted to relatively high RTG/EZG concentrations. These studies fueled initial suggestions that RTG/EZG possessed a new and unique MoA (Rundfeldt, 1997, 1999; Rundfeldt & Netzer et al., 2000a). The first data linking RTG/EZG to the modulation of potassium (K+) channels—a previously untapped superfamily of ion channels that could conceptually deliver an inhibitory effect on neurotransmission, and hence provide a new means to treat epilepsy—was published in 1997 (Rundfeldt, 1997). However, the crucial breakthrough was the discovery and cloning of a new family of K+ channels – KCNQ (Kv7 by more recent accepted nomenclature) (Alexander et al., 2008)—that was found to be genetically linked to a form of inherited human epilepsy known as benign familial neonatal convulsions (BFNC) (Biervert et al., 1998; Charlier et al., 1998; Singh et al., 1998). The potent and selective pharmacologic action of RTG/EZG as a positive allosteric modulator (opener) of key members of this family, such as KCNQ2/3, was published by multiple groups shortly thereafter (Main et al., 2000; Rundfeldt & Netzer, 2000b; Wickenden et al., 2000). Furthermore, the fact that these channels were also demonstrated to underlie the native ‘M-current’, a K+ conductance negatively regulated by muscarinic ligands, led to a newfound appreciation for the MoA of RTG/EZG at the cellular level (Tatulian et al., 2001).

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Figure 1.   The unique MoA of retigabine (RTG)/ezogabine (EZG) (A) Schematic representation of a typical neurone in the central nervous system highlighting the primary site and mechanism of action (MoA) of currently approved antiepileptic drugs (AEDs) for the treatment of partial epilepsy. (B) AEDs can be broadly divided into two classes: drugs that inhibit neuronal excitation or increase inhibition. Briefly, the MoAs associated with these classes are as follows: Sodium (Na+) channel blockers inhibit neuronal action potential firing and transmission by promoting inactivation and reducing contributions to electrical activity at the axon initial segment (AIS) as well as on the axon itself. Calcium (Ca2+) channel modulators reduce excitatory transmission by reducing presynaptic neurotransmitter release, a Ca2+-dependent process. Drugs that bind SV2A may also cause this effect whereas glutamatergic drugs will reduce the effects of this neurotransmitter on AMPA or NMDA receptors on the postsynaptic membrane. Drugs may affect GABA receptors in a number of ways: via direct positive allosteric modulation of GABAA receptor activity (e.g., benzodiazepines), or indirectly, by increasing levels of GABA via inhibition of GABA transaminase (e.g., vigabatrin) or GABA transporter-1 (GAT1, e.g., tiagabine). RTG/EZG is unique in that it acts as a positive allosteric modulator of KCNQ potassium (K+) channels leading to an inhibition of high-frequency action potential firing at the AIS due to increased hyperpolarization. KCNQ channels are also present on dendrites and the axon (not shown), the increased activity of which may also contribute to the anticonvulsant efficacy of RTG/EZG. N.B. Some AEDs also have multiple pharmacological activities that may contribute to efficacy. In the case of RTG/EZG, positive allosteric modulation of GABAA receptors may also occur at high concentrations. AMPA, α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid; Cl, chloride; GABA, γ-aminobutyric acid; NMDA, N-methyl-d-aspartate; SV2A, synaptic vesicle 2A.

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After two decades of research and >100 publications on RTG/EZG, our understanding of RTG/EZG pharmacology at the receptor, cellular, and network level has advanced significantly. We now have a greater understanding of the role of KCNQ channels in the control of neuronal excitability (Gribkoff, 2003; Surti & Jan, 2005; Maljevic et al., 2008; Brown & Passmore, 2009) that provides a rigorous foundation from which to interpret the broad anticonvulsant efficacy of RTG/EZG defined in preclinical seizure models (Large et al., 2012). Crucially, during this timeframe, these preclinical findings have also been shown to translate into significant efficacy for patients in a full development program that culminated in two large-scale phase 3 double-blind placebo-controlled trials in patients with partial (focal) epilepsy [RESTORE 1 (Study 301) and RESTORE 2 (Study 302) (Brodie et al., 2010; French et al., 2011)]. These data come full circle in defining KCNQ K+ channels as a fully validated human target for the treatment of this major disorder which, even with the availability of a large number of approved agents (Fig. 1B), still presents a high unmet medical need. Indeed, due to underlying differences in disease pathology and the acknowledged limitations regarding effectiveness or tolerability of previously approved agents, up to a third of patients are considered refractory to treatment and a further third are in need of better treatment options (Kwan & Brodie, 2000; Kwan et al., 2010). Such findings have led to a constant call for new AEDs with novel MoA. In this article, we will consider the effects of RTG/EZG pharmacology at the receptor, cellular and network level as a means to understand the efficacy and utility of this new AED with a novel MoA for the treatment of epilepsy.

KCNQ Channels, a Family of K+ Channels That Control Cellular Excitability in Humans

  1. Top of page
  2. Summary
  3. KCNQ Channels, a Family of K+ Channels That Control Cellular Excitability in Humans
  4. RTG/EZG Is a Positive Allosteric Modulator (Opener) of KCNQ2–5
  5. RTG/EZG Selectivity of Action: Primary MoA Is at KCNQ Channels
  6. Beyond EC50: Insights into RTG/EZG–KCNQ Pharmacology at the Receptor, Cellular, and Network Level
  7. Discussion
  8. Acknowledgments
  9. Disclosure
  10. References
  11. Supporting Information

We now know that KCNQ channels comprise a family of five related genes (Fig. 2A). Each gene encodes a KCNQ subunit with a 6-transmembrane topography and a pore loop that can assemble to form homotetrameric channels, and in some cases, heterotetrameric channels. The key importance of these channels as modulators of cellular excitability in the central and peripheral nervous system and other organs in the body where they are expressed (Supporting Information Table S1) is clear from the occurrence of a range of human genetic mutations in KCNQ (Kv7) K+ channels that underlie several inheritable disorders. These include long QT syndrome that arises from loss of function of KCNQ1, a predominantly cardiac ion channel that contributes to the repolarization of the cardiac action potential (Jentsch, 2000; Shieh et al., 2000) and a rare form of dominant deafness that is thought to arise from impaired function of KCNQ4 in the outer hair cells of the cochlea organ in the inner ear that disrupts normal K+ balance and excitability (Kharkovets et al., 2000, 2006). In the case of KCNQ2/3, which are the predominant KCNQ channels expressed in the brain, a loss of function due to a range of channel mutations or deletions has been linked to BFNC, a form of epilepsy. The key role that these channels play in maintaining neuronal excitability is further highlighted by the fact that relatively moderate (approximately 25%) reductions in KCNQ channel activity due to impaired expression or function in either subunit, can lead to a loss of control over neuronal excitability in the brain resulting in a seizure predisposition (Maljevic et al., 2008). This finding is in concordance with preclinical data in which transgenic KCNQ2+/− mice, deficient in one copy of the Kcnq2 gene, show increased sensitivity to the chemo-convulsive agent pentylenetetrazole (Watanabe et al., 2000). Consequently, it has been widely suggested that strategies to increase activity of KCNQ2/3 represents a novel targeted means to restore the control of neuronal excitability in patients with epilepsy.

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Figure 2.   Pharmacological action and selectivity of retigabine (RTG)/ezogabine (EZG) at KCNQ2–5 channels. (A) The KCNQ potassium (K+) ion channel family consists of five genes with the indicated phylogeny. Each gene encodes a KCNQ subunit with a 6-transmembrane structure and a pore loop that can assemble to form homo-, and in some cases, heterotetrameric channels. (B) A summary of the pharmacological potency of RTG/EZG on known homo- and heteromeric KCNQ channels defining its selective action at KCNQ2–5 (see text for further details). (C) Illustrates the architecture of a KCNQ channel assembled from four subunits (I–IV, blue) where two transmembrane domains (S5, S6) from each are highlighted in magenta and line the aqueous integral ion channel pore that is selective for K+ ions (adapted with kind permission from Springer Science + Business Media: Meldrum & Rogawski (2007, p. 59), Figure 3). Detailed modeling and mutagenesis studies have shown that RTG/EZG binds to KCNQ2–5 channels at this location near the channel gate leading to a stabilization of the channel open state. Key amino acid residues glycine (G)301 and tryptophan (W)236 for RTG/EZG binding are indicated. The lack of W236 in KCNQ1 explains the lack of activity of RTG/EZG at this cardiac channel. EC50, half maximal effective concentration; ECM, extracellular matrix; IC50, half maximal inhibitory concentration.

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RTG/EZG Is a Positive Allosteric Modulator (Opener) of KCNQ2–5

  1. Top of page
  2. Summary
  3. KCNQ Channels, a Family of K+ Channels That Control Cellular Excitability in Humans
  4. RTG/EZG Is a Positive Allosteric Modulator (Opener) of KCNQ2–5
  5. RTG/EZG Selectivity of Action: Primary MoA Is at KCNQ Channels
  6. Beyond EC50: Insights into RTG/EZG–KCNQ Pharmacology at the Receptor, Cellular, and Network Level
  7. Discussion
  8. Acknowledgments
  9. Disclosure
  10. References
  11. Supporting Information

In vitro pharmacological approaches combined with structural modeling and site-directed mutagenesis studies have now determined the pharmacological action of RTG/EZG to be at the KCNQ2–5 K+ channels. Initial insight into the novel action of RTG/EZG was derived from studies on native neuronal K+ channels. Electrophysiological studies on NG108-15 neuroblastoma, hNT cells (a cell line derived from human neuronal cells) and mouse cortical neurons in culture identified a pronounced effect of low concentrations of RTG/EZG (≥0.1 μm) on the enhancement of an unidentified K+ current in these cells (Rundfeldt, 1997). These same studies also provided first data on the selective and more potent action of RTG/EZG on K+ channels than on other targets associated with the pharmacology or MoA of AEDs (Fig. 1B and see below).

Independently from the elucidation of the precise K+ channel entities targeted by RTG/EZG, other research led to the cloning and characterization of the KCNQ channels (Jentsch, 2000). The clear potential for these targets to represent a novel approach to the treatment of epilepsy catalyzed the formation of an immediate hypothesis regarding whether the drug’s anticonvulsant properties were mediated via a direct pharmacological action on these channels. Independent results from a number of separate groups provided clear and consistent confirmation that RTG/EZG exhibited potent action as a positive allosteric modulator (i.e., opener of KCNQ channels) that would serve to reduce neuronal excitability (Main et al., 2000; Rundfeldt & Netzer, 2000b; Wickenden et al., 2000; Otto et al., 2002). Detailed pharmacological studies have demonstrated the drug’s selectivity of action on KCNQ channels (Fig. 2B). RTG/EZG is effectively devoid of activity at KCNQ1, the cardiac KCNQ channel, and exhibits a higher potency at channels assembled from KCNQ2/3, which are linked to human epilepsy, over those assembled from KCNQ4 or KCNQ5 (Main et al., 2000; Rundfeldt & Netzer, 2000b; Wickenden et al., 2000, 2001; Tatulian et al., 2001; Yeung et al., 2008). One could therefore cite the overall rank order of potency of RTG/EZG based on determinations of the half maximal effective concentration (EC50) as KCNQ3 > KCNQ2/3 = KCNQ3/5 > KCNQ2 > KCNQ4 = KCNQ5, covering the concentration range from 0.6 to 6.4 μm (Fig. 2B). However, in interpreting the relevance of this pharmacology for the effects in vivo, one must also be cognizant of the fact that the KCNQ heteromers KCNQ2/3 and KCNQ3/5 are assembled more efficiently in cells than their homomeric counterparts and can contribute an order of magnitude more K+ current than homomeric channels (Lerche et al., 2000; Main et al., 2000; Wickenden et al., 2000). These heteromeric channels will therefore likely predominate amongst KCNQ channels in delivering the cellular effects and efficacy of RTG/EZG. As a consequence, RTG/EZG is perhaps best considered as a KCNQ2–5 active drug that delivers its predominant anticonvulsant MoA through the heteromeric KCNQ2/3 and KCNQ3/5 channels that are the main constituents of the neuronal M-current.

The in vitro functional characterizations of RTG/EZG–KCNQ pharmacology are now wholly supported by the results of elegant modeling and structure-function studies using site-directed mutagenesis. These approaches, based on the recent advances in our structural knowledge of K+ channel function (Swartz, 2004) have identified the precise location and key amino acids such as glycine (G)301 and tryptophan (W)236 contributing to the RTG/EZG binding site, a hydrophobic pocket near the channel gate (Fig. 2C) (Schenzer et al., 2005; Wuttke et al., 2005; Lange et al., 2009). Crucially, these key amino acids involved in RTG/EZG binding are conserved in KCNQ2–5 and their location near the channel gate provides an explanation for why RTG/EZG binding can increase KCNQ channel function: namely a stabilization of the ion channel in the open K+ conducting form (Schenzer et al., 2005; Wuttke et al., 2005). These data are also informative regarding the lack of RTG/EZG opener action at KCNQ1; the lack of key glycine and tryptophan residues means that the RTG/EZG binding site is absent in this family member (Fig. 2C). Rather, a weak inhibitory effect of RTG/EZG at high concentrations (half maximal inhibitory concentration [IC50] values approximately 100 μm) is reported (Fig. 2B) that most likely reflects a direct occlusion of the KCNQ1 channel pore, similar to the action of chromanol or some benzodiazepines (Seebohm et al., 2003; Lerche et al., 2007).

RTG/EZG Selectivity of Action: Primary MoA Is at KCNQ Channels

  1. Top of page
  2. Summary
  3. KCNQ Channels, a Family of K+ Channels That Control Cellular Excitability in Humans
  4. RTG/EZG Is a Positive Allosteric Modulator (Opener) of KCNQ2–5
  5. RTG/EZG Selectivity of Action: Primary MoA Is at KCNQ Channels
  6. Beyond EC50: Insights into RTG/EZG–KCNQ Pharmacology at the Receptor, Cellular, and Network Level
  7. Discussion
  8. Acknowledgments
  9. Disclosure
  10. References
  11. Supporting Information

A large number of studies have been conducted to explore the pharmacology of RTG/EZG, initially as a search for an underlying MoA to explain its anticonvulsant efficacy, and subsequently to further elucidate the broader selectivity of this agent for consideration as a potential medicinal agent. The results of these studies are summarized below and compared in Table 1, with their likely relevance for therapeutic effect in epilepsy patients where, at the maximum daily dose of 1,200 mg (achieved using divided doses of 400 mg three times daily [t.i.d.]), mean free average plasma concentrations (Cave) of RTG/EZG were 0.83 μm and maximum meanfree plasma concentrations (Cmax) were predicted to be approximately 1 μm (based on a population pharmacokinetic analysis of data obtained from the RESTORE trials (Tompson et al., in preparation). Since RTG/EZG is highly permeable and not subject to P-glycoproteinactive transport across the blood–brain barrier, free plasma concentrations provide an accurate indication of the free brain concentrations achieved.

Table 1.   KCNQ channels: the primary site for RTG/EZG MoA
Pharmacological actionEffectLevel of activity or EC50 or IC50 where determinedaRatio of activity : Free Cmax or Cave at 1,200 mg/day in patients with epilepsyb
  1. AMPA, α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid; Cave, average plasma concentration; Cmax, maximum plasma concentration at steady state; Cmin, minimum plasma concentration at steady state; EC50, half maximal effective concentration; GABA, γ-aminobutyric acid; IC50, half maximal inhibitory concentration; NMDA, N-methyl-d-aspartate.

  2. bEstimated as 1.0 μm based on mean Cmax at 1,200 mg = 1,520 ng/mL from population pharmacokinetic analysis of the RESTORE trial data. Mean Cave concentrations at this dose were 1,261 ng/mL or 0.83 μm (GSK/Valeant data on file; Plasma protein binding = 79–80%; RTG/EZG molecular weight = 303.3 Da).

  3. cSee Table S2 for supporting information.

KCNQPositive allosteric modulatorEC50 = 1.6 μm at KCNQ2/3∼1
GABAPositive allosteric modulator at GABAA receptors (non–benzodiazepine site)Significant effects at ≥10 μm in the majority of studies≥10-fold
Effects on GABA metabolismSignificant effects at 20 μm∼20-fold
Calcium channelsWeak inhibitorIC50 > 100 μm at neuronal Cav channels (29% inhibition at 100 μm)>100-fold
Sodium channelsWeak inhibitorIC50 > 100 μm at neuronal Nav channels (25% at 100 μm)>100-fold
Glutamate receptorsNo effect at NMDA, AMPA, or kainate receptorsNo effect up to 10 μm>10-fold
Other: Broad selectivity profileNo additional activities detectedNo significant interactions in 62 assays of ion channels, transporters, enzymes, and 2nd-messenger systems at 10 μmc>10-fold

Effects on other K+ channels

To ascertain whether the effects of RTG/EZG were selective for KCNQ channels over other K+ channels, a range of functional electrophysiological studies have been conducted on exemplar channels from other families that make up the K+ channel superfamily (Alexander et al., 2008). RTG/EZG was shown to be without significant activity at members of the 2TM K+ family of channels (e.g., inward rectifiers KIR 2.1 [IRK1] or KIR 3.1) as well as 4TM family and the 2-pore domain channels, such as K2P1.1 (TWIK1) at concentrations ≤100 μm. Members of the 6TM family e.g., Kv1.5, hERG, which includes voltage-gated [Kv] channels and EAG and Ca2+ activated channels (in addition to KCNQ channels), were also relatively unaffected, with IC50 values of >50 μm and 59 μm, respectively (GSK/Valeant data on file, personal communications). These data emphasize the remarkably selective action of RTG/EZGon a subset of K+ channels, namely KCNQ2–5 over the broader superfamily of K+ channels that occur in humans.

Effects on GABA receptors and GABAergic neurotransmission

The effects of RTG/EZG on GABAA receptors and other aspects of GABAergic neurotransmission have been examined in a range of in vitro studies, initially based on the exploration of the compound’s MoA. Electrophysiological studies on GABAA-receptor-mediated currents in cultured cortical neurons identified a potentiating effect (Rundfeldt & Netzer, 2000a; Otto et al., 2002). RTG/EZG can therefore additionally act as a positive allosteric modulator of the GABAA receptor; however, higher concentrations of RTG/EZG than are active at KCNQ channels are typically required to produce significant potentiation (i.e., ≥10 μm). More detailed assessment of the effects of RTG/EZG has also been undertaken on a range of different GABAA receptor subtypes expressed in Xenopus oocytes (GSK/Valeant data on file, personal communication). The results from these studies showed that concentrations of RTG/EZG≥10 μm were required to cause significant augmentation of the GABAA receptor response. The potency of RTG/EZG differed somewhat depending on the GABAA receptor subunit combination, with the following rank order: α1β3γ2 = α1β2γ2 > α3β2γ2 = α2β2γ2 > α5β2γ2 = α1β2(N265S)γ2 = α1β1γ2 (GSK/Valeant data on file, personal communication). RTG/EZG can therefore act at the major GABAA receptor isoforms in the brain (e.g., α1β3γ2 and α1β2γ2 together comprise approximately 50% of the CNS receptors (Wafford, 2005). However, in all cases, the potentiating effects were not inhibited by the benzodiazepine site antagonist flumazenil, indicating that the action of RTG/EZG is not through the benzodiazepine site on the receptor, consistent with results from the radioligand binding studies using a range of GABA receptor ligands (see below) and published studies (Rundfeldt & Netzer, 2000a).

In a separate line of investigation, Kapetanovic et al. (1995) uncovered an apparent effect of RTG/EZG on GABA metabolism whereby levels of newly synthesized GABA in rat hippocampal slices were increased following exposure to 20 μm RTG/EZG. This effect was greater than seen for other AEDs and different to flupirtine, a compound that is also active at the KCNQ and GABAA channels in the range of 10–30 μm (Popovici et al., 2008); however, no further studies have shed light on the particular mechanism underlying this effect and in vivo microdialysis studies did not provide any evidence of increases in GABA occurring in vivo (Rundfeldt C, personal communication October, 2010; see also Straub et al., 2001).

Further studies regarding the MoA of RTG/EZG at the GABAA receptor have concluded that RTG/EZG interacts with a novel site on the GABAA receptor complex that is allosterically coupled with the binding sites for the agonist GABA and Org 20549 (a neuroactive steroid); EC50 values for the displacement of these GABAA receptor ligands were ≥23 μm (van Rijn & Willems-van Bree, 2003; van Rijn & Willems-van Bree, 2004). Studies by Otto et al. (2002) have also demonstrated that RTG/EZG can potentiate inhibitory postsynaptic currents mediated by direct activation of GABAA receptors in mouse cortical neurons. Again, significant effects on peak current or charge transfer required concentrations of RTG/EZG ≥10 μm, and were markedly larger at 50 μm indicating that this was the start of the concentration-response curve (Otto et al., 2002). Overall, these data demonstrate that RTG/EZG can modulate GABAergic neurotransmission. Although in vitro concentrations do not necessarily translate directly to in vivo concentrations, the concentrations required to drive GABA effects are thought to be higher than those that are effective at KCNQ channels or attained in patients with epilepsy (Table 1).

Effects on Na+ and Ca2+ channels

The effects of RTG/EZG on Nav channels has been assessed using whole-cell patch clamp electrophysiology on differentiated NG108-15 neuroblastoma cells. Only weak inhibition was detected with 9.1 ± 3.4%, 20.1 ± 2.9%, and 24.9 ± 5.9% reduction in Nav current measured following incubation with either 1, 10, or 100 μm RTG/EZG, respectively. Similar studies on mixed Cav currents studied in differentiated NG108-15 neuroblastoma cells also detected weak activity of RTG/EZG, with significant effects only observed at concentrations above 10 μm. The greatest inhibition of mixed N-, T- and L-type currents was determined to be 15.7 ± 3.0%, 47.3 ± 10%, and 60.1 ± 7.3% reduction following incubation with 10, 30, and 100 μm RTG/EZG, respectively (GSK/Valeant data on file, personal communication). In conclusion, RTG/EZG exhibited only weak inhibitory effects at voltage-gated Nav and Cav channel currents at predominantly supratherapeutic concentrations.

Effects on glutamate receptors

The potential effects of RTG/EZG on glutamatergic neurotransmission were evaluated in a range of functional electrophysiological studies on recombinant receptors expressed in Xenopus oocytes or HEK293 cells and native receptors in cortical neurones in culture. No effect of RTG/EZG was observed on N-methyl-d-aspartate (NMDA)-induced currents in cortical neurons at 10 μm. RTG/EZG was also inactive versus currents induced by 300 μm kainate at a concentration of 10 μm, but did reduce currents by 17% at 100 μm, suggesting a small inhibitory effect at high concentrations. Although a significant block of kainate (10 μm)-induced currents was observed in these studies, more detailed follow-up investigations examining the effect of RTG/EZG on different cloned alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) and kainate receptors, combined with additional studies on kainate-induced currents in neurons did not detect any significant inhibitory effects of RTG/EZG (GSK/Valeant data on file, personal communication). These data, in conjunction with the radioligand binding assay data summarized below, support the conclusion that RTG/EZG does not interact significantly with glutamate receptors.

Cross-screening selectivity profile: radioligand binding assays and additional electrophysiological studies

Radioligand binding studies with RTG/EZG at concentrations of 10−9, 10−7 or 10−5 M conducted at NovaScreen Biosciences (Hanover, MD, U.S.A.) according to standard methods (GSK/Valeant data on file, personal communication) did not show significant interaction with the known modulator binding sites on 62 receptors, ion channels, transporters, enzymes, and second messengers (percent inhibition <50% at 10−5 M; supporting information, Table S2). This panel included receptors for opioids, dopamine, serotonin, glutamate, histamine, acetylcholine, GABA, adenosine, noradrenaline as well as binding sites on voltage-gated Ca2+, K+ and Na+ channels and a range of second messenger system targets/enzymes. Some additional studies at a concentration of 10−4 M were also conducted to further assess potential for interaction with GABA and NMDA receptors at high concentrations. Consistent with the functional studies detailed in the sections above, RTG/EZG demonstrated no significant affinity for benzodiazepine and GABA binding sites on the GABAA receptor complex, nor did it show affinity for the GABA reuptake site, or the glutamate, glycine, and ion channel binding sites on the NMDA receptor complex (percent inhibition <50% at 10−4 M).

Conclusion: pharmacology of RTG/EZG in the context of the clinical setting

Considering in vitro pharmacological data in the context of the mean free Cmax and Cave achieved with RTG/EZG in humans (Table 1) highlights the striking overlap between the efficacious concentrations of the drug and its most potent activity at KCNQ channels such as KCNQ2/3 (Fig. 2B; see also Fig. 5 and below). Indeed, concentrations some 10-fold higher than achieved at the maximum dose of 1,200 mg/day used in patients with epilepsy could be required for significant effects on other receptor systems. Modulation of GABAergic neurotransmission is an MoA relevant to the efficacy of several other AEDs (Fig. 1); however, in in-vivo efficacy studies of RTG/EZG conducted in the mouse MES model or a rapid kindling model of epilepsy in the rat, co-administration of the KCNQ antagonist XE-991 reversed the anticonvulsant effects of RTG/EZG, suggesting a lack of contribution by additional mechanisms (GSK/Valeant data on file, personal communication; Sankar et al., 2008; see Large et al., 2012 for further discussion). Overall, these data combined with the clinical setting assessments clearly support the conclusion that the primary MoA of RTG/EZG involves activity at KCNQ channels such as KCNQ2/3.

Beyond EC50: Insights into RTG/EZG–KCNQ Pharmacology at the Receptor, Cellular, and Network Level

  1. Top of page
  2. Summary
  3. KCNQ Channels, a Family of K+ Channels That Control Cellular Excitability in Humans
  4. RTG/EZG Is a Positive Allosteric Modulator (Opener) of KCNQ2–5
  5. RTG/EZG Selectivity of Action: Primary MoA Is at KCNQ Channels
  6. Beyond EC50: Insights into RTG/EZG–KCNQ Pharmacology at the Receptor, Cellular, and Network Level
  7. Discussion
  8. Acknowledgments
  9. Disclosure
  10. References
  11. Supporting Information

RTG/EZG alters KCNQ channel gating and open-channel probability

The positive allosteric modulation or opener action of RTG/EZG at KCNQ2–5 is underscored by a number of distinct effects on KCNQ channel function uncovered by detailed MoA studies at the receptor level that have impacted our understanding of this agent’s anticonvulsant efficacy. The most prominent effect identified by electrophysiological studies is a marked concentration-dependent shift in the voltage-dependent activation curve of KCNQ channels to more hyperpolarized potentials (Fig. 3A[i–iii]). Quantification of this effect (the EC50 to shift this curve to 50% of its maximum [V1/2] is 1.6 μm; Fig. 3A[iv]) can be used to deduce the potent action of RTG/EZG that explains why more KCNQ channels are open at a given membrane potential in the presence of the drug, particularly following membrane depolarization (e.g., compare the magnitude of response obtained at −60 mV, close to the normal cell RMP, or at a more depolarized level such as −30 mV that may occur following neuronal activity, in the presence or absence of RTG/EZG (Fig. 3A[iii]). This effect translates to larger K+ outward currents and an increased hyperpolarizing effect on the cell (Main et al., 2000; Rundfeldt & Netzer, 2000b; Wickenden et al., 2000; Tatulian et al., 2001). It is worth noting that the increase in K+ current is achieved solely by this means of increasing the probability of a KCNQ channel being open, as RTG/EZG does not alter the single-channel conductance of individual KCNQ2/3 channels (Tatulian & Brown, 2003).

image

Figure 3.   Retigabine (RTG)/ezogabine (EZG) enhances magnitude and duration of KCNQ channel action to resist depolarization, and shifts voltage-dependence (VD) of KCNQ activation, leading to more rapid, prolonged, and increased levels of KCNQ channel opening in response to depolarizing stimuli. (A) (i) Whole-cell potassium currents evoked by voltage steps between −100 and +30 mV in KCNQ2/3-expressing cells are significantly larger in the presence of (ii) 10 μm RTG/EZG and these also exhibit faster kinetics of activation and slowed deactivation (blue arrows; see also B). (iii) Increasing concentrations of RTG/EZG from 0.1 to 10 μm (iv) reversibly shifted VD of KCNQ2/3 activation by approximately 30 mV to more hyperpolarized potentials, with an EC50 of 1.6 μm. (Reproduced with permission from the American Society for Pharmacology and Experimental Therapeutics, Wickenden et al. (2000), p. 597) (B) Expanded portions of KCNQ current responses clearly highlight the effect of RTG/EZG on the rate of channel activation/deactivation. (Reproduced with permission from the American Society for Pharmacology and Experimental Therapeutics, Main et al. (2000)) (C) RTG/EZG action on KCNQ channel gating: 10 μm increased the rate of KCNQ activation (τact) and reduced the rate and contribution of KCNQ slow deactivation (τslow). When combined with VD changes, RTG/EZG leads to an overall increase in KCNQ channel open probability (Popen) – i.e., more KCNQ channels will be open at a given voltage in the presence of RTG/EZG. (D) This effect underlies the pronounced hyperpolarizing effect of RTG/EZG on the cell resting membrane potential (RMP). In the example shown, 1 μm RTG/EZG was able to reduce RMP from approximately −63 to −70 mV, which would dramatically reduce excitability (Reproduced with permission from the American Society for Pharmacology and Experimental Therapeutics, Main et al. (2000, p. 259)).

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More careful examination of the KCNQ current traces in response to the step-depolarizations illustrated also indicates that RTG/EZG alters the rate of KCNQ activation and deactivation (blue arrows in Fig. 3A[ii]). This effect has been studied in detail and is clear when viewed on an expanded timeframe (Fig. 3B) (Main et al., 2000; Wickenden et al., 2000; Tatulian et al., 2001). These studies have demonstrated that RTG/EZG increases the rate of KCNQ activation (τact) and reduces the rate of the slower component of a two-step deactivation process (τslow) (Fig. 3C). These kinetic effects yield more KCNQ channels in the open rather than closed positions at a given membrane potential underlying the marked increase in open-channel probability (Popen) and indicate that RTG/EZG also enhances the timeframe over which KCNQ channels impact cellular excitability.

The hyperpolarizing influence of RTG/EZG stabilizes the RMP of neurons

The effects of RTG/EZG on KCNQ channels are consistent with studies of the agent on native M-currents in neurons (Tatulian et al., 2001; Yue & Yaari, 2004), providing confidence in the translation of the measured in vitro pharmacology at recombinant receptors to native systems. Studies on neurons indicate that KCNQ K+ channels are active at the cell RMP and, unlike the majority of voltage-gated channels, do not inactivate such that they contribute a continual hyperpolarizing influence on cells (Tatulian et al., 2001; Otto et al., 2002; Yue & Yaari, 2004). This is evident from studies with the KCNQ antagonists XE-991 or linopirdine that typically cause a depolarization of the RMP and increase in neuronal excitability and action potential firing. The ability of RTG/EZG to achieve the converse through recruitment of additional KCNQ channels resulting in a concentration-dependent and marked hyperpolarizing effect on cell RMP has also been revealed by similar studies (Fig. 3D).

RTG/EZG reduces spike-frequency adaptation, preferentially impacting high-frequency firing

Studies on the firing properties of rodent cortical and hippocampal neurons indicate an important role of M-currents in setting the electroresponsive properties of neurons and controlling their intrinsic firing frequencies and patterns in response to depolarization (Hetka et al., 1999; Otto et al., 2002; Yue & Yaari, 2004). Such studies have highlighted that in addition to enhancement of the neuronal M-current promoting maintenance of the RMP, RTG/EZG can also reduce subthreshold excitability (i.e., it influences the range of underlying electrical activity in the cell that, if sufficiently depolarized, can reach the threshold to trigger the firing of one or more action potentials). It appears that KCNQ channels are particularly important in this regard since they are highly expressed at the axon initial segment (AIS; see Fig. 1A) (Devaux et al., 2004; Chung et al., 2006; Pan et al., 2006), the part of the neuron that integrates the net depolarizing and hyperpolarizing influences on the cell and from which action potentials originate; KCNQ channels are also present on dendrites and the axon (Devaux et al., 2004; Chung et al., 2006; Pan et al., 2006) meaning that they can also impact dendritic integration and neuronal transmission—effects that could also meaningfully contribute to the anticonvulsant efficacy of RTG/EZG.

In studies on rat CA1 pyramidal (hippocampal) neurones, RTG/EZG showed pronounced concentration-dependent (1–10 μm) attenuation of higher frequency or burst firing evoked by prolonged depolarizing stimuli (Fig. 4A) (Yue & Yaari, 2004). In contrast, single action potentials recorded in response to short duration depolarizing stimuli were unaffected by the same concentrations of RTG/EZG. This sparing effect on lower frequency activity by RTG/EZG is in line with the primary contribution of KCNQ channels to the medium afterhyperpolarization (mAHP), an observed conductance event that serves to control the excitability of neurones over a timescale in the order of tens to hundreds of ms (Storm, 1989; Gu et al., 2005). Such an effect reflects the slow kinetics of KCNQ activation that, even in the agent’s presence, are too slow to inhibit the onset of a solitary action potential, but are well placed to impact subsequent excitability. Therefore, the enhanced KCNQ recruitment following the initial depolarization of an action potential in the presence of RTG/EZG does lead to a reduced spike afterdepolarization that is responsible for the attenuation of subsequent action potentials leading to the phenomenon of spike frequency adaptation. Such differential behavior is likely to underlie the tolerability or therapeutic index achieved with RTG/EZG preclinically or in patients with epilepsy since, from a rising dose or concentration-dependence perspective, the higher levels of neuronal activity or burst firing that are likely to accompany seizures will be inhibited prior to more physiological levels of firing.

image

Figure 4.   Retigabine (RTG)/ezogabine (EZG) action on native KCNQ channels can reduce hyperexcitability, high frequency action potential firing and epileptiform activity (EA) in the brain. (A) RTG showed pronounced attenuation of higher frequency or burst firing in studies on rat CA1 pyramidal (hippocampal) neurones. Short-duration depolarizing stimuli (a1–a4) evoked single action potentials that were unaffected by 1–10 μm RTG/EZG. In contrast, prolonged depolarizing stimuli evoked burst firing of neurones and this activity was preferentially reduced by RTG/EZG in a concentration-dependent manner, due to the kinetics of KCNQ activation that are too slow—even in the presence of RTG/EZG—to inhibit solitary action potentials. However, enhanced KCNQ recruitment by the initial depolarization in the presence of RTG/EZG leads to a reduced afterdepolarization that is responsible for the attenuation of subsequent action potentials (‘spike frequency adaptation’). (Adapted with permission from the Society for Neuroscience, Yue & Yaari (2004)) (B) RTG/EZG displayed inhibitory properties on EA recorded in human cortical brain tissue resected from patients undergoing surgery for intractable partial epilepsy. The recordings show spontaneous sharp wave activity ex vivo (note the higher temporal resolution of the upper trace that shows individual events occurring in the longer time-base traces below) that were sensitive to RTG/EZG, highlighting the presence of functional KCNQ channels in these brain regions and hence the potential for RTG/EZG to provide therapeutic benefit for patients suffering from these intractable focal seizures (Adapted from Straub et al. (2001), © 2001, with permission from Elsevier BV).

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RTG/EZG attenuates epileptiform activity in rat and human brain tissue

RTG/EZG has been shown to attenuate epileptiform activity recorded ex vivo in a number of brain slice preparations including rat hippocampal slices treated with proconvulsant agents such as the K+ channel blocker 4-aminopiridine (4-AP), bicuculline, low Mg2+, or NMDA (Armand et al., 1999, 2000; Dost & Rundfeldt, 2000). RTG/EZG also reduced in vitro spontaneous bursting in the entorhinal cortex of rats that had been treated with kainate to induce status epilepticus (Smith et al., 2007) and attenuated recurrent epileptiform discharges evoked by 4-AP in combination with bicuculline (Armand et al., 1999). Although no clinical data are available, these findings suggest the potential of K+ channel openersfor the treatment of status epilepticus, in particular drug-resistant status (Large et al., 2012 for additional comment). Importantly, from a human translational perspective, similar studies have also been carried out in human brain tissue. Straub et al. (2001) demonstrated antiepileptic effects of RTG/EZG in neocortical slice preparations from 17 patients who underwent surgery for the treatment of intractable (i.e., pharmacoresistant) epilepsy. RTG/EZG decreased the occurrence of spontaneous rhythmic sharp waves that were recorded in these brain slices ex vivo (Fig. 4B) and also suppressed epileptiform field potentials evoked by use of low Mg2+ (Straub et al., 2001). These data provide evidence for the presence of functional KCNQ channels in these brain regions, hence supporting the potential for RTG/EZG to provide therapeutic benefit for patients suffering from such intractable focal seizures. In addition, they emphasize that RTG/EZG can deliver sufficient KCNQ-mediated inhibition to effectively restore physiological levels of neuronal excitability at the network level, an effect that probably underlies its anticonvulsant activity in a broad range of seizure types in the intact brain (Rostock et al., 1996; Tober et al., 1996; Mazarati et al., 2008) (see Large et al., 2012 for review).

Discussion

  1. Top of page
  2. Summary
  3. KCNQ Channels, a Family of K+ Channels That Control Cellular Excitability in Humans
  4. RTG/EZG Is a Positive Allosteric Modulator (Opener) of KCNQ2–5
  5. RTG/EZG Selectivity of Action: Primary MoA Is at KCNQ Channels
  6. Beyond EC50: Insights into RTG/EZG–KCNQ Pharmacology at the Receptor, Cellular, and Network Level
  7. Discussion
  8. Acknowledgments
  9. Disclosure
  10. References
  11. Supporting Information

In this review we have considered the novel MoA of RTG/EZG, originally discovered as a compound with anticonvulsant activity but unknown MoA, and now defined as a selective positive allosteric modulator (opener) of KCNQ2–5 channels. Significant research efforts have contributed to the two main aspects of this pairing: (1) the continued development of RTG/EZG as a potential treatment for epilepsy through its clinical development over the last decade and (2) the fundamental research into the physiological role of KCNQ channels and their potential contribution to diseases such as epilepsy. With the completion of pivotal clinical studies defining the dose-dependent efficacy and tolerability of RTG/EZG in patients with partial epilepsy, both of these avenues of research validate the KCNQ channels as a useful therapeutic target for the treatment of epilepsy (Fig. 5).

image

Figure 5.   Concentrations of retigabine (RTG)/ezogabine (EZG) associated with anticonvulsant efficacy in patients with epilepsy and the pharmacological effects measured at KCNQ2/3 in vitro. Bar height represents the median reduction in seizure frequency (right hand y-axis) endpoint achieved in the two Phase 3 RESTORE trials of RTG/EZG administered t.i.d. (Brodie et al., 2010; French et al., 2011) that assessed 600, 900, or 1,200 mg/day (similar data were achieved with the alternative endpoint assessing the proportion of patients achieving ≥50% reductions in seizures). Bar width indicates the range of concentrations based on the range of mean free Cmin to mean Cmax for each dose calculated by population pharmacokinetic analysis. The mean Cave values achieved at each dose were 0.40, 0.60, and 0.83 μm at 600, 900, and 1,200 mg, respectively, and are indicated by the white dotted line superimposed on each bar. The in vitro RTG/EZG concentration–response profile (black squares and associated curve fit) for the shift in V1/2 of KCNQ channel activation (left hand y-axis) is also overlaid for direct comparison. Cave, average plasma concentration; Cmax, maximum plasma concentration at steady state; Cmin, minimum plasma concentration at steady state.

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At the receptor, cellular and network level, the importance of KCNQ channels as key regulators of neuronal excitability is clear: they are able to work as a powerful inhibitory force in the brain by providing a continual hyperpolarizing influence to maintain or control the cell RMP and reduce subthreshold excitability. This key function provides a clear underlying explanation for the MoA of RTG/EZG that stabilizes KCNQ2–5 channels in the open position, increasing the recruitment of such channels at rest, and particularly following depolarization, that act to exert a hyperpolarizing effect on the cell. RTG/EZG therefore effectively primes the cell to resist firing bursts of action potentials that occur during the sustained depolarizations associated with the initiation and generalization of seizures. These mechanistic effects are consistent with the anticonvulsant properties of RTG/EZG demonstrated in in-vitro and in-vivo models of epilepsy and in patients with epilepsy (Fig. 5) (see Large et al., 2012).

The pharmacological profile of RTG/EZG is therefore different from all currently approved AEDs and may offer an additional treatment option for patients. Initial clinical trials were focused on the adjunctive use of RTG/EZG in patients with partial epilepsy; however, based on the broad-spectrum antiepileptic potential for this agent defined in a wide range of epilepsy models of partial (focal), generalized, idiopathic, and refractory epilepsy (see Large et al., 2012), further clinical investigation is warranted to explore the utility of RTG/EZG for use in other seizure types.

A further avenue for investigation will be the use of RTG/EZG in combination with other AEDs, as is common in clinical practice. An agent with a fundamentally different MoA such as RTG/EZG may offer good potential for use in combination with AEDs that work in other ways (Fig. 1). In this regard, combination studies conducted in animal models are encouraging, demonstrating additive or even synergistic activity with commonly used AEDs such as valproate and lamotrigine (Luszczki et al., 2009). Further preclinical and clinical studies are now warranted to address this specific aspect of AED therapy, and to understand which AEDs may best complement RTG/EZG to maximize efficacy and reduce the potential for side-effects in patients.

Acknowledgments

  1. Top of page
  2. Summary
  3. KCNQ Channels, a Family of K+ Channels That Control Cellular Excitability in Humans
  4. RTG/EZG Is a Positive Allosteric Modulator (Opener) of KCNQ2–5
  5. RTG/EZG Selectivity of Action: Primary MoA Is at KCNQ Channels
  6. Beyond EC50: Insights into RTG/EZG–KCNQ Pharmacology at the Receptor, Cellular, and Network Level
  7. Discussion
  8. Acknowledgments
  9. Disclosure
  10. References
  11. Supporting Information

The authors thank David Gibson PhD, CMPP of Caudex Medical, New York, NY, USA (supported by GlaxoSmithKline and Valeant Pharmaceuticals International) for providing editorial assistance in the preparation of the manuscript, Chris Crean (Valeant Pharmaceuticals International) for expert input regarding the clinical pharmacokinetic data cited, and Steve White PhD (University of Utah, Salt Lake City, UT, USA) for initial discussions regarding content and a review of the manuscript.

Disclosure

  1. Top of page
  2. Summary
  3. KCNQ Channels, a Family of K+ Channels That Control Cellular Excitability in Humans
  4. RTG/EZG Is a Positive Allosteric Modulator (Opener) of KCNQ2–5
  5. RTG/EZG Selectivity of Action: Primary MoA Is at KCNQ Channels
  6. Beyond EC50: Insights into RTG/EZG–KCNQ Pharmacology at the Receptor, Cellular, and Network Level
  7. Discussion
  8. Acknowledgments
  9. Disclosure
  10. References
  11. Supporting Information

We confirm that we have read the Journal’s position on issues involved in ethical publication and affirm that this article is consistent with those guidelines. Martin J. Gunthorpe and Charles H. Large are former employees of GlaxoSmithKline. Raman Sankar has received basic research support from Valeant Pharmaceuticals and NTP, has participated in clinical trials sponsored by Pfizer, and has served as a speaker and/or paid consultant for GSK, UCB, Lundbeck, NTP, and Sunovion.

References

  1. Top of page
  2. Summary
  3. KCNQ Channels, a Family of K+ Channels That Control Cellular Excitability in Humans
  4. RTG/EZG Is a Positive Allosteric Modulator (Opener) of KCNQ2–5
  5. RTG/EZG Selectivity of Action: Primary MoA Is at KCNQ Channels
  6. Beyond EC50: Insights into RTG/EZG–KCNQ Pharmacology at the Receptor, Cellular, and Network Level
  7. Discussion
  8. Acknowledgments
  9. Disclosure
  10. References
  11. Supporting Information

Supporting Information

  1. Top of page
  2. Summary
  3. KCNQ Channels, a Family of K+ Channels That Control Cellular Excitability in Humans
  4. RTG/EZG Is a Positive Allosteric Modulator (Opener) of KCNQ2–5
  5. RTG/EZG Selectivity of Action: Primary MoA Is at KCNQ Channels
  6. Beyond EC50: Insights into RTG/EZG–KCNQ Pharmacology at the Receptor, Cellular, and Network Level
  7. Discussion
  8. Acknowledgments
  9. Disclosure
  10. References
  11. Supporting Information

Table S1. KCNQ channel distribution and expression.

Table S2. Broad receptor screening of RTG/EZG by radioligand binding versus a panel of selected molecular targets (NovaScreen panel).

FilenameFormatSizeDescription
EPI_3365_sm_TableS1-S2.doc107KSupporting info item

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