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BEHAVIORAL PHARMACOLOGY

Anxiolytic Effects of Maxipost (BMS-204352) and Retigabine via Activation of Neuronal K_v7 Channels

NeuroSearch A/S, Ballerup, Denmark

Received January 21, 2005; accepted March 21, 2005.

	Abstract

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Neuronal K_v7 channels are recognized as potential drug targets for treating hyperexcitability disorders such as pain, epilepsy, and mania. Hyperactivity of the amygdala has been described in clinical and preclinical studies of anxiety, and therefore, neuronal K_v7 channels may be a relevant target for this indication. In patch-clamp electrophysiology on cell lines expressing K_v7 channel subtypes, Maxipost (BMS-204352) exerted positive modulation of all neuronal K_v7 channels, whereas its R-enantiomer was a negative modulator. By contrast, at the K_v7.1 and the large conductance Ca²⁺-activated potassium channels, the two enantiomers showed the same effect, namely, negative and positive modulation at the two channels, respectively. At GABA_A receptors (_122s and _222s) expressed in Xenopus oocytes, BMS-204352 was a negative modulator, and the R-enantiomer was a positive modulator. The observation that the S- and R-forms exhibited opposing effects on neuronal K_v7 channel subtypes allowed us to assess the potential role of K_v7 channels in anxiety. In vivo, BMS-204352 (3-30 mg/kg) was anxiolytic in the mouse zero maze and marble burying models of anxiety, with the effect in the burying model antagonized by the R-enantiomer (3 mg/kg). Likewise, the positive K_v7 channel modulator retigabine was anxiolytic in both models, and its effect in the burying model was blocked by the K_v7 channel inhibitor 10,10-bis-pyridin-4-ylmethyl-10H-anthracen-9-one (XE-991) (1 mg/kg). Doses at which BMS-204352 and retigabine induce anxiolysis could be dissociated from effects on sedation or memory impairment. In conclusion, these in vitro and in vivo studies provide compelling evidence that neuronal K_v7 channels are a target for developing novel anxiolytics.

The tetrameric complexes of neuronal K_v7 channels can be homo- or heteromeric with grossly comparable biophysical and pharmacological properties. The threshold for activation of K_v7 channels is around -60 mV (Yang et al., 1998), which is approximately the resting membrane potential of many neurons. A range of pharmacological tools elucidates the importance of K_v7 channels in controlling neuronal excitability. Retigabine, an atypical anticonvulsant in development for epilepsy (Rostock et al., 1996; Ferron et al., 2002), is a positive modulator of K_v7.2 to K_v7.5 channels at concentrations of 1 to 10 µM (Main et al., 2000; Schrøder et al., 2001; Tatulian et al., 2001; Dupuis et al., 2002; Passmore et al., 2003), whereas it has no effect on K_v7.1 (Tatulian et al., 2001). A structural analog of retigabine, the analgesic flupirtine (Katadolon), also enhances K_v7 channel function, although with lower potency (Ilyin et al., 2002; Korsgaard et al., 2003). The racemic mixture of Maxipost (BMS-204352) and the R-enantiomer activates K_v7.4 and K_v7.5 channels with an EC₅₀ of 2.4 µM (Schrøder et al., 2001; Dupuis et al., 2002). BMS-204352 is a positive BK channel modulator (Gribkoff et al., 2001) that has been in development for stroke. Finally, two enhancers of neurotransmitter release (Zaczek et al., 1998), XE-991 and linopirdine, inhibit neuronal K_v7 channels with low micromolar affinity (Wang et al., 2000; Schrøder et al., 2001).

K_v7 channel activators tend to hyperpolarize or prevent depolarization of a cell and are therefore expected to affect pathological conditions underpinned by hyperexcitability. A direct correspondence between neuronal hyperexcitability and anxiety is seen in amygdala-kindled rats (Racine, 1972), which show a lowered threshold for neuronal excitability and enhanced anxiety in various tests (Rosen and Schulkin, 1998). A large body of research indicates that the amygdala is a key structure in fear/anxiety (Davis, 1992), including an induction of the neuronal marker c-fos in rats exposed to anxiety tests (Sullivan et al., 2003). Compared with most central nervous system neurons (for review, see Steriade and McCarley, 1990), the spontaneous firing rate of amygdala neurons are among the lowest in the brain, e.g., the spontaneous firing rate of some projection neurons in the amygdala are typically <1 Hz (Pare and Collins, 2000). This suggests a powerful inhibitory tone in the amygdala, preventing inappropriate fear responses (Davis, 1992; Pare and Collins, 2000). Also, provoking fear in anxiety patients increases glucose utilization, i.e., activity, in the amygdala and leads to an exaggerated fear response (Anand and Shekhar, 2003; Rauch et al., 2003).

The neuronal K_v7 channels are widely expressed in the brain, including the amygdala (Saganich et al., 2001), and the electrophysiological correlate of K_v7 channels, known as the M current (Brown and Adams, 1980), has been described in the amygdala (Womble and Moises, 1992). In this study, it is demonstrated that BMS-204352 enhances, whereas the R-enantiomer inhibits, currents mediated through neuronal K_v7 channels expressed in HEK293 cells. The observation that the S- and R-forms exhibited opposing effects on neuronal K_v7 channel subtypes was surprising and proved to be a powerful in vivo tool in determining the potential role of K_v7 channels in anxiety. BMS-204352 and the R-enantiomer were compared in various anxiety tests, with the former compound showing anxiolytic effects and the latter antagonizing these effects. This led us to conclude that K_v7 channels are novel targets for treating anxiety. Additional studies reported here support this conclusion.

	Materials and Methods

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In Vitro
Molecular Biology. Stable cell lines for K_v7.2/3 and 7.3/4 (Schrøder et al., 2001), K_v7.4 (Søgaard et al., 2001), K_v7.5 (Dupuis et al., 2002), the large conductance Ca²⁺-activated potassium (BK) channel (Ahring et al., 1997), and the GABA_A receptors (Jensen et al., 2002) have been described previously. In short, for stable expression of K_v7 channels the coding regions were subcloned into mammalian expression vectors belonging to the pNS series (NeuroSearch custom-made expression vectors derived from pcDNA3 from Invitrogen, Carlsbad, CA). Host cells were HEK293 (American Type Culture Collection, Manassas, VA), and transfections were carried out using 2 µg of the appropriate construct(s) and LipofectAmine (Invitrogen) according to manufacturer's instructions. Transfected HEK293 cells were cultured in Dulbecco's modified Eagle medium (Invitrogen) at 37°C in 5% CO₂ and 95% air supplemented with 10% fetal bovine serum and the appropriate selection agent(s). When the selection medium resulted in single colonies, these were picked and propagated. Expression of ion channels was confirmed functionally with patch-clamp electrophysiology.

Patch-Clamp Electrophysiology. Experiments with the HEK293 cell lines were performed in the whole-cell configuration. On the day of experiment, cells were plated on cover slips (Ø = 3.5 mm) and allowed to attach. The cover slip was then placed in a custom-made 15-µl perfusion chamber (flow rate, 1 ml/min) mounted on an Olympus IX-70 microscope (Tokyo, Japan). The microscope was placed on a vibration-free table (TMC, Peabody, MA) in a grounded Faraday cage. All experiments were performed at room temperature (20-22°C). An EPC-9 patch-clamp amplifier (HEKA, Lambrecht, Germany) was connected to a personal computer via an ITC16 interface and controlled by Pulse software (HEKA). Data were stored directly on the hard disc and analyzed by the IGOR software (Wavemetrics, Lake Oswego, OR). The pipette capacitance (C_fast) was compensated when a giga-ohm seal was obtained, and cell capacitance (C_slow) as well as series resistance (R_s) were compensated electronically before every application of the voltage protocol. Usually, C_slow ranged from 5 to 20 pF, and R_s was in the range 2 to 5 M. All experiments with drifting R_s values (more than 100%) were discarded, and calculated voltage errors due to R_s were always <5 mV. After the establishment of the whole-cell configuration, the voltage protocol was applied to the cell every 5 s from a holding potential of -90 mV. Since the holding potential was very close to the equilibrium potential for potassium (E_K,20°C = -91 mV), and since the studied channels are closed at negative potentials, a brief hyperpolarizing step to -150 mV was used to assess the degree of leak current. Due to the length (>1.5 s) of the protocol, this leak assessment was performed instead of traditional leak subtraction with a number of downscaled sweeps, and it also allowed the study of drug effects on the leak currents. The experiment was discontinued if the leak current in the control situation exceeded 10% of the voltage-activated current. Since the leak was measured at a potential with approximately the same driving force on K⁺-ions as the activating step, it was subtracted postexperimentally when appropriate.

Xenopus laevis Oocyte Electrophysiology. Selected oocytes were injected with 25 to 50 nl of cRNA mixture using a Pico Pump (WPI, Sarasota, FL). The cRNA mixture was GABA_AR subunits _1/2, ₂, and _2s in the ratio of 2:2:3 and in a total concentration of 1 µg/µl. For two-electrode voltage-clamp experiments, an oocyte was placed in an RC26Z recording chamber (Warner Instruments, Hamden, CT) that was continuously perfused with 2.5 ml/min OR2. Recording electrodes were fabricated from BF150-110-10 borosilicate glass with filament (Sutter Instrument Company, Novato, CA) using a DMZ-Universal puller (Zeitz, Augsburg, Germany). Electrodes were initially filled with a small plug of 0.2% agarose made in 3 M KCl and then backfilled with 3 M KCl and stored in 3 M KCl until needed. Typical electrode resistances were 0.4 to 0.8 M. Currents were amplified by a Geneclamp 500B (Axon Instruments, Inc., Union City, CA), low-pass filtered at 20 Hz, digitized at 500 Hz by a Digidata 1322A (Axon Instruments), and then recorded as well as analyzed by a personal computer (Compaq Evo) using the pClamp8 suite (Axon Instruments). Drug application was controlled by placing a capillary tube with an inner diameter of 1.5 mm (Modulohm 214813; Modulohm, Herlev, Denmark) 1 to 2 mm from the oocyte and connecting this through Teflon tubing to a Gilson 233XL autosampler (Gilson Medical Electric, Middleton, WI). A flow of 2.5 ml/min through the capillary tube ensured a rapid exchange of liquid surrounding the oocyte in the matter of few seconds. During the time interval between recordings, which was set at 5 min, the oocyte was perfused with OR2 through the capillary tube as well as the bath perfusion. Effects of compounds were evaluated by comparing the currents recorded from an EC₂₀ concentration of GABA with or without compound.

Solutions: Patch-Clamp Experiments. The extracellular (bath) solution was 140 mM NaCl, 4 mM KCl, 2 mM CaCl₂, 1 mM MgCl₂, and 10 mM HEPES (pH = 7.40 with NaOH). Test compounds (see Chemistry) were dissolved in the extracellular ringer by dilution at least 1000 times from DMSO stock solutions (10 mM). The less than 1%thou DMSO did not have any effect on channel function. The intracellular (pipette) solution was 144 mM KCl, 5.37 mM CaCl₂, 1.75 mM MgCl₂, 4 mM NaATP, 0.4 mM NaGTP, 10 mM EGTA, and 10 mM HEPES (pH = 7.20 with KOH). By use of the program EqCal (Biosoft, Ferguson, MO), the concentration of free Ca²⁺ was calculated to 100 nM, the free Mg²⁺ was 0.1 mM, and MgATP was 1.45 mM.

Solutions: Two-Electrode Oocyte Experiments. The OR2 solution was 90 mM NaCl, 2.5 mM KCl, 2.5 mM CaCl₂, 1 mM MgCl₂, and 5 mM HEPES pH 7.4. GABA was dissolved in OR2 to a final concentration of 2 µM. Test compounds were initially dissolved at 10 mM in 100% DMSO and then diluted in the experimental solution (OR2 with 2 µM GABA).

Fitting Procedures. As previously described (Strøbæk et al., 2000), K_i values were calculated based on current versus time plots and the kinetics of drug-induced reduction in the current. This is fitted with the nonequilibrium version of the Michaelis-Menten equation:

(1)

The current I at time t is a function of basal current I₀ and concentration C of inhibitor. k_off is the off-rate (s^-1), k_on is the on-rate (M^-1 · s^-1) and K_i = k_off · k_on^-1 is the inhibitory constant.

The activation curves were fitted according to Boltzmann formalism;

(2)

allowing an initial voltage-independent component (I_leak) and then a voltage-dependent rise to I_max with a slope of k and midpoint V_0.5.

Chemistry. All compounds used in this study were synthesized in the Medicinal Chemistry department, NeuroSearch A/S (Ballerup, Denmark). The structures of the compounds synthesized are depicted in Fig. 1.

View larger version (18K): [in this window] [in a new window]   Fig. 1. Structures of compounds.

Retigabine [D23129; N-(2-amino-4-(4-fluorobenzylamino)-phenyl)-carbamic acid ethyl ester] was obtained in an overall yield of 30% as the bis-hydrochloride by following the synthesis described in the literature (Dieter et al., 1996). The free base was isolated as a white crystalline compound by column chromatography. Retigabine as a free base is a stable compound with a molecular weight of 303.3 g/mol, provided it is stored in the dark. Retigabine has a cLogP of 2.0 and pK_a of 10.8.

The R-enantiomer [(R)-3-(5-chloro-2-methoxyphenyl)-3-fluoro-6-(trifluoromethyl)-1,3-dihydro-2H-indole-2-one] was obtained from the mother liquor of the synthesis of BMS-204352 by a standard procedure (Pendri et al., 1998), giving an overall yield of 19% and an enantiomeric excess of 94.2%.

XE-991 (10,10-bis-pyridin-4-ylmethyl-10H-anthracen-9-one) was synthesized in a one-step procedure (Earl et al., 1989) from anthrone and 4-picolylchloride hydrochloride to give a yield of 30%. The yellowish crystalline compound has a molecular weight of 376.5 g/mol and a cLogP of 4.6.

In Vivo
Animals. Female NMRI or male C57 mice (20-25 g), and male Wistar rats (150-180 g) were used in behavioral studies (Taconic M&B Breeding Center, Germantown, NY). Animals were housed and habituated for at least 7 days before experiments in Macrolon III cages (20 x 40 x 18 cm) with eight mice or two rats per cage. Food (Altromin, Lage, Germany) and tap water were available ad libitum. Animals were housed on a 12-h light/dark cycle (lights on at 7:00 AM and off at 7:00 PM). All experiments were performed according to the guidelines of the Danish Committee for Experiments on Animals.

Drugs and Solutions. All drugs were administered i.p. dissolved in 5% Tween 80 15 min prior to a behavioral test unless otherwise stated. In mice, substances were administered in a volume of 10 ml/kg and in rats as 1 ml/kg volume.

General in Vivo Models
Mouse Exploratory Motility. Effect of test compounds on exploratory motility in mice was measured in automated cages (20 cm x 30 cm; TSE Systems, Bad Homburg, Germany) equipped with infrared sensors (6 x 2). Mice were individually placed in cages for 30 min, and interruptions of infrared sensor pairs were detected by a TEST control unit with the data recorded on a computer running ActiMot software (TSE Systems). Data shown are the mean distance ± S.E.M. animals traveled during the 30-min test.

Mouse Wire Grip Test. The wire grip test was used to determine the effect of test compounds on muscle relaxation/strength. Mice were suspended with both front paws on a horizontal steel wire (50 cm long, diameter 1.5 mm) attached to two poles and elevated 28 cm above a bench. The number of mice unable to maintain grip of the steel wire with both front paws and one hind paw in one of three trials was determined.

Mouse Rotarod Ataxia. Effect of test compounds on sensorimotor function was evaluated using the mouse Rotarod. The Rotarod consisted of a wooden rod (length, 50 cm; diameter, 4 cm) partitioned into sections by plastic discs (diameter, 17 cm), allowing up to eight mice to be tested simultaneously. Mice were pretrained on the day of testing, and only mice capable of walking on the rotating rod (6 revolutions/min) for 2 min were selected for drug testing. After drug administration, the mice falling off the Rotarod twice in a 2-min period were classified as ataxic.

Mouse Anxiety Models
Mouse Zero Maze. The potential of test compounds to affect anxiety levels was evaluated in the mouse zero maze (TSE Systems), an unconditioned model of anxiety (Shepherd et al., 1994). The zero maze had a total diameter of 53 cm and was elevated 50 cm above the ground. The platform (width, 2.7 cm) consisted of two open areas (wall height, 6 mm) and two closed areas (wall height, 11 cm). The walls were made of clear Perspex. The mouse was placed in the closed area facing the closed area and was allowed to explore the maze for 5 min. The initial latency to enter the open area with all four paws placed in an open area was measured. Thereafter, the Noldus Observer program (version 2.01; Noldus Information Technology, Wageningen, The Netherlands) was used to register the following five events: 1) time spent in the open area, 2) number of entries into the open areas (an open area entry was defined as the mouse having all four paws in an open area), 3) number of stretched-attend postures (defined as the mouse, while situated in a closed area, first exhibiting an elongated body posture resulting in both front paws and snout crossing over the closed-open divide, followed by rapid retraction of the whole body back to the original position), 4) number of rearings (rising on the hind paws) in the closed area, and 5) number of head dips in the open areas (scanning over the side of the maze). Data for each measure are presented as the mean ± S.E.M. Experiments were conducted in rooms where light levels were set at 20 lux (YF-1065; YFE, Taipei, Taiwan). The experimenter was blind to the treatments given to animals in all studies.

Mouse Marble Burying. Mice were placed for 1 h in novel cages (one mouse per cage, 20 x 30 cm) in which there were 20 glass marbles (15 mm in diameter) situated in four rows of five on top of 5 cm of sawdust. The mean number of glass marbles buried ± S.E.M. between 10 to 60 min was taken as an index of "anxiety", i.e., the more marbles buried the more anxious the mouse-a marble was classified as buried when at least two-thirds was covered by sawdust (Broekkamp et al., 1986). The experimenter was blind to the treatments given to animals in all studies.

Rat Anxiety Models
Rat-Conditioned Emotional Response. Coulbourn operant chambers (Coulbourn Instruments, Allentown, PA) with associated Coulbourn pellet dispensers and operant levers were used. These chambers were housed in outer sound- and light-attenuating shells equipped with a ventilation fan that also helped to mask external noise. A single operant lever was positioned on the left side of the front panel of the operant chamber, approximately 2 cm above the grid floor. Formula P Noyes food pellets (45 mg; Research Diets, Inc., New Brunswick, NJ) could be delivered to the food magazine centered on the front wall of the chamber approximately 2 cm above the floor. A houselight (4.0 mA, 28 V) was positioned on the front wall of the chamber, above the food magazine, approximately 1 cm below the ceiling. The operant chambers were controlled and the number of lever presses recorded by MED Associates (St. Albans, VT) software.

Rats were water-deprived 24 h prior to being placed in the operant chambers and initially trained to associate lever pressing with water reward on a fixed ratio 1 reinforcement schedule. The schedule of requirement was progressed until rats had been trained to lever press for water reward on a variable interval 60-s schedule (Lattal, 1991). Training took place 5 days a week, and conditioning sessions lasted 20 min. Every training session consisted of 5-min light (L) and 2.5-min dark (D) periods in the fixed sequence L D L D L. During both L and D periods, water was delivered on the variable interval 60-s schedule. In the D periods, lever presses also elicited mild scrambled foot shocks (0.4 mA, 0.1 s, Lafayette Master shocker; Lafayette Instrument Co., Lafayette, IN) through the grid floor on an independent variable interval 20-s schedule. After approximately 10 weeks of training, lever pressing was almost completely suppressed during the D periods, and testing began.

Testing. Rats were tested with drugs twice a week on Tuesdays and Thursdays (shock off), and the other three workdays were used as baseline training sessions (shock on). Prior to every baseline session, rats were injected with physiological saline to eliminate any cues deriving from the injection. The number of lever presses during the L and D periods was determined, and an increase in response rate during the D period with no effect on response rate in the L period was indicative of an anxiolytic effect. In addition, these response rates were used to calculate a suppression ratio (SR) according to the following formula:

(3)

A suppression ratio of 0 indicates that the D has evoked conditioned fear and has completely suppressed lever pressing, whereas a suppression ratio of 0.5 indicates the response rate is unchanged by the D, i.e., the complete absence of fear.

Data Analysis. All data were analyzed using SigmaStat version 2.0 (SPSS Inc., Chicago, IL). For zero maze, marble burying, and exploratory motility, data were analyzed by one-way analysis of variance followed by Dunnett's or Tukey's honest significant difference tests where appropriate. For the conditioned emotional response test in rats, separate two-way analyses of variance for the L, D, and suppression ratio measures were conducted with treatment as the between-subjects factor and trial as the within-subjects factor. Dunnett's post hoc test was used in the case of a significant main effect of treatment. In no conditioned emotional response study was a significant treatment x trial interaction found and, therefore, is not reported. Significance was set at P < 0.05.

	Results

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It was previously shown, that the racemic mixture of BMS-204352 and the R-enantiomer activates K_v7.4 channels expressed in HEK293 cells (Schrøder et al., 2001, 2003). In continuation of these studies and as a prerequisite to the in vivo studies, the resolved enantiomers were initially tested for their effects on K_v7 channels.

In Vitro Effects
Opposing Effects of the Enantiomers on K_v7.4 Channels. Figure 2 shows the effect of both enantiomers in comparison with retigabine on K_v7.4 channels expressed in HEK293 cells. The top panel depicts the voltage protocol. The holding potential was -90 mV, and a brief (50 ms) hyperpolarizing step to -150 mV was performed to assess an eventual leak component before the K_v7.4 channels were activated by a depolarizing step to -30 mV. Interestingly, the effects of the enantiomers were opposing (middle panel); BMS-204352 activated whereas the R-enantiomer inhibited the voltage-activated current. The bottom panel in Fig. 2 shows the time course of a representative experiment. The K_v7.4 current was measured at the end of the depolarizing step and plotted as a function of time. The K_v7.4 currents were, in general, small just after establishment of the whole-cell configuration but increased within the first 1 to 5 min due to an unknown process, and usually 5 to 10 min were allowed to pass before a stable baseline was obtained. In the experiment shown, 10 µM retigabine was first added as a control, and a fast and prominent increase in current was induced. After a washing period, 10 µM BMS-204352 was applied, and a similar effect was obtained although the washout was somewhat slower compared with retigabine. It was noted (bottom panel) that after an initial peak effect of the compounds, there was a slight desensitization to the drugs. This was due to an acceleration of the activation kinetics, which imposed a dynamic effect on the current trace (not shown). This meant that the effect at measuring point (arrow in top panel in Fig. 2) would overshoot before reaching equilibrium. In addition to the increase in steady-state current, deactivation was dramatically slower in the presence of BMS-204352, and a prominent current was induced at -150 mV (see traces in middle panel). The current induced by BMS-204352 at the step to -150 mV appeared similar to the previously described voltage-independent K_v7.4 current induced by the racemate (Schrøder et al., 2003). The following application of the R-enantiomer (10 µM) was neither similar nor inert but induced a slow reversible inhibition of the current. Note that the kinetics of the drug-channel interaction (bottom panel) differed among the enantiomers in that the activator was faster than the inhibitor. Furthermore, as described for the racemate, the drug-channel kinetics for activation induced by BMS-204352 were slower at the voltage-independent current than at the voltage-dependent current (data not shown).

View larger version (20K): [in this window] [in a new window]   Fig. 2. Enantiomer-specific modulation of Kv7.4 channels by BMS-204352 and the R-enantiomer. Kv7.4 channels were activated by a step to -30 mV from a holding potential of -90 mV. The top panel shows the entire voltage protocol that consists of 100 ms at -90 mV, 50 ms at -150 mV, 50 ms at -90 mV, 1 s at -30 mV, and finally 500 ms at -90 mV. The middle panel shows a control trace and traces obtained in the presence of either 10 µM retigabine, 10 µM BMS-204352, or 10 µM of the R-enantiomer of BMS-204352. The voltage protocol was applied every 5 s, and the bottom panel shows the current obtained at the end of the activating step (arrow in protocol) as a function of time. Compounds were present in the extracellular solution during the periods indicated by the bars.

Effects of the Enantiomers on the Activation Curves of K_v7.4 Channels. We then wanted to test the effects of BMS-204352 and the R-enantiomer on the activation curve of K_v7.4 measured as tail currents (Fig. 3). The channels were exposed to potentials ranging from -160 mV to 40 mV for 1 s before stepping to -120 mV (top panel). BMS-204352 lowered the threshold for activation, accelerated the rate of activation, and slowed the deactivation at -120 mV. In contrast, the R-enantiomer suppressed the current at all potentials. The peak tail current as a function of the preceding potential is shown in the bottom panel. The activation curve in the control situation has a threshold for activation of -60 mV and a half-maximal activation potential (V_0.5)of -24 mV (interval: -33 to -21 mV, n = 5). The activation curve was shifted in the hyperpolarized direction by 10 µM BMS-204352, resulting in a threshold of approximately -85 mV and a V_0.5 = -43 mV (interval, -54 to -36 mV, n = 4) and a maximal voltage-dependent current approximately 80% above the control situation. The R-enantiomer gave approximately the same threshold of activation as the control and an insignificantly different V_0.5 of -20 mV (interval, -28 to -11 mV, n = 3, P = 0.2).

View larger version (20K): [in this window] [in a new window]   Fig. 3. Enantiomer-specific shift in the activation curve for Kv7.4. A series of voltage steps (1-s duration) ranging from -120 mV to +40 mV each followed by a step to -120 mV was applied every 5 s from a holding potential of -90 mV. Top panel shows the currents obtained before (control, left traces) and after application of 10 µM BMS-204352 (middle traces) as well as the R-enantiomer of BMS-204352 (right traces). Bottom panel shows the peak tail currents measured at the step to -120 mV as a function of the potential of the preceding voltage step. Data obtained before () and after application of 10 µM BMS-204352 () or the R-enantiomer () are shown. The amplitude of the tail current represents a qualitative measure of the degree of channel activation. A Boltzmann fitting procedure (eq. 2) to the data points resulted in the solid curves shown and resulted in half-maximal activation potentials (V0.5)of -25.9, -35.2, and -19.7 mV for control, BMS-204352, and R-enantiomer, respectively.

Subtype-Dependent Effects Induced by the Enantiomers. To test whether the opposing effects of this pair of enantiomers were an isolated phenomenon on K_v7.4 channels, similar experiments were performed on other -subunits of the K_v7 family (Fig. 4A). For the neuronal K_v7 channel subtypes expressed as homo- or heteromers, the effect of the enantiomers were qualitatively like that on K_v7.4 with respect to the voltage-dependent current: an activation by BMS-204352 and an inhibition by the R-enantiomer. In Table 1, the activating effect of BMS-204352 has been calculated as percentage of baseline current, and K_i values for the inhibition by the R-enantiomer have been calculated according to eq. 1. Across the subunit combinations, K_v7.5 and K_v7.3/5 were the most sensitive (605 and 931% of baseline, respectively), K_v7.4 and K_v7.3/4 exhibited medium sensitivity (230-245%), whereas K_v7.2/3 appeared to be least sensitive (125% of baseline at 10 µM). The potentiation of K_v7.5 containing channels showed rather large variations. The inhibition by the R-enantiomer was very similar for channels containing K_v7.4 or K_v7.5, whereas the inhibition was weaker for K_v7.2/3 channels (K_i = 24 µM compared with 3.4 to 5.4 µM for the other subunit combinations tested). When the enantiomers were applied to K_v7.1, the effect was surprisingly an inhibition of the current for both BMS-204352 and the R-enantiomer, amounting to K_i values of 3.7 and 4.3 µM, respectively.

View larger version (15K): [in this window] [in a new window]   Fig. 4. Differential effects of BMS-204352 and the R-enantiomer on neuronal Kv7 subtypes, Kv7.1 and BK. A, whole-cell currents (VH = -90 mV) obtained by activation at -30 mV of various Kv7 channel subtypes before (left panels) and after application of 10 µM BMS-204352 (middle panels) or 10 µM of the R-enantiomer (right panels). B, whole-cell currents (VH = -90 mV) obtained by activation at 0 mV of BK channels. The traces were obtained before (control) and after application of 10 µM BMS-204352 (top panel) or the R-enantiomer (bottom panel).

Effects of Enantiomers on BK and GABA Receptors. BMS-204352 is a positive modulator of the large conductance calcium-activated K⁺ channel (BK), (Gribkoff et al., 2001). The enantiomers were tested on BK channels expressed in HEK293 cells, and it was found that both enhanced the BK-mediated currents at 0 mV by approximately 3-fold (Fig. 4B) with identical kinetics (not shown). The compounds were also tested on GABA_A receptors using two different subunit combinations: ₁₂₂ and a₂₂₂ expressed in Xenopus oocytes (data not shown). In combination with an EC₂₀ concentration of GABA, BMS-204352 (10 µM) reduced the current by 20% (n = 3-5) on both receptor subtypes, whereas the R-enantiomer (10 µM) on average enhanced the current by 13% (n = 2-7). These effects were concentration-dependent, reaching maximum inhibition of 46% (n = 5) on ₁ (100 µM BMS-204352) and a maximum potentiation of 46% (n = 2) on ₁ (100 µM R-enantiomer). For comparison, retigabine was tested on the same subunit combinations and showed concentration-dependent potentiations up to 100% (n = 2, 30 µM retigabine).

In Vivo Effects
Ancillary Models (Mouse). To aid in the interpretation of drug effects in the anxiety models described further below, we initially tested the drugs for any nonspecific sensorimotor effects using standard motility cages, a Rotarod, and a simple index of muscle strength.

Exploratory Motility (See Fig. 5). BMS-204352 (3-30 mg/kg) had no effect on exploratory activity [F(3,24) = 1.7], whereas the R-enantiomer (3-30 mg/kg) significantly reduced distance traveled by the mice [F(3,24) = 20.8], with a modest reduction compared with the vehicle group at 10 to 30 mg/kg (P < 0.05). Retigabine had a significant effect on exploratory activity [F(3,24) = 30.2], with a significant reduction in distance traveled at 30 mg/kg compared with vehicle (P < 0.05).

View larger version (11K): [in this window] [in a new window]   Fig. 5. Effect of neuronal Kv7 channel modulators on exploratory motility in mice. A, retigabine; B, BMS-204352; and C, R-enantiomer 15 min after i.p. administration. Data are expressed as mean distance traveled (minutes) ± S.E.M. (n = 8). *, P < 0.05 compared with the vehicle control group (Dunnett's test).

Anxiolytic Models (Mouse)
Zero Maze (See Fig. 6 and Table 2). In this unconditioned animal model of anxiety, both positive modulators of neuronal K_v7 channels, BMS-204352 and retigabine, induced a dose-dependent anxiolytic effect. BMS-204352 significantly and dose dependently increased time spent in the open areas [F(3,27) = 18.2], reduced latency to enter an open area [F(3,27) = 6.9], and increased the number of entries in to the open areas [F(3,27) = 5.0], with a significant difference at doses of 10 to 30 mg/kg on these measures relative to the vehicle control group (P < 0.05). Furthermore, at 30 mg/kg, BMS-204352 also significantly reduced stretch-attend postures [F(3,27) = 5.7] and increased head dips [F(3,27) = 3.9], with no effect on rearing activity [F(3,27) = 0.8].

View larger version (31K): [in this window] [in a new window]   Fig. 6. Effect of neuronal Kv7 channel modulators in the mouse zero maze. A, retigabine; B, BMS-204352; and C, R-enantiomer 15 min after i.p. administration on time in the open areas (seconds), latency to enter the open areas (seconds), and number of entries to the open areas (note the different scale on the y-axis for the R-enantiomer for the measures of time in open and latency to enter open). Data are expressed as means ± S.E.M. (n = 7-8). *, P < 0.05 compared with the vehicle control group (Dunnett's test).

Retigabine dose dependently and significantly increased time spent in the open areas [F(3,25) = 4.9], reduced latency to enter an open area [F(3,25) = 5.5], and increased the number of head dips [F(3,25) = 4.3], at doses between 10 to 30 mg/kg relative to the vehicle control group (P < 0.05). There was no significant effect on the number of entries to the open areas [F(3,25) = 2.7] or stretched-attend postures [F(3,25) = 2.3]. However, there was a significant main effect of treatment on the number of rearings [F(3,25) = 3.0], although Dunnett's tests showed that no drug treatment group differed from the vehicle group.

In contrast to the two positive modulators of neuronal K_v7 channels, the R-enantiomer, a negative modulator of these channels, was inactive up to 30 mg/kg on any measure in the zero maze [F(3,25), range of 1.0-2.2 for all measures]. There was a tendency for the R-enantiomer to reduce time spent in the open areas and increase latency to enter an open area, i.e., effects opposite to those seen with BMS-204352 on these parameters.

Marble Burying (See Figs. 7 and 8). In this unconditioned model of anxiety in mice, in which animals bury harmless glass marbles placed on top of sawdust in an observation cage, both BMS-204352 and retigabine reduced burying behavior, indicating an anxiolytic effect. BMS-204352 significantly reduced marble burying behavior in a dose-dependent manner [F(3,28) = 15.2], with a significant difference (P < 0.05) from vehicle at the highest dose of 30 mg/kg. Likewise, retigabine dose dependently and significantly [F(3,28) = 25.8] reduced burying behavior, with a significant reduction compared with vehicle at 3 to 10 mg/kg (P < 0.05). From the data in the section on ancillary models described above, it is clear that at the doses effective in the marble burying test, neither retigabine (1-10 mg/kg) nor BMS-204352 (3-30 mg/kg) affected exploratory activity in mice, suggesting that the reduction in marble burying seen with these two compounds at these doses cannot easily be attributed to nonspecific motor deficits (compare Figs. 5 and 7).

View larger version (21K): [in this window] [in a new window]   Fig. 7. Effect of neuronal Kv7 channel modulators on marble burying in mice. A, retigabine; B, BMS-204352; C, R-enantiomer; and D, XE-991 15 min after i.p. administration. Data are expressed as mean burying behavior ± S.E.M. (n = 8). *, P < 0.05 compared with the vehicle control group (Dunnett's test).

View larger version (12K): [in this window] [in a new window]   Fig. 8. Antagonism of the reduction in marble burying induced by retigabine and BMS-204352 by the neuronal Kv7 inhibitors XE-991 and the R-enantiomer. A, retigabine (10 mg/kg) alone and retigabine (10 mg/kg) + XE-991 (1 mg/kg); B, BMS-204352 (30 mg/kg) alone and BMS-204352 (30 mg/kg) + R-enantiomer (3 mg/kg). Data are expressed as mean burying behavior ± S.E.M. (n = 8). *, P < 0.05 compared with the vehicle control group; #, P < 0.05 compared with the retigabine/BMS-204352 alone group (Tukey's honest significant difference multiple comparison test).

In the following studies where the K_v7 blockers were used to antagonize the effects of the positive modulators of neuronal K_v7 channels, we selected a dose of 1 mg/kg XE-991 and 3 mg/kg of the R-enantiomer so as to avoid any adverse effects of the blockers themselves as indicated in the exploratory motility and/or Irwin studies described above. Furthermore, since XE-991 has a short t_1/2 (Earl et al., 1998), we administered this compound 5 min before the marble burying test and 10 min after administration of 10 mg/kg retigabine. In Fig. 8A, it is clear that XE-991 at 1 mg/kg completely reversed the reduction in marble burying induced by retigabine (10 mg/kg). There was a main effect of treatment [F(2,21) = 8.4], with the post hoc multiple comparison test showing significant differences between the vehicle and retigabine alone treated groups, as well as between the XE-991 + retigabine and retigabine alone groups (P < 0.05). Likewise, Fig. 8B shows that the R-enantiomer (3 mg/kg) antagonized the reduction in marble burying induced by BMS-204352 (30 mg/kg). There was a main effect of treatment [F(2,21) = 5.8], with the post hoc multiple comparison test showing a significant difference between the control and BMS-204352 alone groups, as well as between the BMS-204352 alone and BMS-204352 + R-enantiomer groups.

Anxiolytic Models (Rat)
Conditioned Emotional Response (See Table 3). In this shock-based conditioned model of anxiety in rats, BMS-204352 engendered an anxiolytic profile, although this was not seen with retigabine. During baseline responding in the light period, retigabine completely disrupted lever pressing at 10 mg/kg, and therefore, this dose was excluded from further analysis. Neither retigabine (1-3 mg/kg) nor BMS-204352 (3-60 mg/kg) significantly affected response rate in the dark (anxiety) period of the test [main effect of treatment: F(2,25) = 1.5 and F(4,41) = 1.8, respectively] or during baseline responding in the light period of the test [main effect of treatment: F(2,25) = 0.8 and F(4,41) = 1.0, respectively]. By contrast, whereas retigabine had no effect on the suppression ratio [F(2,25) = 0.1], BMS-204352 significantly affected the ratio between light and dark responses [F(4,41) = 2.6, P < 0.05], indicative of an anxiolytic profile.

	Discussion

Top Abstract Materials and Methods Results Discussion References

 
In this study we show that 1) BMS-204352 is a positive modulator of all neuronal K_v7 channel subtypes expressed in HEK293 cells, whereas the R-enantiomer is a negative modulator; 2) both enantiomers are negative modulators of K_v7.1 channels; 3) BMS-204352 and the R-enantiomer are positive modulators of BK channels; 4) whereas BMS-204352 is a negative modulator of GABA_A receptors, the R-enantiomer is a positive modulator; 5) BMS-204352 as well as retigabine show clear dose-related anxiolytic efficacy in the mouse zero maze; 6) in another unconditioned model of anxiety, the marble burying test (Broekkamp et al., 1986), BMS-204352 and retigabine reduced burying behavior at doses not affecting general activity levels; and 7) these effects on burying behavior can be antagonized by a K_v7 channel blocker, either XE-991 or the R-enantiomer of BMS-204352. These behavioral and electrophysiological data suggest that the anxiolytic efficacy of BMS-204352 can be ascribed to positive modulation of neuronal K_v7 channels, indicating that this is a novel target for developing anxiolytics. Finally, neither BMS-204352 (3-30 mg/kg) nor retigabine (1-10 mg/kg) impaired the performance of rats in a delayed nonmatching to position task (Dunnett et al., 1989), a test for memory (data not shown).

The racemic mixture of BMS-204352 and its R-enantiomer was previously shown to activate voltage-dependent and voltage-independent K_v7.4 currents (Schrøder et al., 2003). In this study, we investigated whether these two effects were separately induced by one enantiomer. We found that BMS-204352 induced activation of the voltage-dependent as well as the voltage-independent current and that the R-enantiomer inhibited both K_v7 current components. This profile holds true for all the neuronal K_v7 channel subtypes, whereas for the K_v7.1 subtype both enantiomers block the current. In light of the presence of K_v7.2/7.3 (Saganich et al., 2001) and K_v7.5 (Schroeder et al., 2000a) in the amygdala, and the absence of K_v7.1 in the brain, this implies positive modulators of these channels have a potential as anxiolytics.

To ascertain if an effect at K_v7 channels specifically mediates the anxiolytic effects of BMS-204352 and retigabine observed in this study, we additionally tested the compounds on BK and GABA_A channels. The BK channel is found in the basolateral amygdala (Meis and Pape, 1997) but is unlikely to mediate the anxiolytic effects of BMS-204352, since both this and the R-enantiomer are positive modulators of the BK channel. GABA_A receptors are the targets of traditional anxiolytics belonging to the benzodiazepine class. Retigabine has been reported to affect GABA_A currents (Rundfeldt and Netzer, 2000), which in part could account for its antiepileptic effects. We also describe a positive modulatory effect in vitro of retigabine on GABA_A receptors, but in line with others (Rundfeldt and Netzer, 2000), we found that the concentration required was at least one order of magnitude above that of the K_v7 effect. Moreover, we have seen that retigabine (0.3-10 mg/kg) shows no generalization to the chlordiazepoxide discriminative cue (data not shown), a cue based on the ability of benzodiazepines to allosterically enhance the effects of GABA. The negative modulatory effect of BMS-204352 and the positive modulatory effect of the R-enantiomer at GABA_A receptors, respectively, suggest that if an effect at GABA_A receptors is relevant to our observations in the anxiety tests then the R-enantiomer rather than BMS-204352 would have been predicted to be anxiolytic.

In the zero maze unconditioned anxiety model we saw dose-dependent effects of both BMS-204352 and retigabine. In addition, we saw a tendency for the R-enantiomer to show the opposite effect of BMS-204352. In the marble burying model, BMS-204352 had an anxiolytic effect, which could be fully antagonized by the R-enantiomer, which argues in favor of a neuronal K_v7 channel-mediated mechanism. Additionally, with the K_v7 channel blocker, XE-991 (1 mg/kg), we were able to reverse the reduction in marble burying behavior induced by retigabine, although this dose of XE-991 in itself reduced marble burying. In contrast to these effects in unconditioned models of anxiety in mice, we saw no affect of retigabine in the conditioned emotional response model of anxiety in rats. Shock-based models like the conditioned emotional response are highly sensitive to benzodiazepines (Green, 1991), and either this factor or a species difference between mouse and rat may explain the lack of efficacy with retigabine. By contrast, BMS-204352 engendered an anxiolytic response at 60 mg/kg in the conditioned emotional response test. Looking closely at the data (Table 3), it appears that the basis for the increase in suppression ratio after BMS-204352 was an increase in response rate in the dark period rather than a decrease in light period response rate, suggesting a true anxiolytic profile. Nonetheless, the effect is not equivalent to the efficacy attained with a benzodiazepine in this model (Stanhope and Dourish, 1996).

The in vitro profiles of BMS-204352 and the R-enantiomer provide powerful tools with which to delineate behavioral effects mediated by neuronal K_v7 channels. Here we have demonstrated that the positive modulator of neuronal K_v7 channels, BMS-204352, is anxiolytic in two unconditioned models of anxiety in mice and that the R-enantiomer antagonizes the effect in one model. Moreover, BMS-204352 is also active in a rat-conditioned model of anxiety, although the effect is not of the magnitude seen with benzodiazepines in this test. Further supporting data with retigabine and XE-991 demonstrate that this channel family may be a target for the development of novel anxiolytics. Clearly, further studies are needed to address potential tolerance, abuse, or other side effect liabilities that may be associated with this target. Thus far, our data suggest that separation between sedative/cognitive side effects and anxiolytic efficacy can be claimed for drugs acting at this target.

	Footnotes

 
Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.

doi:10.1124/jpet.105.083923.

ABBREVIATIONS: BK, large conductance Ca²⁺-activated potassium; DMSO, dimethyl sulfoxide.

Address correspondence to: Mads Peder Gersdorff Korsgaard, NeuroSearch A/S, 93 Pederstrupvej, Ballerup, DK-2750, Denmark. E-mail: mgk{at}neurosearch.dk

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