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BEHAVIORAL PHARMACOLOGY

Characterization in Rats of the Anxiolytic Potential of ELB139 [1-(4-Chlorophenyl)-4-piperidin-1-yl-1,5-dihydro-imidazol-2-on], a New Agonist at the Benzodiazepine Binding Site of the GABA_A Receptor

elbion AG, Radebeul, Germany

Received February 10, 2005; accepted April 26, 2005.

	Abstract

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Benzodiazepines are among the most effective drugs for the treatment of anxiety disorders. However, their use is limited by undesired side effects, including sedation, development of tolerance, and drug abuse. The aim of this study was to evaluate the pharmacological profile of ELB139 [1-(4-chlorophenyl)-4-piperidin-1-yl-1,5-dihydro-imidazol-2-on] in different models of anxiety and to correlate these effects with its activity in vitro. ELB139 binds with an IC₅₀ of 1390 nM to the flunitrazepam binding site in rat forebrain cortical membranes. In rat hippocampal neurons, ELB139 potentiated GABA-induced currents without reaching the maximum effect of diazepam, indicating a partial benzodiazepine agonism. The potentiation was antagonized by the benzodiazepine antagonist flumazenil. ELB139 (10 and 30 mg/kg p.o.) was active in three different animal models of anxiety, i.e., in the elevated plus-maze, the light and dark box, and the Vogel conflict test. The anxiolytic activity in the elevated plus-maze was almost completely reversed by flumazenil (5 mg/kg i.p.), indicating that interaction with the benzodiazepine binding site is central to the pharmacological activity. No hint of sedation was observed at the doses tested in the three anxiety models and the open field. Also, no development of tolerance was observed within 6 weeks b.i.d. treatment with ELB139 in the elevated plus-maze test. In summary, ELB139 elicits strong effects on anxiety-related behavior in rats mediated by its benzodiazepine-like activity without showing sedation or the development of tolerance, a major side effect of benzodiazepines. These characteristics make the compound a prime candidate for clinical development.

Benzodiazepines, still regarded as the most effective drugs for the treatment of anxiety disorders, show a fast onset of the anxiolytic activity. However, they also show undesired side effects, such as ataxia, sedation, skeletal muscle relaxation, amnesia, and interactions with ethanol and barbiturates. Other major problems are the development of tolerance to therapeutic effects and the potential for drug abuse (Costa and Guidotti, 1996; Dubinsky et al., 2002). Several attempts were made to reduce the major side effects of drugs binding to the benzodiazepine recognition site at the GABA_A receptor, namely, sedation and the development of tolerance.

One approach has been made by partial, nonselective agonists at the benzodiazepine binding site with high affinity to the GABA_A receptor. Compared with full agonists, imidazenil (for example) did not induce cognitive deficits nor was development of tolerance observed after long-term administration in animal models (Auta et al., 2000; Costa and Guidotti, 1996). However, the anxiolytic activity of imidazenil found in animals could not be reproduced in humans and was not clearly separate from sedation (Atack, 2003). Similar difficulties occurred with abecarnil and bretazenil, partial agonists developed by Schering AG (Berlin, Germany) and Roche Diagnostics (Basel, Switzerland), respectively (Costa and Guidotti, 1996; Atack, 2003). RWJ-51204, a newer compound, shows a separation between anxiolysis and sedation in mice and monkeys but not in rats (Dubinsky et al., 2002).

Another approach is to develop agonists that are highly selective for GABA_A receptor subtypes containing 2- and 3-subunits but not 1-subunits, the latter possibly being responsible for sedation (Low et al., 2000; Griebel et al., 2001). The degree of subtype selectivity seems still to be limited (Griebel et al., 2001), but few compounds of that kind have been described. Ocinaplon has shown anxiolytic effects in clinical trials, but the initiation of phase III trials was initially put on hold by the Food and Drug Administration pending the acquisition of further safety data; clinical testing was later recommenced but at a lower dose than the one tested in phase II. NS2710, developed by NeuroSearch (Ballerup, Denmark), showed a sedative effect and affected cognitive function (Atack, 2003). An undisclosed lead compound from the research and development program of Merck had already reached clinical testing when its development was discontinued for unstated reasons; nevertheless, the research program is continuing (Atack, 2003). Another compound, SL651498, which was found to act as a high-affinity ligand with functional subtype selectivity for ₂-containing GABA receptors, has still to prove its ideal profile in humans (Griebel et al., 2001, 2003).

A different class of drugs used for the treatment of anxiety is the SSRIs. These drugs are well established as antidepressants and do not induce the major side effects typical of benzodiazepines, such as tolerance or drug abuse; however, the late onset of their anxiolytic and antidepressive effect limits their therapeutic benefit (Nutt et al., 1999). Besides, their therapeutic use is affected by weight gain and sexual dysfunction, which lead patients to discontinue the therapy (Perna et al., 2001). A combination of the positive effects of benzodiazepine receptor ligands and SSRIs, i.e., rapid onset of action and potent anxiolytic activity plus the absence of tolerance, abuse potential, and sedative potential, could serve as a template for an ideal anxiolytic. However, the search for the ideal anxiolytic is ongoing.

ELB139 is a new chemical entity emerging from a research and development program for anticonvulsants based on in vivo screening in cooperation with the National Institutes of Health (Rostock et al., 1998) and on pharmacophore modeling (Fig. 1). The present study was initiated to evaluate the anxiolytic activity of the compound as well as to obtain first insight into its possible mechanism of action. The anxiolytic potential was tested in three different animal models. Anxiety-related and locomotor-activity-related parameters were recorded in each test, to obtain more detailed information about the separation between the drug's sedative and anxiolytic effects. To gain initial insight into the mechanism of action of ELB139, its affinity to the benzodiazepine binding site and its effect in vitro on GABA-induced current were determined and compared with its effects in vivo and their reversibility by the benzodiazepine antagonist flumazenil. In addition, ELB139 was administered subacutely and chronically, to assess the risk of tolerance development. Diazepam was used as reference compound.

View larger version (8K): [in this window] [in a new window]   Fig. 1. Chemical structure of ELB139.

	Materials and Methods

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Animals
Male Wistar rats [Crl:(WI)BR; Charles River, Sulzfeld, Germany], weighing 250 to 350 g, were used. They were housed in groups of five under standard conditions on a 12-h light/dark cycle (light on at 6:00 AM) with ad libitum access to water and food (ssniff M/R 15; Spezialdiäten GmbH, Soest/Westfalen, Germany). The experiments were approved by the Committee on Animal Care and Use of the Federal State of Saxony and carried out following the German Law on the Protection of Animals.

Chemicals
ELB139 and diazepam were obtained from elbion AG (Radebeul, Germany) (formerly AWD). Flumazenil was obtained from Tocris Cookson, Inc. (Bristol, UK). [³H]Flunitrazepam was purchased from Amersham Biosciences, Inc. (Freiburg, Germany). For patch-clamp experiments, salts were purchased from Sigma Chemie (Deisenhofen, Germany). All other compounds were obtained from Merck (Darmstadt, Germany).

Inhibition of Specific [³H]Flunitrazepam Binding to Benzodiazepine Binding Site
Neuronal membrane fraction from rat forebrain (excluding the cerebellum) were prepared using standard techniques described by Borbe and Zierenberg (1985). The membrane fraction (150 µl) was incubated with 0.5 nM [³H]flunitrazepam and an appropriate concentration of the test compound for 30 min at 4°C. Nonspecific binding was determined in the presence of 10 µM diazepam. Binding was terminated by filtration of the incubated membrane fraction using Filtermat A (Pfizer, Inc., New York, NY) presoaked with 1% polyethylene imine and a Micro Cell harvester (Skatron, Liver, Norway). Then, the Filtermat A was carefully washed with 0.05 M Tris/HCl buffer, pH 7.7, to eliminate unbound radioactivity. The filters were counted in a scintillation counter (Betaplate 1205; Berthold Technologies, Bad Wildbad, Germany) to determine the specific binding of [³H]flunitrazepam. Compounds were screened at 6 to 10 concentrations to determine IC₅₀ and K_i. In the assay, the dissociation constant of [³H]flunitrazepam was found to be 1.5 nM, its greatest number of binding sites was 0.38 nM, and the specific binding was 90%.

Determination of GABA-Induced Currents in Rat Hippocampal Neurons
Cell Culture. For GABA-induced current recordings, rat hippocampal neurons were obtained from hippocampal tissue of 18-day-old embryos. Cells were cultivated together with astrocytes, on glass coverslips coated with poly-L-lysine, at a density of 5 x 10⁵ cells/cm². The cells were cultivated in basal medium Eagle supplemented with 10% horse serum, 10% fetal calf serum, and 2 mM glutamine. After 3 days, 5 µM cytosine-1-D-arabinofuranoside was added to inhibit astrocyte propagation and consequent concealment of neurons by large numbers of astrocytes. The cultivation was continued with basal medium Eagle supplemented with 5% horse serum, 5% fetal calf serum, and 2 mM glutamine. For the experiments, neurons were used between days 7 and 8 in culture.

Patch-Clamp Recording. The whole-cell variant of the patch-clamp technique was used for the voltage-clamp experiments (Hamill et al., 1981). The micropipettes were drawn (Sutter Instrument Company, Novato, CA) from borosilicate glass capillaries (Science Products, Hofheim, Germany) and heat-polished at the tip with equipment from ALA Scientific Instruments (Westbury, New York). The pipette resistances were between 2 and 5 M. The composition of the internal solution was 140 mM CsCl, 1 mM MgCl₂, 1 mM CaCl₂, 10 mM HEPES, and 11 mM EGTA. The osmolality was adjusted to about 300 mOsM. The internal solution was prepared in advance and deep-frozen in aliquots of 1 ml. To fill the recording pipettes, the solution was thawed every morning, and 2 mM Na₂ATP was added.

Current signals were amplified with an EPC-9 amplifier and were digitized, stored, and analyzed by using the TIDA system (HEKA, Lambrecht/Pfalz, Germany). The data were sampled, digitally filtered (Bessel 10 kHz), and stored on a computer disc at a frequency of 2 kHz. For recording, a coverslip was transferred into the recording chamber and permanently superfused with modified extracellular solutions containing 140 mM NaCl, 5 mM KCl, 2 mM CsCl, 1 mM MgCl₂, 10 mM HEPES, 5 mM D-glucose, and 0.003 mM tetrodotoxin, pH 7.3 to 7.4 (NaOH).

The cells were clamped at a potential of -80 mV. The concentration of GABA was selected to elicit approximately 20% of the maximum current induced by high concentrations of GABA. This concentration was found to be 3 µM and elicited a current of -268 ± 35 pA (n = 16). The GABA solution or the test compounds (at concentrations between 1 and 100 µM) together with GABA were applied locally onto the clamped cell by using an eight-channel rapid solution exchanger (DAD8; ALA Scientific Instruments). For evaluation of the drug effect, the maximum current amplitude induced by application of 3 µM GABA was set to 100%, and the relative maximum current amplitude induced by the test compound and GABA in relation to the GABA-induced maximum current amplitude in the same cell was calculated.

Animal Models of Anxiety
Drug Administration. ELB139 and diazepam suspended in 0.5% hydroxyethylcellulose were administered orally, respectively, 1 h and 30 min before the test. The pretreatment time was chosen due to previous epilepsy experiments evaluating the time of peak effects (data not shown). For subacute administration, the two compounds were administered twice daily for five consecutive days, with the last administrations 1 h and 30 min before the test, respectively. For long-term exposure, ELB139 (10 and 30 mg/kg p.o.) was administered twice daily on 5 days per week for 6 weeks. For antagonism tests, flumazenil was administered at a dose of 5 mg/kg i.p. 20 min before the test.

Elevated Plus-Maze. The elevated plus-maze used was a plusshaped maze made of gray polyvinyl chloride with a black floor. It consisted of two closed arms (14 x 50 cm) with protective walls (27 cm in height), two open arms (14 x 50 cm) opposite each other, and a central arena of 14 x 14 cm. The maze was elevated 100 cm off the ground. The apparatus was situated in a separate small shielded square room within the laboratory so that animals were not disturbed by environmental stimuli. The light intensity was 200 ± 50 lux, the light being more intense in the open arms. To start the experiment, the rat was placed in the central arena of the apparatus with the head pointing midway between a closed and an open arm. The test lasted for 5 min. Behavior was videotaped during the session, and the following parameters were recorded by the computer program "VideoMot" (DOS and type II-version; TSE, Bad Homburg, Germany): number of entries into the open arms/total number of entries (percentage), time spent in the open arms/time spent in all arms (percentage), and the total number of entries into the arms (n), or, alternatively, the total distance traveled (centimeters) with the new system as having a higher loading on locomotor activity (Ramos et al., 1997).

Light and Dark Box. The apparatus consisted of two chambers (21 x 22 cm) with a grid floor that were connected by a 6.5 x 6-cm opening. One chamber had black walls, and the other had white walls. The white chamber was additionally brightly lit. For the experiment, the rat was placed in the white chamber and was allowed to explore the two-chamber area for 5 min. The following parameters were recorded by a laboratory assistant: number of transitions between the two chambers (n) and time spent in the light chamber (seconds).

Vogel Conflict. A modification of the method of Vogel et al. (1971) was used. For the test operant behavior boxes (30 x 23 x 19 cm) with a stainless steel grid floor were used (habitest operant cage; Coulbourn Instruments). A water bottle with a metal drinking tube was fitted from the outside to the box so that only the drinking tube extended into the box. This tube was connected to an electric shock generator (precision-regulated animal shocker; Coulbourn Instruments), which could produce a 0.5-mA shock when the tongue of the rat touched the tube.

The test required three consecutive days in which drinking sessions were performed once a day, always at the same time. On the first day, rats were allowed to drink water for 15 min without being punished, to accustom them to the operant box. They were then put back into their home cage and left without water for 24 h. On the second day, water was replaced by a 5.0% glucose solution, and rats were allowed to drink for 5 min during the training session. They were again left without water for the next 24 h in their home cage. On the last day, the rats were again offered drinking water. The whole test session lasted for 210 s. For the first 30 s, rats were allowed to drink water without the licks being punished. During the remaining 180 s, rats received a mild electric shock when they touched the drinking tube. The number of unpunished (n) and punished (n) licks were counted by the computer program Graphic state notation (Coulbourn Instruments).

Open Field. The MotiTest apparatus (TSE) was used for this experiment. The test area consisted of a squared arena (45 x 45 cm) with protective Plexiglas walls (20 cm in height) where rats could move freely. Horizontal movements were recorded by 32 infrared photocells arranged along the bottom of each wall of the arena. Vertical movements (rearings) were recorded by a horizontal row of 32 infrared photocells 12 cm above the floor. The light intensity was 150 ± 50 lux. The center of the arena was defined as the central 50% of the arena by the computer program. To start the experiment, the animals were placed in the middle of the squared arena, and movements were recorded for 10 min. The following parameters were recorded by the computer program ActiMot (TSE): active time (seconds), total distance traveled (meters), number of rearings (n), distance traveled in the center/total distance (percentage), and active time spent in the center/total activity time (percentage).

Statistical Analysis
Results are shown as mean ± S.E.M. The in vivo results were analyzed by one-way analysis of variance. The Tukey test was used for individual comparison. p < 0.05 was regarded as significant.

	Results

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Inhibition of Specific [³H]Flunitrazepam Binding to Benzodiazepine Binding Site
ELB139 inhibited the binding of the specific radioligand [³H]flunitrazepam to the benzodiazepine receptor with an IC₅₀ value of 1390 ± 32.1 nM and a K_i value of 1040 ± 24.1 nM (mean ± S.E.M.). The IC₅₀ and the K_i of diazepam accounted for 9.1 ± 0.7 and 6.8 ± 0.6 nM, respectively (mean ± S.E.M.).

GABA-Induced Currents
ELB139 was tested at the concentration of 1, 10, and 100 µM. Administered onto the neurons in the absence of GABA, ELB139 elicited no current. Administration of GABA alone (3 µM) induced a chloride current of -268 ± 35 pA (n = 16), which is approximately 20 to 30% of the maximum current inducible by high concentrations of GABA. If ELB139 at the above-mentioned concentrations was administered with 3 µM GABA, the compound enhanced the current concentration dependently to 108.8 ± 13.3, 162.7 ± 17.5, and 165.9 ± 19.1%, respectively, of the value without ELB139. A value of 100% represents the current elicited by administration of 3 µM GABA alone (Fig. 2).

View larger version (10K): [in this window] [in a new window]   Fig. 2. Effect of 1, 10, and 100 µM ELB139 on GABA-induced currents. The GABA-induced current elicited by 3 µM GABA was set to 100%, and the percentage of the current elicited by the substance during GABA application was calculated. Effects were tested for significance using the paired t test.

View larger version (12K): [in this window] [in a new window]   Fig. 3. Flumazenil antagonism of GABA-induced currents. GABA or GABA and substance (10 µM) were supplied for 10 s followed by a washout of 30 s. Flumazenil (10 µM) was administered 30 s before the application of additional GABA and the test substance (ELB139 or diazepam). The currents were normalized to the current elicited by GABA (100%). Effects were tested for significance by the paired t test (***, p < 0.001).

View larger version (18K): [in this window] [in a new window]   Fig. 4. Results from the elevated plus-maze after acute administration. ELB139 at 10 and 30 mg/kg and diazepam at 6 mg/kg were administered once p.o. 1 h and 30 min, respectively, before the test. Rats were placed on the elevated plus-maze for 10 min (n = 12). Data are shown as mean ± S.E.M. Significant to control, *, p < 0.05 and ***, p < 0.001; significance between 10 and 30 mg/kg ELB139, #, p < 0.05.

Development of tolerance. To evaluate the risk of tolerance development toward the anxiolytic effect, the elevated plusmaze test was used after repeated administration of both ELB139 and diazepam (Fig. 5). After five consecutive days of b.i.d. administration, ELB139 at 30 mg/kg p.o. still significantly increased the two anxiety-related parameters of the elevated plus-maze, the percentage of entries into open arms [F(3.81) = 4.24; p = 0.39; n = 5], and the percentage of time spent in them [F(3.81) = 6.70; p = 0.015; n = 5], whereas diazepam at 4 mg/kg p.o. b.i.d. lost its effect on these two anxiety-related parameters [F(3.81) = 0.75 and 0.44, respectively; N.S.; n = 5]. There was no effect on the total number of entries for both compounds [F(3.81) = 0.10 (ELB139) and 0.60 (diazepam); N.S.; n = 5].

View larger version (26K): [in this window] [in a new window]   Fig. 5. Results of the elevated plus-maze after repeated administration. Subacutely, ELB139 at 10 and 30 mg/kg and diazepam at 4 mg/kg (diaz) were administered p.o. twice daily for 4 days and 1 h or 30 min, respectively, before the test (n = 5). Chronically, ELB139 at 10 and 30 mg/kg p.o. was administered twice daily 5 days/week for 6 weeks (n = 10). Rats were placed on the elevated plus-maze for 10 min. Data are shown as mean ± S.E.M. *, p < 0.05.

Reversal of the acute effect by flumazenil. To assess whether, and to what extent, the low-affinity partial agonistic effect of ELB139 at the benzodiazepine binding site might contribute to the anxiolytic effect, the benzodiazepine antagonist flumazenil was used in a different experiment. The results are shown in Fig. 6. At 30 mg/kg p.o. ELB139 again significantly increased the percentage of entries into open arms [F(3.32) = 4.23; p = 0.023; n = 10] and the time spent in them [F(3.32) = 14.37; p < 0.001; n = 10], further confirming the anxiolytic potential of the compound in this model. The total distance traveled was unchanged by ELB139 at this dose [F(3.32) = 1.99; N.S.; n = 11]. This anxiolytic effect was almost completely reversed by flumazenil at 5 mg/kg i.p.; the percentage entries into the open arms did not differ from vehicle-treated rats [F(3.32) = 4.23; p = 0.780; n = 11]. However, the time spent in the open arms remained slightly but significantly increased in comparison with vehicle-treated rats [F(3.32) = 14.37; p = 0.020; n = 11].

View larger version (19K): [in this window] [in a new window]   Fig. 6. Reversal of the acute anxiolytic effect of ELB139 and diazepam by flumazenil. Reversal of the acute anxiolytic effect of 4 mg/kg p.o. diazepam (D) and 30 mg/kg p.o. ELB139 (139) by flumazenil (F; 5 mg/kg i.p., 20 min before testing) in the elevated plus-maze. Data are shown as mean ± S.E.M. Statistical significance was determined by one-way analysis of variance and Tukey testing. Significantly different from control with *, p < 0.05; **, p < 0.01; and ***, p < 0.001; significant difference between compound group and flumazenil group, #, p < 0.05.

Diazepam at 4 mg/kg p.o. also increased the percentage time spent in the open arms significantly [F(3.32) = 8.51; p < 0.001; n = 10] and the percentage of entries into them distinctly [F(3.32) = 2.29; N.S.; n = 10]. The anxiolytic effect of 4 mg/kg p.o. diazepam was also reversed by flumazenil. The entries into the open arms did not differ between ELB139-treated and vehicle-treated rats [F(3.32) = 2.29; N.S.; n = 11]. The percentage of time spent in the open arms also remained slightly increased, but it did not reach level of significance [F(3.32) = 8.51; p = 0.155; n = 11]. The significant increase of the total distance traveled induced by diazepam at 4 mg/kg p.o. [F(3.32) = 11.12; p < 0.001; n = 10] was totally reversed by flumazenil [F(3.32) = 11.12; N.S.; n = 11].

Light and Dark Box. ELB139 significantly increased the number of transitions between the light and the dark chamber, starting at 10 mg/kg p.o. [F(2.87) = 3.43; p = 0.037; n = 10]. Diazepam at 6 mg/kg p.o., significantly increased the number of transitions [F(2.87) = 3.43; p = 0.021; n = 10]. The time spent in the light chamber was distinctly increased with diazepam and ELB139 at 6 and 10 mg/kg p.o., respectively [F(2.87) = 1.97; N.S.; n = 10].

Vogel Conflict Test. Data are shown in Fig. 7. ELB139 showed an effect on the number of punished licks starting at 10 mg/kg p.o. It significantly increased the punished licks at 30 mg/kg p.o. [F(2.83) = 3.38; p = 0.043; n = 12] without changing the unpunished licks [F(2.83) = 3.37; N.S.; n = 12]. Diazepam significantly increased the number of punished licks at 10 mg/kg p.o. [F(2.83) = 3.38; p = 0.041; n = 12]. However, the number of unpunished licks was significantly decreased to the control group at this dose [F(2.83) = 3.37; p = 0.042; n = 12].

View larger version (16K): [in this window] [in a new window]   Fig. 7. Results of the Vogel conflict test. Diazepam (6 mg/kg) and 10 and 30 mg/kg p.o. ELB139 were administered orally, respectively, 30 min and 1 h before testing. Data are shown as mean ± S.E.M. *, p < 0.05.

	Discussion

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The aim of the present study was to evaluate the pharmacological profile of ELB139 in different models of anxiety and to correlate these effects with its activity in vitro. The in vitro studies showed ELB139 to be a low-affinity, partial agonist at the benzodiazepine binding site. Its IC₅₀ for the inhibition of specific [³H]flunitrazepam binding was about 150 times higher than that of diazepam (Costa and Guidotti, 1996; Nazar et al., 1997; Dubinsky et al., 2002). Electrophysiological experiments in hippocampal neurons characterize the compound as a partial agonist because the potentiation of GABA-induced currents did not reach the potentiation obtained with diazepam, even after administration of 100 µM, a dose approximately 95-fold higher than the IC₅₀. The potentiation of GABA-induced currents is completely antagonized by flumazenil, further supporting the benzodiazepine binding site interaction.

The anxiolytic activity of ELB139 was evaluated in three animal models of anxiety, two ethological models (elevated plus-maze and light and dark box) and one based on conditioned fear (Vogel conflict). This approach allowed us to test the effect of the compound on a broader spectrum of behavioral facets belonging to different anxiety disorders. It has not yet been tested in the social interaction test, an animal model of social phobia (Rodgers et al., 1997). However, although the social interaction test is described as detecting another type of anxiety that is differentially modulated by GABA_A receptors, benzodiazepines have been shown to be active in this test, too (Gonzalez et al., 1998). In all tests used, ELB139 starting at 10 mg/kg p.o. showed an effect on anxiety-related parameters (Pellow et al., 1985; Pellow and File, 1986; Kennett et al., 2000, Choleris et al., 2001). It increased the number of transitions significantly and the time spent in the light chamber distinctly in the light and dark box and the percentage of time in the center of the open field at 10 mg/kg, whereas in the other anxiety tests significant effects were consistently seen at 30 mg/kg. Diazepam, used as reference compound in all experiments, showed significant effects at 4 to 10 mg/kg p.o. These doses lie in the range described for diazepam in other studies, i.e., between 2 and 10 mg/kg, depending on the test and the rat strains used (Wada and Fukuda, 1991).

These data indicate a discrepancy between the low affinity of ELB139 for the benzodiazepine binding site of rat forebrain membranes in combination with the partial agonism and the effective dose in comparison with diazepam. The 150-fold lower affinity is not reflected in a similar separation of the active in vivo doses, which were only 3- to 7-fold higher, depending on the model and the vehicle used. Furthermore, in spite of the partial agonism, ELB139 showed a reproducible and consistent anxiolytic effect at 30 mg/kg, the extent of which was comparable with that of diazepam, as seen with the five elevated plus-maze runs throughout the year, with 11 to 12 animals/group (Figs. 4, 5, 6). This may raise the question of whether other mechanisms may contribute to the potent anxiolytic activity of ELB139. Because the activity of ELB139 could be antagonized with flumazenil to an extent similar to that published for diazepam (Wada and Fukuda, 1991), the benzodiazepine interaction is very likely to be central to the anxiolytic activity. Initial data, however, indicate that ELB139 may be highly subtype-selective for the 3-subunit of the benzodiazepine binding site. Because the 3-subunit is not the major subunit in the hippocampus or the forebrain, both the binding and the electrophysiological experiments may fail to reflect the actual potency of ELB139 on GABA subunits. Studies to evaluate further the subtype selectivity are underway (Rabe et al., 2005).

A major problem of benzodiazepine-like anxiolytics is that their anxiolytic activity cannot be clearly separated from sedation (File, 1990; Costa and Guidotti, 1996; Dubinsky et al., 2002; Atack, 2003). Thus, at high doses diazepam starts to reduce the activity of the rats, as hinted at by the significantly reduced unpunished licks in the Vogel conflict test, a parameter related to locomotor activity (Nazar et al., 1997; Kennett et al., 2000). To evaluate the safety profile of ELB139, in each test of the present study the parameters predominately related to activity were recorded in parallel to the anxiety-related parameters. We determined the unpunished licks in the Vogel conflict test and the total number of entries and total distance traveled in the elevated plus-maze test (Ramos et al., 1997). In both tests, these parameters were not significantly affected by ELB139 at 30 mg/kg p.o. For the light and dark box, it is more difficult to separate anxiety- and locomotor activity-related parameters, because the number of transitions between the two chambers is influenced both by anxiety and by activity (Hascoet and Bourin, 1998). The distance traveled in the dark chamber is a more closely related to activity but could not be detected in our system (Hascoet and Bourin, 1998). These results indicate that the anxiolytic effect of ELB139 at 30 mg/kg p.o. is not significantly affected by sedation. Only in the open field at 30 mg/kg p.o. was a slight reduction of the activity to be seen, although the total distance traveled was unchanged at this dose. The activity comprises active locomotion and static movements, whereas the total distance traveled almost exclusively reflects locomotion. Therefore this reduction of activity seems to be due to a decrease of the static movements rather than to locomotion. This can be supported by data obtained in the RotaRod test and the alcohol interaction test. The impairment of RotaRod performance has an ED₅₀ of 265 mg/kg p.o. for ELB139, indicating that motor activity is not significantly affected at 30 mg/kg orally. Likewise, the compound was not found to amplify significantly the depressant effects of ethanol at 100 mg/kg p.o. (Dost et al., 2004). Because static movements comprise different types of activity, such as breathing and grooming, it is difficult to put them down to a certain behavioral response. Sedation would reduce both static and locomotor activity. Interestingly, Blanchard et al. (1991) and Homberg et al. (2002) have found that enhanced anxiety-related behavior is to a certain extent accompanied by an increase in grooming behavior.

Diazepam at lower anxiolytic doses (2–6 mg/kg p.o.) is described as significantly increasing locomotor activity (Dawson et al., 1995). Here, this is predominantly seen in the open field (Table 1). This hyperactivity is discussed differently. Partly, it is associated with an increased exploratory activity due to its anxiolytic effect. However, it is also described as a nonspecific hyperlocomotion, which, because the anxiety-related parameters are influenced by the locomotor activity of the animals, may further enhance these parameters and thus the anxiolytic activity (Soderpalm et al., 1991; Dawson et al., 1995). Additionally, the hyperlocomotion is discussed as a reflection of the psychostimulant effect of diazepam (Ikemoto, 2004). Such an obvious stimulating effect on locomotor activity was not seen with ELB139.

Further problems of benzodiazepine-like compounds are their potential to induce tolerance toward their therapeutic effect and physical dependence, resulting in withdrawal symptoms after cessation of the therapy (Follesa et al., 2001; Atack, 2003). The tolerance is discussed as being caused by a decrease in GABA_A receptors and also by a rearrangement of the GABA_A receptor subunits (Costa and Guidotti, 1996; Fernandes et al., 1999). This phenomenon is seen mainly with full, but also with partial high-affinity agonists (Follesa et al., 2001; Atack, 2003). An exception among the partial agonists is imidazenil, which retains its anticonvulsant activity, even after a prolonged period of administration (Costa and Guidotti, 1996). Developing a partial, low-affinity agonist of the benzodiazepine binding site was conceived as a new approach to avoid the development of tolerance and physical dependence and the occurrence of withdrawal symptoms. In the present study, ELB139, in contrast to diazepam (Fernandes et al., 1999), retained its significant effect on anxiety-related behavior, even after chronic administration, indicating that it does not induce tolerance to its anxiolytic activity at least with the administration schedule used in the present experiment.

In summary, in the present study ELB139 elicited strong effects on anxiety-related behavior mediated predominantly by its benzodiazepine-like activity. The extent of anxiolytic activity was comparable with that of diazepam. At efficacious doses, the compound was devoid of major side effects such as development of tolerance or sedation in rats. These characteristics make the compound a prime candidate for new anxiolytic drug. Indeed, the drug is currently undergoing phase II clinical testing as an anxiolytic.

	Footnotes

 
This study was supported by the European Fund for Regional Development 2000-2006, sector technology support, and by Federal State of Saxony Grant SAB 8093.

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

doi:10.1124/jpet.105.084681.

ABBREVIATIONS: SSRI, selective serotonin reuptake inhibitor; RWJ-51204, 5-ethoxymethyl-7-fluoro-N-(2-fluorophenyl)-3-oxo-1,2,3,5-tetrahydropyrido[1,2-a]benzimidazole-4-carboxamide; SL651498, 6-fluoro-9-methyl-2-phenyl-4-(pyrrolidin-1-yl-carbonyl)-2,9-dihydro-1H-pyrido[3,4-b]-indol-1-one; ELB139, 1-(4-chlorophenyl)-4-piperidin-1-yl-1,5-dihydro-imidazol-2-on; NS2710, 1-[1-[3-(3-pyridyl)phenyl]benzimidazol-5-yl]ethanone O-ethyloxime.

Address correspondence to: Dr. Barbara Langen, Department of Pharmacology, elbion AG, Meissner Str. 191, D-01445 Radebeul, Germany. E-mail: barbara.langen{at}elbion.de

	References

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