Introduction

Top Abstract Introduction Materials and Methods Results Discussion Appendix 1 References

Anxiety disorders are highly prevalent worldwide, and although most people contend with anxiety as a normal and periodic component of daily life, the emotional responses of some people are greatly exaggerated. Anxious traits can interfere with life's daily activities and may be associated with comorbid medical conditions. The benzodiazepines (BZs) are generally regarded as the most effective class of drugs for treating anxiety disorders (Woods et al., 1992, 1995). They provide a prompt onset of anxiolysis, and patients rarely exhibit tolerance to their anxiolytic actions (Rickels et al., 1987). However, currently marketed BZ drugs cause undesirable effects such as sedation, motor and memory impairment, skeletal muscle relaxation, and physical dependence. Although the BZs have a high margin of safety when used therapeutically, their unintentional or intentional use with other CNS depressant drugs such as ethanol and barbiturates is hazardous. Therefore, these limitations have inspired a search for non-BZ drugs that are as efficacious as the BZs without these undesirable effects and that do not cause physical dependence.

The anxiolytic efficacy of benzodiazepines arises from their ability to modulate the physiological activity of GABA at GABA_A receptors. GABA_A receptors are heteropentameric, ligand-gated chloride ion channels that constitute the predominant inhibitory neurotransmitter system in the central nervous system of mammals. Many subtypes of GABA_A receptors exist with different pharmacological properties (Olsen and Tobin, 1990; Burt and Kamatchi, 1991; Doble and Martin, 1992; Stephenson, 1995; Chen et al., 1996) Although the specific type(s) of GABA_A receptors associated with anxiolytic activity or other CNS effects of GABA_A receptor modulators have not been precisely identified, some associations have been postulated (Mehta and Shank, 1995a,b; McKernan and Whiting, 1996), and some experiments have implicated the ₁-subunit and the ₂-subunit of the GABA_A receptor in the sedative and anxiolytic activities of the BZs, respectively (Low et al., 2000; McKernan et al., 2000). However, a compound with an ideal profile has not been identified, based solely on its affinity for different subtypes of GABA_A receptors and its intrinsic modulatory activity at the subtypes.

An alternative approach is the development of partial agonist modulators of the GABA_A receptor. Conceptually, partial agonist modulators have a lower intrinsic activity, and, therefore, their potential maximal effect is lower than that of the full agonists. Partial GABA_A receptor agonists include the imidazole benzodiazepines bretazenil and imidazenil and the nonbenzodiazepine pazinaclone.

These agents were first tested to determine their affinity for the BZ site on GABA_A receptors and for their intrinsic modulatory activity, as judged by the GABA shift. The GABA shift serves as a quantitative indicator of intrinsic modulatory activity and is derived by dividing the IC₅₀ obtained with GABA present into the IC₅₀ obtained in the absence of GABA. These experiments were performed in vitro using ligand-receptor binding techniques, in which a subcellular fraction enriched in synaptic membranes prepared from discrete regions of the CNS of rats served as the source of GABA_A receptors. Subsequently, compounds active in vitro were evaluated in a battery of in vivo tests that provided empirical evidence for anxiolytic efficacy or unwanted CNS-related effects. In vivo tests were performed using rats, mice, and monkeys.

The present investigation compares and contrasts the in vitro and in vivo preclinical pharmacological properties of RWJ-51204 with those of full and partial agonist GABA_A receptor modulators. RWJ-51204 was compared in detail with the full agonist anxiolytic lorazepam because this compound is used extensively in anxiety disorders. RWJ-51204 is a partial agonist at BZ receptors and a member of a novel class of compounds collectively referred to as pyrido[1,2a] benzimidazoles. The chemical name for RWJ-51204 is 5-ethoxymethyl-7-fluoro-3-oxo-1,2,3,5-tetrahydrobenzo[4,5] imidazo[1,2a]pyridine-4-[N-(2-fluorophenyl)]carboxamide (Fig. 1). RWJ-51204 was discovered through original research at Johnson & Johnson Pharmaceutical Research and Development, L.L.C. (Reitz et al., 1998).

View larger version (10K): [in this window] [in a new window]   Fig. 1.   Chemical structure of RWJ-51204.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion Appendix 1 References

Test Animals

This study complied with the protection guaranteed to animals in accordance with the Guide for the Care of Laboratory Animals as adopted and promulgated by the National Institutes of Health and the regulations implementing the Animal Welfare Act, as amended. Adult male Long-Evans rats and adult male CD-1 albino mice were obtained from Charles River Laboratories, Inc. (Wilmington, MA). Rodents were allowed a quarantine period of 5 days. Rodents were housed in community cages with approved bedding materials. They had free access to standard rodent diet (Purina Chow; Purina, St. Louis, MO). Pair-housed male feral squirrel monkeys weighing 0.7 to 1 kg were obtained from Charles River Laboratories, Inc., and the University of Miami. While these experiments were conducted, they were fed a diet of standard monkey chow (Purina Chow) and fruit to maintain their body weight at approximately 85% of their free-feeding weight. Animals were kept under standard laboratory conditions (approximately 22°C, 12-h light/dark cycle) and had free access to clean drinking water.

Neurochemistry

Affinity for BZ Site on GABA_A Receptors. Rats were killed by cervical dislocation, and the whole brain plus cervical region of the spinal cord was excised and placed in an ice-cold isotonic, buffered, aqueous sucrose (0.3 M) solution. The brain plus spinal cord was dissected into regions, including the cerebral cortex, hippocampus, cerebellum, and medulla/spinal cord. The tissue from several rats was pooled and homogenized, from which a well washed membrane fraction was prepared and used as the biological material in the assay (Mehta and Shank, 1995). For each sample, a portion of the membrane fraction containing 0.1 to 0.2 mg of protein was incubated in 2 ml of a 3 mM phosphate-buffered solution containing 0.1 M NaCl and 0.01 to 0.03 µCi of a ³H-labeled ligand [³H]Ro15-4513, [³H]flumazenil (PerkinElmer Life Sciences, Boston, MA). The receptor-ligand binding reaction was allowed to reach equilibrium at an ambient temperature of 21-23°C (30 min) and then the reaction was terminated by vacuum filtration to separate the incubation medium from the biological membranes. The membrane samples were washed to remove unbound ligand. The ³H bound to each membrane sample was quantified using liquid scintillation spectrometry. Compounds were dissolved in dimethyl sulfoxide at 10 mM. The stock solutions were diluted 500-fold into water to make the highest working concentration; lower concentrations were made by serial dilution into water. For individual experiments, a set of samples included a vehicle control (water); a reference compound at a saturating concentration, e.g., flunitrazepam at 10 µM (to determine nonspecific binding); and 5, 10, or 11 concentrations of one or more test compounds. Each experiment was performed in duplicate or quadruplicate and the raw data were averaged (cpm bound).

Intrinsic Activity for Modulating GABA_A Receptors. The assay procedure is the same as the one described above, except that GABA is included at 0.1 mM.

Anticonvulsant Activity

Inhibition of Pentylenetetrazole-Induced (PTZ) Convulsions. Compounds that positively modulate the activity of GABA at GABA_A receptors can block these convulsions (Swinyard, 1949). Mice were fasted overnight, no longer than 16 h, but were allowed access to water except during the period of observation. PTZ (85 mg/kg) was administered s.c. 1 h after p.o. (10 ml/kg) administration of compounds or their vehicle. Mice were housed individually for 30 min for observation of clonic convulsions (present or absent). The dose of PTZ administered in these experiments was determined from the dose-response curve to cause 90% of vehicle-treated mice to exhibit clonic convulsions. The effectiveness of compounds in the PTZ-induced convulsion assay was determined from the number of mice that did not exhibit convulsions at each dose.

Anxiolytic Activity

Water-Lick Conflict (Vogel Test). The anxiolytic activity of compounds was assessed by determining their ability to release (disinhibit) behavior that had been suppressed by punishment (Vogel et al., 1971). Adult rats were deprived of water for 48 h and were deprived of food for at least 16 h before testing. After the first 24 h of water deprivation, they were placed in a sound-attenuating chamber for a training period, in which they were allowed 200 licks from a bottle containing tap water. The experiment was performed the next day. Vehicle or compounds were administered orally by gavage, and at specified times after dosing, rats were placed in the chamber and allowed access to tap water. The first lick at the stainless steel sipper tube of a water bottle initiated a 3-min test session in which every 20th lick was punished by a 0.2-s, 0.5-mA shock (root mean square, measured across the electrodes) delivered via the sipper tube. If rats failed to drink within 5 min, the experiment was terminated, and they were evaluated for signs of CNS depression. Rats were not reused in this experiment. The anxiolytic effectiveness of a compound in this assay was determined from the number of rats, at each dose, that received a number of shocks that was equal to or greater than the calculated 90th percentile of the number of shocks received by approximately 600 vehicle-treated rats. This criterion was eight shocks when rats were tested 1 h after administration and 10 shocks when rats were tested 4 h after administration.

Effect of Flumazenil on Anxiolytic Activity. RWJ-51204 was tested to determine whether its anxiolytic activity in the Vogel test could be antagonized by pretreatment with flumazenil. Antagonism of the anxiolytic activity of drugs by flumazenil indicates that this activity is due to a modulation of the activity of GABA via an interaction with the BZ site on GABA_A receptors. RWJ-51204, lorazepam (positive control anxiolytic agonist), or their vehicle was administered orally to separate groups of rats. Flumazenil (10 mg/kg s.c. at 1 ml/kg) or its vehicle was administered 45 min after the compound, and rats were placed in the conflict chamber 15 min later.

Elevated Plus-Maze. This behavioral assay is based on the innate aversion that rats have of illuminated open spaces. Compounds with anxiolytic activity have been shown to increase the frequency with which rats venture into open spaces (Pellow et al., 1985). Vehicle or compounds were administered orally to adult rats that were deprived of food but not water for 18 h before use. Rats were not reused in this experiment. At a specified time after dosing, the rats were placed on an open arm of the elevated plus-maze, facing the center. The 10-min test was initiated when the rat entered the center of the apparatus. Each black plastic maze had two open arms and two arms with 40-cm-high walls (enclosed arms) of equal length (50 cm) extending from the center at right angles, such that arms of similar type were opposite each other. Each elevated plus-maze was elevated approximately 60 cm above the floor. Infrared photo-beams that crossed the entrance of each arm and the center of the maze detected the exploratory activity of an animal. Data collection was automated. The anxiolytic effects of compounds in the elevated plus-maze were quantified by calculating the percentage of entries into the open arms as follows: percentage of open arm entries = 100 × (entries into open arms/[entries into the open arms + entries into the closed arms + entries into the center]).

Conflict-Induced State Anxiety in Monkeys. The anxiolytic activity of compounds was assessed by determining their ability to release (disinhibit) behavior that had been suppressed by punishment (Gleeson and Barrett, 1990). Specifically, this procedure was designed to induce a state of behavioral "conflict" between approach and avoidance tendencies. Squirrel monkeys were fasted overnight before each experiment but allowed access to water except during the test procedure. Experiments were conducted with a monkey seated in a primate chair inside a light- and sound-attenuating ventilated cubicle (66 cm in height × 71 cm in width × 40.6 cm in depth). Each session consisted of ten 3-min trials, separated by a 30-s time-out after each pellet delivery or time of availability. At the end of each 3-min trial, a lever press (made between 3 and 3.5 min) produced the reward, a banana-flavored pellet (300 mg). A punishment contingency was superimposed upon each 3-min trial by delivering a brief (0.2-s) electric shock to the tail (up to 3 mA) after each 30th lever press (fixed ratio-30). Compounds were administered p.o. and testing commenced either 45 min (1-h test) or 3 h 45 min (4-h test) after dosing.

2

Ancillary CNS-Related Effects

Observations of General Behavior in Squirrel Monkeys. Vehicle or compounds were administered orally, and behavior was observed after dosing at approximately 1 and 4 h after administration and intermittently throughout the day for up to 6 h. CNS behavioral effects and other activities were recorded by two observers using a checklist of behaviors. After the 4-h observation, monkeys were given access to food and were observed at 24 h after administration for long-lasting or late-appearing behavioral and physical signs. During the period of observation, monkeys were housed two per cage. The general behavior of each monkey after administration of a compound was compared with its own behavior before administration. Emergent behavioral and physical signs were recorded. Five behavioral and physical signs were routinely and closely monitored and documented; these include 1) ataxia (unsteady and wobbly gait, indicating severe motor impairment); 2) soft body tone (decrease in normal body and limb muscular tone, indicating skeletal muscle relaxation); 3) decreased agility (decrease in hand-eye coordination and cautious movements, indicating sedation and impaired fine motor coordination); 4) slow ambulation (a reduction in the rate of movement, indicating sedation); and 5) decreased spontaneous activity (a reduction in the frequency and duration of ambulatory activity, indicating sedation).

Motor Impairment (Horizontal Screen). Mice were placed on a horizontally held screen (mesh size 6.4 mm, wire diameter approximately 1.0 mm). The screen was inverted and mice that successfully climbed to the top side of the screen within 1 min were selected for testing. Compounds or vehicle was administered p.o. by gavage and 1 h after dosing, the mice were tested for their ability to climb to the top side of the screen (pass the test). Compounds that impair coordination or depress motor function can prevent mice from performing this task (Coughenour et al., 1977). The number of mice that did or did not pass the test was recorded. ED₅₀ values were calculated by the method of probits.

Motor Impairment (Rotarod). Rats were trained to walk upon a rotating (9 rpm) rod (rotarod; diameter 12 cm). Those rats able to walk on the rotarod for 3 min were selected for use. One hour after oral (5 ml/kg) administration of the compounds or the vehicle, rats were placed on the rotarod, and the time they were able to remain on the rotarod (up to a maximum of 1 min) was recorded. Rats were tested a second time if they failed the first trial. Rats that did not remain ambulatory on the rotarod for 1 min during either the first or second trial were scored as impaired. Compounds that impair motor coordination or depress motor function decrease performance in this test (Dunham and Miya, 1956).

Sedation (Automated Horizontal Motor Activity). This is an automated procedure for measuring effects of compounds on spontaneous motor activity (Dews, 1953). A drug-induced decrease in spontaneous horizontal motor activity is regarded as an indication of sedation. Some compounds decrease horizontal motor activity at doses below those that impair forced motor activity, and may be referred to as sedatives (Kinnard and Carr, 1957). The Digiscan apparatus (AccuScan, Inc., Columbus, OH) used in the present study differed from that used by Dews (1953); it consists of a plastic cubicle (40.6 cm in length × 40.6 cm in width × 30.5 cm in height) placed in the center of a main frame. Photocell sensors (eight beams from front to back and eight beams from side to side) were built into the sides of the frame for monitoring horizontal movement. The photocells were located at right angles to each other, projecting horizontal infrared beams of light 5 cm apart and 2 cm above the floor. Vehicle or compounds were administered orally by gavage to groups of rats. Each rat was placed into a separate plastic cubicle 50 min after dosing, and spontaneous exploratory activity was recorded for 10 min. Horizontal movement of the rats was recorded by counting the number of times the beams of light were interrupted (counts). Collection of the data and preliminary data analysis were automated. The sedative effect of compounds in the horizontal motor activity test was quantified from the number of rats whose counts were less than or equal to the calculated 10th percentile (744 counts) of that in 473 vehicle-treated rats.

Sedation (Total Entries in the Elevated Plus-Maze). The total number of entries into all arms and the center of the elevated plus-maze serves as a measure of spontaneous motor activity. Compounds with sedative activity reduce the total number of entries in the elevated plus-maze. These data were obtained from the behavioral assay described above. The sedative effect of compounds in the elevated plus-maze assay was quantified from the number of rats whose total entries into all arms and the center, at each dose, was less than or equal to the calculated 10th percentile (67.1 total entries) of the total entries in 359 vehicle-treated rats.

Interaction with Ethanol (Narcosis Assay). Vehicle or compounds were administered orally by gavage, and 1 h after administration of the compound, a soporific dose of ethanol (2 g/kg) was administered to rats i.p. After a rat became immobile, it was placed on its back and if it remained on its back (loss of righting reflex), it was observed for return of the righting reflex. The time the righting reflex was lost and then regained was recorded for each rat. To ensure that the righting reflex was fully recovered, each rat was required to right itself three times. A range of doses of each compound was used to determine their effective dose (ED₅₀) for ethanol potentiation. This was quantified from the number of rats whose duration of sleep, at each dose of the compound, was equal to or greater than the calculated 90th percentile (41.5 min) of the duration of sleep in 621 vehicle-treated rats.

Effect on Chlordiazepoxide-Induced Motor Impairment. The ability of RWJ-51204 to antagonize motor impairment induced by chlordiazepoxide, a full agonist BZ, was evaluated to provide evidence for or against the concept that RWJ-51204 is a partial agonist modulator of GABA_A receptors at the BZ site. RWJ-51204, (CGS-8216; antagonist/partial inverse agonist used as a positive control), or their vehicle was administered to separate groups of mice p.o. 1 h before testing, and chlordiazepoxide (45 mg/kg s.c.) was administered 30 min before testing. Mice were tested for their ability to climb to the top side of the inverted screen as described previously (Coughenour et al., 1977). Activity as an antagonist of chlordiazepoxide-induced motor impairment was defined by the percentage of reduction in the proportion of mice that failed the test in each group that received RWJ-51204 and chlordiazepoxide relative to the control group that received the vehicle and chlordiazepoxide. The dose of chlordiazepoxide (45 mg/kg s.c.) approximated the ED₉₀ dose (90% of mice failed the test). A compound was considered active if impairment in the group that received the test compound with chlordiazepoxide was statistically significantly less than that in the group that received the vehicle with chlordiazepoxide (P < 0.05; Fisher's exact test, one-tailed).

Drugs. PTZ (Sigma-Aldrich, St. Louis, MO), flunitrazepam, diazepam, clonazepam, bretazenil (all supplied by F. Hoffman-La Roche, Nutley, NJ), alprazolam, panadiplon (Upjohn, Kalamazoo, MI), lorazepam (Wyeth, Philadelphia, PA), abecarnil (Schering AG, Berlin, Germany), and imidazenil (Fidia, Washington, DC) were from suppliers indicated in parentheses. Drugs were dissolved or suspended in an aqueous vehicle comprised of 0.5% (w/v) methylcellulose (15 centipoises) solution containing 0.4% (v/v) Tween 80 for p.o. administration. Flumazenil was dissolved in polyethylene glycol 200. Chlordiazepoxide (as the HCl salt) or PTZ was dissolved in 0.9% (w/v) NaCl aqueous solution (saline). Solutions of chlordiazepoxide were used within 30 min. Drugs were administered orally in a volume that was equivalent to 10 ml/kg for mice and 5 ml/kg for rats or monkeys. Volumes administered parenterally were 10 ml/kg for mice and 1 ml/kg for rats or monkeys. Ethanol [100% (w/w); specific gravity at 25°C = 0.7871] was diluted to 20% (w/v) with saline and was administered i.p. to rats at 10 ml/kg.

Data Analysis. For determinations of the affinity of compounds for the BZ site on GABA_A receptors, specific binding (SB) of the ligand to the receptor was calculated by subtracting the cpm for nonspecific binding from the cpm for the vehicle control samples and the samples containing the compound. The percentage of inhibition of control SB caused by the compound was calculated using the following equation: percentage of inhibition = [1  (SB for compound/SB for vehicle control)] × 100. IC₅₀ values were calculated from an analysis of the concentration-inhibition data using either a nonlinear curve-fit analysis of the untransformed data (Mehta and Shank, 1995) or a linear regression analysis of the data transformed to the log-logit format. K_i values were calculated from IC₅₀ values using the Cheng-Prusoff equation (Cheng and Prusoff, 1973). A quantitative indicator of the intrinsic activity of compounds for modulating GABA_A receptors is the GABA shift (GS), which is calculated by dividing the IC₅₀ obtained with GABA present into the IC₅₀ obtained in the absence of GABA.

	Results

Top Abstract Introduction Materials and Methods Results Discussion Appendix 1 References

Neurochemistry

Affinity for BZ Site on GABA_A Receptors. K_i values for RWJ-51204 and eight reference compounds were determined using synaptosomal membranes from discrete regions of the rat brain. The concentration of labeled ligand ([³H]flumazenil or [³H]Ro-15-4513) in the reaction medium was 2- to 5-fold lower than the K_d for the ligand as determined in separate experiments (Mehta and Shank, 1995a,b). For hippocampal membranes, [³H]Ro-15-4513 was used as the ligand because Ro-15-4513 binds with exceptionally high affinity to a GABA_A receptor that is much more highly expressed in the hippocampus than other areas of the brain. By using a low concentration of [³H]Ro-15-4513 (0.06 nM), it was possible to selectively label this receptor subtype (Mehta and Shank, 1995a,b).

View this table: [in this window] [in a new window]   TABLE 1 Affinity and intrinsic activity (as measured by the GABA shift) of RWJ-51204 and eight GABA-based reference anxiolytics for benzodiazepine sites in four areas of the rat CNS using [3H]Flumazenil or [3H]Ro15-4513 as ligands The values represent the results of one or two experiments performed in quadruplicate. Where the results are from two experiments the average ± the range around the average are shown. For each experiment, 11 concentrations of the compound tested were used to generate concentration-inhibition (ligand displacement) curves. Not shown are data obtained using a reference compound (flunitrazepam) at a saturating concentration (10 µM) to determine nonspecific binding. Ki values were calculated by fitting a substrate saturation equation to the percentage of inhibition of specific ligand bound as described in detail in Mehta and Shank (1995a).

Intrinsic Activity for Modulating GABA_A Receptors. The ratio of K_i values obtained without and with GABA present in the incubation medium is considered to be an in vitro measure of the intrinsic modulatory activity of compounds that bind to the BZ site on GABA_A receptors (Burt and Kamatchi, 1991; Doble and Martin, 1992; Mehta and Shank, 1995a,b; Chen et al., 1996; McKernan and Whiting, 1996). This ratio is referred to as the GS. A GS greater than 1.0 is indicative of positive modulatory (agonist) activity, whereas a GS less than 1.0 is indicative of negative modulatory (inverse agonist) activity, and a GS near 1.0 is indicative of no intrinsic modulatory (antagonist) activity. In our experiments, lorazepam, clonazepam, alprazolam, and diazepam nearly always exhibited GS values greater than 2 (Table 1). These compounds have frequently been termed full agonists, but this term may be simplistic and misleading because there is no established absolute measure of intrinsic modulatory activity. The reference compounds developed more recently, i.e., bretazenil, abecarnil, panadiplon, and imidazenil exhibit GS values lower than the full agonists and are often referred to as partial agonists. In our experiments, the GS for these compounds was always between 1 and 2, as were the values for RWJ-51204 (Table 1).

Anticonvulsant Activity

Inhibition of PTZ Convulsions. RWJ 51204 dose-relatedly antagonized PTZ-induced clonic convulsions when administered orally (ED₅₀ = 0.04 mg/kg; Table 2). RWJ-51204 had a lower oral ED₅₀ that was approximately 260-fold lower than that of the partial agonist anxiolytic bretazenil.

Anxiolytic Activity

Water-Lick Conflict (Vogel Test). RWJ-51204, administered orally, increased the median number of shocks received over a wide range of doses (0.1-10 mg/kg). Based on the number of rats that met the criterion of activity at each dose, the ED₅₀ for RWJ-51204 (0.4 mg/kg) was slightly, but not significantly, lower than that for lorazepam (ED₅₀ = 0.6 mg/kg) 1 h after administration (Fig. 2; Table 3); however, the slope of the dose-response line for RWJ-51204 (0.67) was lower than that for lorazepam (2.04). RWJ-51204 (1 and 10 mg/kg) and lorazepam (10 mg/kg) were effective when tested 4 h after oral administration.

View larger version (19K): [in this window] [in a new window]   Fig. 2.   Anxiolytic (Vogel conflict) and sedative (horizontal locomotion) effects of RWJ-51204 and seven GABA-based reference anxiolytics. Lines were plotted from the linear portion of the dose response lines. Drugs were dissolved or suspended in an aqueous vehicle comprised of 0.5% (w/v) methylcellulose (15 centipoises) solution containing 0.4% (v/v) Tween 80 and administered p.o. (5 ml/kg) 1 h before the test (see Materials and Methods). Defined criteria were used to determine the effectiveness of compounds. The number of animals that met the criterion at each dose was quantified, and ED50 and 95% FL for each compound were calculated by the method of probits (Finney, 1971) and are shown in Table 3. The anxiolytic effectiveness of a compound in Vogel conflict was determined from the percentage of rats, at each dose, that received a number of shocks that was equal to or greater than the calculated 90th percentile of the number of shocks received by approximately 600 vehicle-treated rats. This criterion was eight shocks when rats were tested 1 h after administration. The sedative effect of compounds in the horizontal motor activity test was quantified from the percentage of rats whose counts were less than or equal to the calculated 10th percentile (744 counts) of that in 473 vehicle-treated rats. Abbott's probit correction (Finney, 1971) was used to account for instances in which vehicle-treated animals exhibited an effect that was within the criterion used to define drug activity.

Effect of Flumazenil on Anxiolytic Activity. When RWJ-51204 was administered with the vehicle (polyethylene glycol) for flumazenil, significant anxiolytic activity occurred at 1 to 100 mg/kg; but when administered with 3 mg/kg i.m. flumazenil, 100 mg/kg RWJ-51204 was required for similar activity (Table 4). This suggests that RWJ-51204 was 100 times less effective in rats that were given flumazenil. An active dose of lorazepam was also antagonized by this dose of flumazenil.

Elevated Plus-Maze. RWJ-51204 and lorazepam increased percentage of entries into the open arms of the elevated plus-maze over a dose range of 0.1 to 10 mg/kg p.o. 1 h after oral administration (Table 5). Compared with their respective vehicle control groups, higher doses of the other anxiolytics were required to produce an increase in percentage of entries. Bretazenil did not exhibit activity at the highest dose tested (3 mg/kg) in the elevated plus-maze test.

Conflict-Induced State Anxiety in Monkeys. RWJ-51204 and eight reference drugs were tested orally at several doses and their effective anxiolytic doses and corresponding anxiolytic activities were compared by two methods of data analysis. 1) Anxiolytic activity was defined by the number of monkeys in each experiment that met an efficacy criterion at each dose, and a probit analysis of the dose-response data from combined experiments was performed to provide an ED₅₀ value and probit slope. 2) The percentage of increase in the lever-press response rate above the forecasted control rate was used to quantitate anxiolytic activity and the slope of the regression (Fig. 3).

View larger version (25K): [in this window] [in a new window]   Fig. 3.   A, dose-response lines for RWJ-51204 and four full agonist, GABA-based reference anxiolytics for increasing conflict responding by squirrel monkeys. Data values were determined by linear regression of the percentage of increase in response rate in individual monkeys after logarithmic transformation of each dose. Each dose of drug was tested in from 4 to 12 monkeys. The number of monkeys used at each dose is shown in Table 5. Drugs were dissolved or suspended in an aqueous vehicle comprised of 0.5% (w/v) methylcellulose (15 centipoises) solution containing 0.4% (v/v) Tween 80 and administered p.o. (5 ml/kg) 1 h before the test (see Materials and Methods). The slopes of the dose-response lines for the full agonist anxiolytics generally were steeper than for RWJ-51204. The slope of the dose-response line for alprazolam is probably artificially low due to marked sedative effects (Table 5). B, dose-response lines for RWJ-51204 and four partial-agonist GABA-based reference anxiolytics for increasing conflict responding by squirrel monkeys. Each dose of drug was tested in from four to six monkeys. Data values were determined and methods used are specified in A. The slopes of the dose-response lines for the partial-agonist anxiolytics were more shallow than for RWJ-51204.

Ancillary CNS-Related Effects

Observations of General Behavior in Squirrel Monkeys. RWJ-51204 caused slight-to-mild, and at most, moderate CNS side effects in less than 50% of monkeys. No effects were observed 1 h after administration of 0.3 or 1 mg/kg. Sporadic slight or mild effects were observed at 0.1 and 3 mg/kg. At 10 and 20 mg/kg, effects were more frequent but were slight, or, at most, moderate (Table 7). The only effect observed 4 h after administration was mild ataxia in one monkey that had received an oral dose of 10 mg/kg. In contrast to RWJ-51204, the dose for 100% increase in conflict responding (taken from Fig. 3, the linear regression of the dose-response lines, and shown in Table 7) of the full agonists alprazolam (moderate to marked), clonazepam (slight), and diazepan (moderate to marked), and the partial agonists bretazenil (moderate to marked) and imidazenil (slight) produced CNS side effects in more than 50% of monkeys tested in the conflict procedure.

Motor Impairment (Horizontal Screen). RWJ-51204 impaired motor coordination in mice at a much higher oral dose (26.9 mg/kg; Table 2) than the effective dose for blockade of PTZ-induced seizures (Table 2). For RWJ-51204, the ratio of the oral ED₅₀ value for motor impairment to the ED₅₀ for block of PTZ-induced seizures was 726, whereas the corresponding ratio for lorazepam and most other reference anxiolytics was much lower. RWJ-51204 exhibited a shallow dose-response curve in the motor impairment test (slope = 0.56) compared with lorazepam (slope = 0.98).

Motor Impairment (Rotarod). RWJ-51204 (0.1-30 mg/kg; n = 3-32) impaired motor coordination in rats as determined by the rotarod test. However, little or no impairment was observed at higher doses (100-1000 mg/kg; n = 3/dose). Therefore, the ED₅₀ value of RWJ-51204 (0.12 mg/kg) was calculated from the linear part of the dose-response curve (Table 8). The slope of the dose-response curve of RWJ-51204 (0.83) was much lower than that of lorazepam (2.36). RWJ-51204 had a lower oral ED₅₀ than lorazepam (ED₅₀ = 0.44 mg/kg). All rats given RWJ-51204 orally at 30 mg/kg exhibited sedation, reduced skeletal muscle tone, and impairment of rotarod performance. However, rats given doses from 100 to 1000 mg/kg also exhibited a mild and transient dose-related increase in the startle reflex.

Sedation. RWJ-51204 reduced spontaneous motor activity in rats as measured by two methods: quantifying horizontal motor activity (Fig. 2; Table 3) and counting the total number of entries into each arm and the center of the elevated plus-maze (Table 5). Although spontaneous motor activity was reduced by oral doses of lorazepam (horizontal motor activity ED₅₀ = 0.44 mg/kg) that were similar to that of RWJ-51204 (horizontal motor activity ED₅₀ = 0.32), the slope of the RWJ-51204 dose-response curve for reduction of horizontal motor activity (slope = 0.62) was much more shallow than that of lorazepam (slope = 1.79). Sedative ED₅₀ values for other anxiolytics for reduction of horizontal motor activity are shown in Table 3. In the elevated plus-maze, the minimal effective dose of RWJ-51204 (0.1 mg/kg) for increasing open arm entries and decreasing total arm entries were similar, whereas the minimal effective doses of lorazepam were 0.1 and 0.3 mg/kg, respectively.

Interaction with Ethanol (Narcosis Assay). RWJ-51204 (0.1-10 mg/kg; n = 6-20) prolonged the soporific effect of ethanol. The oral ED₅₀ for RWJ-51204 (ED₅₀ = 0.26 mg/kg) in the ethanol-induced narcosis assay was virtually the same as that for clonazepam (ED₅₀ = 0.27 mg/kg), but was 2 to 40 times lower than corresponding values for eight other reference anxiolytics to which it was compared (Table 8). The slope of the dose-response curve was much lower for RWJ-51204 (1.10) than for lorazepam (2.06).

Effect on Chlordiazepoxide-Induced Motor Impairment in Mice. RWJ-51204 antagonized chlordiazepoxide-induced discoordination in mice when administered orally at 100 mg/kg (Table 9). CGS 8216 (2.5 mg/kg), a partial inverse agonist/antagonist, antagonized the locomotor discoordinating property of chlordiazepoxide under these conditions of testing.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion Appendix 1 References

Structurally different compounds can modulate the activity of GABA by interacting with the BZ site on GABA_A receptors and can exert a wide range of intrinsic modulatory effects (Olsen and Tobin, 1990; Burt and Kamatchi, 1991; Doble and Martin, 1992; Mehta and Shank, 1995a,b; Stephenson, 1995; Chen et al., 1996; McKernan and Whiting, 1996). Compounds that modulate GABA_A receptors can be classified into several types; strong positive modulators are termed "full agonists" and strong negative modulators are termed "inverse agonists". Compounds with less than full intrinsic activity are termed "partial agonists" or "partial inverse agonists", and compounds that bind to the receptor but possess no modulatory activity (no intrinsic activity) are termed "antagonists". Because of the multiplicity of GABA_A receptors, and the varied pharmacological properties of drugs having differing degrees of intrinsic activity and affinity for these receptor subunits (Griebel et al., 2001), this analysis is too simplistic to precisely describe the pharmacological profile of compounds that modulate GABA_A receptors.

The ligand receptor binding data obtained in this study demonstrate that RWJ-51204 binds with high affinity to GABA_A receptors at a site common to, or overlapping, the BZ site. Based on the IC₅₀ values, RWJ-51204 has a 2- to 10-fold higher affinity than lorazepam or clonazepam for the BZ site on GABA_A receptors, and a 10- to 100-fold higher affinity than diazepam. A comparison between RWJ-51204 and bretazenil, abecarnil, and imidazenil indicated that these four compounds all possessed similar affinities for the BZ sites on GABA_A receptors in all major areas of the rat CNS. The GABA shift values for RWJ-51204 were lower than those for the full agonists (lorazepam, clonazepam, alprazolam, and diazepam) and similar to those for the partial agonists bretazenil, panadiplon, and imidazenil. Based on these in vitro results, the intrinsic positive modulatory activity of RWJ-51204 seems to be less than that of the full agonists and similar to that of the partial agonists. These results suggest that RWJ-51204 may be broadly classified as a partial agonist.

In acute studies in mice, RWJ-51204 was evaluated for anticonvulsant activity in the PTZ seizure test, and for motor impairment in the horizontal screen test. The oral ED₅₀ for RWJ-51204 against PTZ was 0.04 mg/kg, whereas the ED₅₀ for motor impairment was 27 mg/kg, indicating a 700-fold separation between efficacy and motor impairment. Compared with the other anxiolytic compounds, RWJ-51204 was more potent (lower ED₅₀) against PTZ seizures and exhibited a wider separation between the anti-PTZ dose and the dose causing motor impairment. These results are consistent with the concept that RWJ-51204 in mice is a partial agonist, i.e., it has a lower intrinsic positive modulatory effect than a full agonist. This concept is supported by the observation in mice that RWJ-51204 reduced the motor impairment caused by chlordiazepoxide.

In rats, the anxiolytic activity of RWJ-51204 in the Vogel test, like that of lorazepam, was antagonized by flumazenil, verifying that this activity of RWJ-51204 is mediated by a modulatory effect on one or more types of GABA_A receptors. Comparison of the effective anxiolytic doses of RWJ-51204 and lorazepam in efficacy and side effect tests revealed that anxiolytic efficacy did not occur at doses below those causing CNS side effects. The minimum effective oral dose of RWJ-51204 in the two efficacy tests in rats (Vogel conflict and elevated plus-maze) was 0.1 mg/kg, and the ED₅₀ value in Vogel conflict was 0.4 mg/kg, respectively. The corresponding ED₅₀ value for lorazepam was 0.6 mg/kg. In four tests for CNS side effects (rotarod impairment, decrease in total entries in the elevated plus-maze, decrease in horizontal motor activity in the automated motor activity test, and ethanol narcosis potentiation), the minimum effective dose of RWJ-51204 when tested 1 h after oral administration was 0.3 mg/kg, and the ED₅₀ values ranged between 0.1 and 1 mg/kg. For lorazepam, the minimum effective dose 1 h after oral administration in four tests for CNS side effects was 0.3 mg/kg and the ED₅₀ values in these tests ranged from 0.4 and 1.1 mg/kg, respectively. Hence, for RWJ-51204 and lorazepam there was no separation between minimum effective doses and ED₅₀ doses in the efficacy tests and the corresponding doses in the side effect tests in the rat. The results of these studies show that there is no separation between doses of RWJ-51204 for anxiolytic and CNS side effects.

However, a comparison of the activity of RWJ-51204 and lorazepam in rats revealed that for the anxiolytic efficacy tests and the side effect tests, the slope of the dose-response curves and the maximum responses were usually lower for RWJ-51204. For example, RWJ-51204 potently impaired rotarod performance in rats (ED₅₀ = 0.12 mg/kg), and all rats given RWJ-51204 orally at 30 mg/kg exhibited sedation, reduced skeletal muscle tone, and impairment of rotarod performance. In contrast, virtually no impairment was observed from 100 to 1000 mg/kg p.o. This is consistent with the concept that RWJ-51204 possesses a lower intrinsic activity than lorazepam, i.e., RWJ-51204 exhibits a partial agonist efficacy profile. However, this idea needs to be validated in rats using similar high doses to determine whether these high doses produce anxiolytic effects.

RWJ-51204 was effective in the conflict test in monkeys (ED₅₀ of approximately 0.5 mg/kg p.o.) at doses in which no physical signs of ataxia, soft body tone, or sedation (decreased spontaneous activity or slow ambulation) and no decrease in motor coordination (agility) were observed. By comparison, adverse effects such as ataxia, soft body tone, or decreased agility were slight to moderate in less than 50% of the monkeys tested even at doses of 10 and 20 mg/kg p.o. In the monkey conflict test, RWJ-51204 was approximately 10-fold less potent (higher ED₅₀) than lorazepam, alprazolam, clonazepam, and equipotent with diazepam. However, RWJ-51204 was superior to the reference anxiolytics when compared in terms of the effective anxiolytic dose (ED₅₀) relative to the dose causing appreciable CNS side effects. In addition to a generally wider separation between the effective anxiolytic dose, and the dose causing adverse effects, these effects were much less severe at high doses. The only frequent CNS side effect observed at these doses was a decrease in ambulatory speed, agility, and locomotor activity, an indication of mild sedation. Our results for the partial agonist reference anxiolytics revealed that, as expected, the maximum anxiolytic effect was generally less (lower intrinsic activity) than observed for the full agonist anxiolytics, and CNS side effects were generally less severe. In monkeys, abecarnil did not exhibit significant anxiolytic efficacy or unwanted effects at any dose (0.03, 0.1, 0.3, 1, or 10 mg/kg p.o.). Bretazenil was a potent anxiolytic (ED₅₀ of 0.02 mg/kg p.o.), but occasional CNS side effects were observed at low doses (0.03 and 0.1 mg/kg p.o.). Of the four reference partial agonist anxiolytics, panadiplon exhibited the best profile; however, this compound has been reported to be unacceptable as a development candidate due to hepatotoxic effects (Ulrich et al., 1995).

In vitro and in vivo data obtained in these experiments indicate that RWJ-51204 fits into the class of GABA_A receptor modulators commonly termed partial agonists. RWJ-51204 exhibits a profile of activity in mice and monkeys (but not in rats), suggesting that it has a profile of anxiolyic activity associated with less sedation, motor impairment, or muscle relaxation than currently available GABA_A receptor modulators, i.e., the benzodiazepines. In a preliminary experiment in healthy male human volunteers, a 1-mg oral dose of RWJ-51204 significantly reduced the mean number of (DSM-1, Diagnostic and Statistical Manual of Mental Disorders) panic symptoms-induced by cholecystokinin-4 compared with the placebo (Bradwejn et al., 1999).