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JPET #63487
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In Vivo Pharmacological Characterization of Indiplon, a Novel
Pyrazolopyrimidine Sedative-Hypnotic
Alan C Foster1‡, Mary Ann Pelleymounter1*, Mary Jane Cullen1*, Dacie Lewis1, Margaret
Joppa1, Ta Kung Chen2, Haig P Bozigian2, Raymond S Gross3 and Kathleen R Gogas1
Departments of Neuroscience1, Development2 and Medicinal Chemistry3, Neurocrine
Biosciences Inc., 10555 Science Center Drive, San Diego, CA 92121-1102, USA.
JPET Fast Forward. Published on July 15, 2004 as DOI:10.1124/jpet.103.063487
Copyright 2004 by the American Society for Pharmacology and Experimental Therapeutics.
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Running Title: In Vivo Pharmacology of Indiplon
‡ Correspondence: Alan C Foster, PhD
Department of Neuroscience
Neurocrine Biosciences
10555 Science Center Drive
San Diego, CA 92121-1102
USA
Tel: (858) 658-7760
Fax: (858) 658-7696
Email afoster@neurocrine.com
* Current Address: Department of Metabolic Research
Bristol-Myers Squibb
311 Pennington-Rocky Hill Road
Pennington, NJ 08543, USA
Number of pages: 39
Number of tables: 1
Number of Figures: 11
Number of references: 40
Abstract: 250 words
Introduction: 750 words
Discussion: 1473 words
Abbreviations: GABA: γ-aminobutyric acid; HBC: 2-hydroxypropyl-beta-cyclodextran: HPMC:
hydroxylpropylmethyl cellulose; MED: minimal effective dose.
Section Assignment: Neuropharmacology
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Abstract
Indiplon (NBI 34060; N-methyl-N-[3-[3-(2-thienylcarbonyl)-pyrazolo[1,5-alpha]pyrimidin-7-
yl]phenyl]acetamide), a novel pyrazolopyrimidine and high affinity allosteric potentiator of
GABAA receptor function, was profiled for its effects in rodents after oral administration. In
mice, indiplon inhibited locomotor activity (ED50 = 2.7 mg/kg, p.o.) at doses lower than the non-
benzodiazepine hypnotics zolpidem (ED50 = 6.1 mg/kg, p.o.) and zaleplon (ED50 = 24.6 mg/kg,
p.o.), a sedative effect that was reversed by the benzodiazepine site antagonist, flumazenil.
Indiplon inhibited retention in the mouse passive avoidance paradigm over a dose-range and with
a temporal profile that coincided with its sedative activity. Indiplon, zolpidem and zaleplon were
equally effective in inhibiting locomotor activity in the rat, and produced dose-related deficits on
the rotarod. In a rat vigilance paradigm, indiplon, zolpidem and zaleplon produced performance
deficits over a dose-range consistent with their sedative effects, although indiplon alone showed
no significant increase in response latency. Indiplon produced a small deficit in the delayed non-
match to sample paradigm at a dose where sedative effects became apparent. Indiplon was active
in the rat Vogel test of anxiety, but showed only a sedative profile in the mouse open field test.
The pharmacokinetic profile of indiplon in both rat and mouse was consistent with its
pharmacodynamic properties and indicated a rapid Tmax, short t1/2 and excellent blood-brain
barrier penetration. Therefore, indiplon has the in vivo profile of an efficacious sedative-
hypnotic, in agreement with its in vitro receptor pharmacology as a high affinity allosteric
potentiator of GABAA receptor function, with selectivity for α1 subunit containing GABAA
receptors.
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Benzodiazepine drugs have been used extensively for the treatment of insomnia, in addition to
other central nervous system disorders. Although effective as hypnotics, benzodiazepines also
produce undesirable side effects, such as memory impairment, have durations of action which
can lead to drowsiness and functional impairment upon awakening (“next day hangover”) and
are associated with dependence and abuse liability (Johnson and Chernik, 1982; Lister, 1985;
Roehrs et al., 1994; Woods and Winger, 1995). Consequently, the development of drugs with
sedative/hypnotic activity equivalent to the benzodiazepines, but with an improved side effect
profile, would be of great benefit in the successful management of insomnia, which itself
represents an enormous cost to society (Walsh and Engelhardt, 1999).
The benzodiazepine drugs act through potentiation of the effects of the inhibitory
neurotransmitter, γ-aminobutyric acid (GABA), by binding to a specific site on the GABAA
receptor to produce allosteric enhancement of anion flux through this ligand-gated chloride
channel (McKernan and Whiting, 1996; Barnard et al., 1998; Mohler et al., 2002; Möhler et al.,
2002). GABAA receptors are hetero-oligomers formed primarily by the association of one or
more α1-6, β1-3, and γ1-3 subunits that can assemble in different combinations to form receptor
subtypes that differ in their pharmacology and cellular and regional distribution (Barnard et al.,
1998). The effects of benzodiazepine site ligands arise from their relative affinity and efficacy
for GABAA receptor subtypes. These subtypes are formed by the assembly of different GABAA
receptor subunits as hetero-oligomers containing, in particular, different α subunits (Pritchett et
al., 1989; McKernan and Whiting, 1996; Barnard et al., 1998; Mohler et al., 2002; Möhler et al.,
2002). The diverse pharmacological properties of benzodiazepines, such as sedation, muscle
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relaxation, anxiolysis, anticonvulsant and memory impairment, have been attributed to
interaction with these different receptor subtypes (Mohler et al., 2002; Möhler et al., 2002) and
α1 subunit containing receptors have been associated with sedative/hypnotic activity (Rudolph et
al., 1999).
The “non-benzodiazepine” drugs represent a new generation of sedative/hypnotics (Mitler, 2000)
and are associated with greater selectivity for α1-containing GABAA receptors than earlier
benzodiazepines. Most notable of these are the imidazopyridine, zolpidem (Ambien), the
cyclopyrrolone zopiclone (Imovane) and the pyrazalopyrimidine, zaleplon (Sonata) (Figure
1). The most extensively characterized member of this group is zolpidem. The advantages of
these newer agents over benzodiazepines appear to result from their mode of interaction with the
GABAA receptor combined with a reduced duration of action. Thus, for example, zolpidem has a
moderate affinity for α1-containing GABAA receptors in radioligand binding experiments (10-
100 nM; (Arbilla et al., 1985; Smith et al., 2001; Sullivan et al., 2004) is fully efficacious (Smith
et al., 2001; Sullivan et al., 2004), has selectivity for GABAA receptors containing the α1 subunit
(Damgen and Luddens, 1999; Smith et al., 2001), produces sedative activity in animals which is
mediated through α1 subunit-containing GABAA receptors (Crestani et al., 2000) and has a half-
life in rodents and humans after oral administration of 2-3 hours (Gaudreault et al., 1995;
Greenblatt et al., 1998). Zopiclone displays a similar efficacy profile in animal tests (Perrault et
al., 1990), but has only marginal selectivity for GABAA receptors containing the α1 subunit
(Damgen and Luddens, 1999; Smith et al., 2001) and its half-life in man of 3.5-6.5 hours
(Fernandez et al., 1995) has been associated with next day hangover effects, including impaired
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driving ability (Bocca et al., 1999; Vermeeren et al., 2002). Zaleplon has a half-life of 1 hour in
man after oral administration (Greenblatt et al., 1998), however, this compound has a lower
affinity than zolpidem at benzodiazepine binding sites, and a reduced selectivity for α1 subunit-
containing GABAA receptors (Damgen and Luddens, 1999). It is, therefore, of interest to make
side-by-side comparisons of the in vivo pharmacology of the non-benzodiazepine
sedative/hypnotics in rodents to determine how their relative receptor pharmacology profiles and
pharmacokinetic properties translate into in vivo efficacy.
Indiplon (NBI 34060; N-methyl-N-[3-[3-(2-thienylcarbonyl)-pyrazolo[1,5-a]pyrimidin-7-
yl]phenyl]acetamide; Figure 1) is a novel pyrazolopyrimidine, which is a high affinity allosteric
potentiator of GABAA receptor function, and possesses the characteristics of a compound with
selectivity for α1 subunit containing GABAA receptors (Sullivan et al., 2004). Indiplon is
currently being developmed for the treatment of insomnia and has potential advantages over the
currently marketed benzodiazepine and non-benzodiazepine drugs. We now report the profile of
activity of indiplon in rodent models of sedation, muscle relaxation, vigilance, amnestic liability
and anxiety and its pharmacokinetic profile in rodents. In most cases we have made side-by-side
comparisons between indiplon, zolpidem, zaleplon and triazolam to directly compare the in vivo
pharmacology of these drugs.
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Methods
Materials
Indiplon, N-desmethyl-indiplon and zaleplon were synthesized by the medicinal chemistry
department at Neurocrine Biosciences, Inc. Zolpidem and triazolam were purchased from Sigma-
RBI (Natick, MA). Flumazenil was purchased from Tocris (Bristol, U.K.)
Mouse Studies
Subjects: Adult male CD-1 mice (22-25g) were housed in groups of 15 in the Neurocrine
vivarium, where ambient temperature was 25-27 °C, and a 12 hour light/dark cycle was in effect
(7am-7pm). Mice were allowed food and water ad libitum. All studies were conducted during
the light cycle.
Drug Administration: All compounds were given by oral gavage as suspensions in 45% 2-
hydroxypropyl-beta-cyclodextrin (HBC:Sigma-RBI, Natick, MA). Zolpidem, zaleplon, indiplon
and N-desmethyl-indiplon were given at doses ranging from 0.03-100 mg/kg, and triazolam was
given at doses ranging from 0.0003-10 mg/kg. All drugs were given in a volume of 10 ml/kg.
Locomotor Activity: The sedative properties of zolpidem, zaleplon, indiplon, N-desmethyl-
indiplon and triazolam were compared using non-habituated locomotor activity as an index of
sedation. Thirty minutes after dosing, mice were placed into cages that were similar to their
home cages, but surrounded by an array of 16 photocell assemblies (Columbus Instruments,
“Opto-Varimex-Mini Model B”, Columbus, OH). Total locomotor activity was defined as the
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number of beam breaks averaged over 10 minute bins, and was collected for a total of 10 - 60
minutes, depending on the requirements of the study. The dose response function for each drug
was characterized with a separate cohort of mice. In one study designed to assess the duration of
effect for indiplon, mice were treated with 2 mg/kg, p.o., and immediately placed into the
locomotor activity test chambers. To study the effects of the benzodiazepine antagonist,
flumazenil, on indiplon’s inhibition of locomotor activity, flumazenil was administered at 10, 30
or 45 mg/kg (i.p.) alone or concurrently with 1.5 mg/kg indiplon (p.o.), and locomotor activity
measured at 30-34 minute post-dose interval.
Passive Avoidance Retention: For the dose response studies with 24 hour retention, male CD-1
mice were treated with indiplon (0.03-100 mg/kg), zaleplon (0.03-100 mg/kg), zolpidem (0.03-
100 mg/kg) or triazolam (0.003-10 mg/kg) and, 30 minutes later, placed into the brightly lit
compartment of a passive avoidance apparatus. Latency to enter the dark compartment through a
guillotine door was measured (typically 5-10 seconds). Mice that required more than 180 sec to
enter the compartment were not used in the study. Upon entry into the dark compartment, the
guillotine door was closed, and footshock was administered (0.4 mA, 5 sec). Mice were then
taken out of the chamber and tested 24 hrs later. On the test day, mice were again placed into the
lit portion of the apparatus, and the latency to enter the dark compartment was measured. The
test day latency was used as the measure of retention for the association between the dark
compartment and footshock (Jarvik and Kopp, 1967).
For the studies to determine the importance of time interval length between drug administration
and passive avoidance training in producing a retention deficit 24 hrs later, indiplon (2 mg/kg),
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was given by oral gavage either 30, 60 or 120 minutes prior to passive avoidance training. One
group of animals also received indiplon immediately after training to assess their effects on
memory consolidation (designated as the zero time point in Figure 5).
For the studies which assessed the impact of retention interval (24 or 72 hours) on the indiplon-
induced retention deficit, mice were given indiplon (3 mg/kg, p.o.) 30 minutes prior to passive
avoidance training. They were then tested either 24 or 72 hours after training for retention of the
association between context (dark compartment) and footshock.
Open Field Test: Thirty minutes after administration of indiplon (0.03 – 1 mg/kg, p.o.) mice
were placed into a clear Plexiglas arena (50x50x22cm) surrounded by an array of photocell
beams (Accuscan, Inc., Columbus, Ohio). A lamp directed on the center of the field provided a
light level of 120 lux in the center of the arena. Testing was conducted during the light cycle in a
room with constant white noise. Each animal was placed in the center of the arena to initiate the
10 minute testing session. Horizontal activity and time spent and in the center of the Plexiglas
arena were recorded as photobeam breaks.
Statistics (mouse): Locomotor activity data were analyzed over time using a mixed design,
repeated measures ANOVA, where the between groups variable was drug dose and the within-
groups variable was time. Differences between dose groups at specified time points were
analyzed using simple effects ANOVA followed by Fisher’s Least Significant Difference test as
the post-hoc measure for group differences (provided that the simple effects ANOVA omnibus F
ratio was significant). ED50 estimates were calculated for each drug from the locomotor activity
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counts collected during the 30-40 minute post-dose interval, expressing these as percent
inhibition versus vehicle control and plotting these data against the log10 drug dose. The resulting
curve was analyzed, and ED50 values derived, using the following equation: y = maximum
inhibition/(1 + 10(x – log10
ED50
)), where y is percent inhibition of locomotor activity and x is the
log10 drug dose (Prism, GraphPad, San Diego, CA). Passive avoidance data were analyzed by
one-way ANOVA, with retention latencies as the dependent variable. The independent variable
was dose, time of treatment relative to training, or retention test delay interval. Minimal effective
doses (MED) are defined in this report as the lowest dose to produce a statistically significant
effect.
For the experiment where flumazenil was administered along with indiplon in the locomotor
activity assay, the data were analyzed with a one-way ANOVA with post-hoc analysis using
Fisher's PLSD where appropriate.
Open-field data were analyzed using one-way ANOVA for two variables: horizontal activity and
percent time in center, where Fishers Least Significant Difference test was used as the post-hoc
test for group differences.
Rat Studies
Subjects: For all studies adult male Sprague-Dawley or Long-Evans rats (200-250g) were
housed in groups of 2 in the Neurocrine vivarium, where ambient temperature was 25-27 °C, and
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a 12 hour light/dark cycle was in effect (7am-7pm). Rats were allowed food and water ad
libitum, unless they were subjects in the vigilance and delayed non-match to sample studies,
where they were food restricted so that they maintained 85% of their baseline body weight. Rats
in the Vogel test were water deprived for 24 hours prior to testing. All studies were conducted
during the light cycle.
Drug Administration: All compounds were given by oral gavage as suspensions in 45% HBC
(Sigma-RBI, Inc), unless they were used in studies where food was the reinforcer, such the as
vigilance paradigm. For those studies, compounds were suspended in 1% Tween 180 and water,
a vehicle that, in comparison with 45% HBC, occupies a smaller volume and therefore, is less
likely to distend the stomach. Zolpidem, zaleplon and indiplon were given at doses ranging from
1-30 mg/kg, and triazolam was given at doses ranging from 0.3-10 mg/kg. All drugs were given
in a volume of 1 ml/kg.
Locomotor Activity: Male Sprague-Dawley rats were treated with either indiplon (1-10 mg/kg),
zaleplon (1-10 mg/kg), zolpidem (3-30 mg/kg) or triazolam (0.04-10 mg/kg) or an equal volume
of vehicle (45% HBC) by oral gavage. These dose ranges were based on the activity of the
compounds in the mouse locomotor activity assay. Thirty minutes after dosing, rats were placed
into clean home cages surrounded by an array of 16 photobeam assemblies (Columbus
Instruments, Opto-Varimex-Mini Model B, Columbus, OH). Locomotor activity was defined as
the number of beam breaks averaged over 10 minute bins, and was collected for a total of 30
minutes. The dose-response function for each drug was characterized with a separate cohort of
rats. To compare the sedative effects of compounds under the conditions used in the vigilance
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assays, food-deprived, Long-Evans rats were utilized. Locomotor activity was measured as
described above 30 minutes after a single oral dose of compound in 1% Tween 180 as the
suspension vehicle using the following doses: indiplon, 3 mg/kg; zaleplon 6 mg/kg; zolpidem, 25
mg/kg, triazolam, 50 mg/kg.
Rotarod Latency: Male Sprague-Dawley rats were initially given 3 training trials on the
rotating rod (6 revolutions/minute; Columbus Instruments Economex Accelerating Rotarod for
rats and mice, Columbus, OH). Twenty-four hours later, they were given indiplon (1-10 mg/kg),
zaleplon (1-10 mg/kg), zolpidem (3-30 mg/kg) or triazolam (0.3-10 mg/kg) by oral gavage.
Thirty minutes after drug dosing they were again placed on the rotarod apparatus for 3 trials (60
second intertrial intervals). The average latency to fall off the rod was recorded for each rat.
Dose ranges were based on the initial locomotor activity measurements in the mouse.
Vigilance: Food-restricted male Long-Evans rats were trained in Coulbourn (Allentown, PA)
operant boxes fitted with MED PC Associates (St. Albans, VE) 5-choice nosepoke panel and
software. The methods employed were based upon the Robbins version of Leonard’s 5-choice
serial reaction time task for humans (Cole and Robbins, 1987). The nosepoke panel consisted of
5 openings on the wall opposite to the food hopper. Trials were conducted in the dark, where
illumination of the pellet feeder started a trial. When the feeder was illuminated, the rat was
required to nosepoke in the pellet feeder, then turn to the opposite wall and wait until one of the
5 openings was illuminated. The rat was then required to nosepoke in the illuminated opening in
order to receive the food reward in the pellet feeder. Illumination of the openings was conducted
in a random sequence, with a duration of 3 seconds. If the rat gave a nosepoke in the wrong
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opening, or did not make an immediate nosepoke response, a 7 second dark time-out period was
engaged. If the response was correct, a 3 second inter-trial interval was engaged, ending with the
illumination of the pellet feeder. Rats were trained to a criterion of less than 15 omission errors
(per 100 attempts; an omission error is a failure to nosepoke in response to illumination) and a
nosepoke latency of less than 1.75 seconds prior to testing. On the test day, rats were given
indiplon (1-30 mg/kg), zaleplon (0.3-10 mg/kg) or zolpidem (0.3-30 mg/kg) by oral gavage in
1% Tween 180, 30 minutes prior to the onset of testing. Average latency to nosepoke, number of
correct trials/total trials and omission errors were recorded.(Cole and Robbins, 1987). In a
second study, indiplon (3 mg/kg; p.o.) was administered 10 hours prior to the onset of vigilance
testing. The same animals were tested in the locomotor activity assay 30 minutes after treatment
(9.5 hours prior to vigilance testing) to ascertain that indiplon at the selected dose reduced
locomotor activity by approximately 50% when compared to vehicle controls. Because the
vigilance task required food as a motivation, and the 45% HBC vehicle we had utilized for the
previously discussed studies could have caused stomach distension, we used a 1% Tween
180/water vehicle for this and any other tasks that were appetitively motivated. We tested the
sedative effects of indiplon, zaleplon, zolpidem and triazolam in this vehicle in food restricted
rats compared to a group that had free access to food, and found that the vehicle reduced the
effects of zaleplon and zolpidem and that food restriction reduced the effect of triazolam, but not
the non-benzodiazepine hypnotics. We then titrated the doses of all four hypnotics in the Tween
vehicle and food restricted rats to produce a 50% reduction in locomotor activity compared to
vehicle-treated animals. The following data expresses the average number of photobeams broken
by each group during the first 30 minutes of observations, and for each drug the corresponding
percent inhibition: Vehicle: 2412 ± 192, 3 mg/kg indiplon: 1333 ± 240 (45% inhibition), 6 mg/kg
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zaleplon: 1371 ± 142 (43% inhibition), 25 mg/kg zolpidem: 1216 ± 192 (52% inhibition) and 50
mg/kg triazolam: 1287 ± 96 (47% inhibition). Because the sedative effects of triazolam were so
severely altered by food restriction, a comparison with triazolam was not made in the vigilance
studies.
Delayed non-match to sample: Food-restricted male Long-Evans rats were trained in
Coulbourn (Allentown, PA) operant chambers. Two retractable levers extended on either side of
the food hopper/nosepoke apparatus. The hopper/nosepoke apparatus contained photocells that
monitored pellet delivery and receipt. Rats were initially trained on an FR1 schedule where the
animal would initiate a trial with a nosepoke in the hopper, which produced extension of one
lever. Lever extension varied from left to right throughout the entire session during this initial
training period, where the animal learned to initiate a trial with a nosepoke and to receive a
reward by pressing the extended lever. During the second phase of training, trial initiation again
produced only one lever; this now served as the “study” phase of the trial. After variable delays
of 0.5, 1 or 2 seconds, both levers would extend, and the animal would be required to choose the
lever alternate to that presented during the study phase. Rats continued in this phase of training
until they reached an 80% criterion with 100 obtained reinforcements. Training then proceeded
with variable delays of 1, 2, 4, 8, 12, 16 and 20 seconds, until rats achieved a 70% accuracy
criterion regardless of delay. Delays were presented in a random order, using a list without
replicate scheme. Rats were then assigned to treatment groups based upon their accuracy at the
20 sec delay. Indiplon was administered by oral gavage 30 minutes prior to testing at a dose low
enough to allow at least 70% of vehicle responding (2 mg/kg). During testing, rats were
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presented with variable delays of 1, 4, 16 and 32 seconds. Each test session continued for 30
minutes, with number of trials initiated and number of accurate choices recorded as dependent
variables. Both of these variables were averaged over trials for each delay. In a second study,
indiplon (2 mg/kg, p.o.) was administered 10 hours prior to the onset of delayed non-match to
sample testing.
Vogel Test: Male Sprague-Dawley rats were water deprived for 24 hours prior to testing in
Coulbourn operant chambers. After 200 free (unpunished) licks, rats received a 100ms, 0.4 mA
shock every 10 licks. The test session commenced after the first shock was received and lasted
for 5 minutes. Indiplon was administered by oral gavage in 45% HBC vehicle. Both the number
of shocks taken and the total number of licks over the test session were recorded as indices of
conflict behavior.
Pharmacokinetic studies: Indiplon was administered by oral gavage to CD-1 mice (4 mg/kg;
N=2) or CD rats (5 mg/kg; N=4) in a vehicle of 0.5% hydroxylpropylmethylcellulose (HPMC)
suspension solution. Terminal blood and brain samples were taken at pre-determined time for
composite sampling. All plasma and brain samples were flash frozen in liquid nitrogen
immediately after harvest and stored at -21°C until analysis. The bioanalytical method applied
for the measurement of indiplon in plasma along with added internal standard consisted of
precipitation with 200 µl of acetonitrile from 50 µl of plasma, centrifugation and recovery of the
supernatant, drying down under vacuum then reconstitution in 30:70 acetronitrile:water before
introducing into an HPLC-fluorescence detector system for analysis. The analytical method
applied for the measurement of indiplon in brain tissue consisted of: dividing the brain saggitally
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into 2 halves, homogenizing one half of the brain tissue with a Tissue Tearor, extraction of
indiplon along with added internal standard with 2 ml of 70:30 acetonitrile:water from the
homogenate, centrifugation and recovery of the supernatant, drying down under vacuum then
reconstitution in 30:70 acetronitrile:water before introduction into an LC-MS/MS system for
analysis. The lower limits of quantification (LLOQ) for the analytical methods were 20 ng/ml
and 5 ng/g for plasma and brain samples, respectively. All pharmacokinetic parameters were
calculated from a non-compartmental model using WinNonlin program version 1.2.
Data analysis: Locomotor activity data were analyzed over time using a mixed design, repeated
measures ANOVA, where the between groups variable was drug dose and the within-groups
variable was time. Differences between dose groups at specified time points were analyzed using
simple effects ANOVA followed by Fisher’s Least Significant Difference test as the post-hoc
measure for group differences (provided that the simple effects ANOVA omnibus F ratio was
significant). Curves were fitted and ED50 estimates were calculated for each drug from the
locomotor activity counts during the 30-40 minutes post-dose interval, expressing these as 10
minute locomotor activity averages as percent inhibition of vehicle control and plotting these
data against the log10 drug dose, as described above for the mouse data. Rotarod data were
analyzed by one-way ANOVA, with latency to fall as the dependent variable. The independent
variable was dose. Curves were fitted and ED50 values estimated as described above. Vigilance,
DNMTS and Vogel data were analyzed using one-way ANOVA with dose as the independent
variable, and Fisher’s Least Significant Difference as the post-hoc test for group differences. The
total reinforcement, choice accuracy, omission error and response latency variables for the
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vigilance test were averaged for the 1, 2 and 3 second stimulus durations prior to analysis, since
there were no group differences when these stimulus durations were assessed separately.
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Results
Pharmacokinetics in the rat and mouse
After oral dosing in the mouse, peak plasma levels of indiplon (Tmax) occurred at 30 minutes with
a half-life (t1/2) of 1 hour (Figure 2A). The levels of indiplon were higher in the brain and
paralleled the time course in the plasma, peaking at 30 minutes, with a brain:plasma ratio of 1.7,
indicating excellent blood-brain barrier penetration. A similar plasma profile was observed after
oral dosing of indiplon in the rat (Figure 2B): Tmax was 30 minutes and the estimated t1/2 was 1
hour. On the basis of these data, behavioural tests were conducted 30 minutes after oral
administration of drug, unless otherwise indicated.
Locomotor Activity in the Mouse
Indiplon produced a dose-dependent reduction in non-habituated locomotor activity
[F(8,590)=15.5;p<.0.0001] over the 60 minute observation period, with a maximal effect during
the first 10 minutes of observation [F(8,117)=23.1;p<0.0001] (Figure 3A). Similar effects were
observed for zaleplon [F(8, 126)=9.7;p<0.0001], zolpidem [F(8,106)=13.0;p<0.0001] and
triazolam [F(10,95)=22.2;p<0.0001]. Maximal drug effects were observed early in the
observation period because locomotor activity decreased over time for all animals as a function
of gradual habituation to the test environment [F(5,590)=65.7;p<0.0001].
Dose-response curves derived from these data (Figure 3B), indicated that indiplon was more
effective than zaleplon and zolpidem, ED50 values being: indiplon, 2.7 mg/kg; zaleplon, 6.1
mg/kg and zolpidem, 24.6 mg/kg. Triazolam was the most potent of the drugs tested with an
ED50 value of 0.04 mg/kg. Indiplon, zolpidem and triazolam achieved a similar maximum level
18
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of inhibition (approximately 90% versus vehicle). The maximum inhibition by zaleplon appeared
lower at approximately 70%, although doses higher than 100 mg/kg would be needed to clearly
define the maximal effect of this drug. We also tested desmethyl-indiplon (Figure 1), the major
metabolite of indiplon, which was inactive up to a dose of 100 mg/kg, p.o.
[F(8,125)=0.6;p<0.771] (Figure 3B).
The duration of the sedative activity of indiplon was evaluated in mice at a dose that
approximated its ED50 value in the locomotor activity assay. At 2 mg/kg, p.o., indiplon produced
inhibition of locomotor activity which persisted for 2 hours [F(1,132)=33.8;p<0.0001;
F(2hrs)(1,22)=5.7;p<0.02] (Figure 3C), consistent with the pharmacokinetic profile of the drug
in mice (vide supra).
To confirm that the effects of indiplon on locomotor activity were due to an interaction with the
benzodiazepine site on the GABAA receptor, the effects of the benzodiazepine site antagonist,
flumazenil, on the inhibition of locomotor activity produced by indiplon were examined. An
approximate ED50 dose of indiplon (1.5 mg/kg, p.o.) was administered to mice, along with 3
doses of flumazenil and locomotor activity measured for a 4 min period starting 30 min after the
indiplon dose. This timing was chosen to take into account the rapid clearance of flumazenil
from mouse plasma and brain (d'Argy et al., 1987; Potier et al., 1988). Flumazenil produced a
dose-dependent reversal of the inhibition of locomotor activity produced by indiplon [F(8,91) =
5.4; p < 0.001] (Figure 3D). Flumazenil alone had no effect on locomotor activity at 10, 30 or 45
mg/kg, i.p. (data not shown).
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Passive Avoidance in the Mouse
Indiplon significantly reduced retention latency [F(6,84) = 12.8; p<0.0001] (Figure 4A) with an
MED of 1 mg/kg (p<0.0001). Retention latency was also reduced by zaleplon [F(10,91) = 9.8;
p<0.0001], MED = 3 mg/kg (p<0.001), zolpidem [F(10,103) = 17.4; p<0.0001], MED = 30
mg/kg (p<0.0001) and triazolam [F(10,101) = 18.3; p<0.0001], MED = 0.1 mg/kg (p<0.0001)
(Figure 4B,C,D). A further experiment examined the importance of the time interval between
treatment and training with a 2 mg/kg dose of indiplon (Figure 5). No significant retention deficit
occurred when indiplon was administered 120 or 60 minutes prior to the training session, but a
significant effect was seen with 30 min pretreatment (p<0.03), consistent with the results in
Figure 4A. Treatment with indiplon immediately after the passive avoidance training gave no
significant deficit 24 hours later (Figure 5; zero time point). To examine the effect of the degree
of difficulty on passive avoidance retention, an experiment was carried out where the time period
between training and test periods was lengthened to 72 hours. Vehicle-treated mice showed a
non-significant trend towards reduced retention 72 hours after training relative to the 24 hour
paradigm (p<0.17). Mice treated with 3 mg/kg indiplon 30 minutes prior to training showed a
significant retention deficit at 24 (p<0.0001) and 72 (p<0.01) hours after training. Increasing the
time between training and testing from 24 to 72 hours did not enhance the retention deficit
(p<0.58 at 24 versus 72 hours) in indiplon-treated mice (data not shown).
Locomotor Activity in the Rat
Locomotor activity in the rat was recorded from 30 to 60 min after oral administration of the test
compounds. Indiplon significantly reduced locomotor activity when compared to vehicle in a
dose-dependent manner [F(3,54) = 13.9; p<0.0001] (Figure 6A,B). Dose-dependent inhibition
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was also observed with zaleplon [F(3,56) = 66.0; p<0.0001], zolpidem [F(3,56) = 22.1;
p<0.0001] and triazolam [F(4,70) = 36.5; p<0.0001] (Figure 6B). ED50 values (mg/kg) derived
from the dose-response relationships were: indiplon, 2.5; zaleplon, 0.8; zolpidem, 2.4; and
triazolam, 1.4.
Rotarod Performance in the Rat
Indiplon significantly reduced the latency to fall from the rotarod when compared to
vehicle in a dose-dependent manner [F(3,36) = 12.9; p<0.0001] (Figure 7A). Dose-
dependent reductions in latency were also observed for zaleplon [F(3,28) = 15.2;
p<0.0001] (Figure 7B), zolpidem [F(3,28) = 32.4; p<0.0001] (Figure 7C) and triazolam
[F(3,28) = 21.4; p<0.0001] (Figure 7D). ED50 values (mg/kg) derived from the dose-
response realtionships were: indiplon, 5.9; zaleplon, 5.4; zolpidem 14.8; and triazolam,
4.8.
Vigilance Measures in the Rat
The results of the vigilance studies are shown in Figure 8A-D. Indiplon significantly
reduced the total number of trials initiated [F(4,47) = 5.4; p<0.001], increased the
number of omission errors [F(4,47) = 10.8; p<0.0001] and reduced the proportion of
correct trials in relation to the total number of trials initiated [F(4,47) = 6.5; p<0.0003],
with an MED for all of these measures of 3 mg/kg (p<0.03). Indiplon had no significant
effect on the average latency to respond to a cue light stimulus (nosepoke latency).
Zaleplon significantly reduced the total number of trials initiated [F(4,44) = 8.7;
p<0.0001], increased the number of omission errors [F(4,44) = 5.9; p<0.0006], reduced
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the proportion of correct trials in relation to the total number of trials initiated [F(4,44) =
6.5; p<0.0003] and increased the average latency to nosepoke [F(4,44) = 4.8; p<0.003].
The MED for zaleplon in all measures except latency to respond was 3mg/kg (p<0.04).
Zolpidem significantly reduced the total number of trials initiated [F(5,32) = 10.2;
p<0.0001], increased the number of omission errors [F(5,32) = 21.8; p<0.0001], reduced
the proportion of correct trials in relation to the total number of trials initiated [F(5,32) =
10.2; p<0.0001] and increased the average latency to nosepoke [F(5,32) = 9.7; p<0.0001].
The MED for zolpidem in all measures was 10 mg/kg (p<0.02). When the above
parameters were measured in the vigilance paradigm 10 hours after indiplon
administration (3 mg/kg, p.o., a dose that produced a significant inhibition of locomotor
activity in the same animals from 30 – 60 minutes after dosing [F(1,17) = 10.9;
p<0.004]), no significant difference from vehicle was observed in any parameter (data not
shown), indicating no residual sedative effects 10 hours after treatment.
Delayed Non-Match to Sample Test in the Rat
As shown in Figure 9A, indiplon produced deficits in responding at the 4 and 32 second
delay intervals [F(1,72)=13.3; p<0.001; post-hoc p<0.02], but this was accompanied by a
generalized reduction in responding, as measured by the number of reinforcements
obtained at each delay interval [F(1,72)=13.8; p<0.001]. Reduced responding was
statistically significant even at the 1 second delay interval (p<0.006), even though
accuracy was maintained (Figure 9B). When the task was repeated 10 hours after dosing,
no deficits in response accuracy [F(1,21)=5.5; p<0.051; post-hoc p<0.14] or number of
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reinforcements obtained [F(1,21)=1.4; p<0.27] were observed in the indiplon group
versus vehicle (Figure 9C,D).
Vogel Test in the Rat
Indiplon was active in the punished drinking model of anxiety (Vogel et al., 1971).
Following oral administration, indiplon showed a dose-dependent effect measured either
by the number of shocks received [F(4,39) = 7.6; p<0.0001] (Figure 10) or licks taken
[F(4,39)=7.6; p<0.0001] (data not shown), with an MED of 3 mg/kg (p<0.016).
Open field in the mouse
In an ethological anxiolytic test, the open field paradigm in mice, indiplon showed no
evidence of anxiolytic activity, as would have been indicated by increased percent time in
the center of the open field rather than a reduction in these measures [F(4,45)=8.1;
p<0.0001], as we observed. The reduction in these measures was likely a function of
suppressed locomotor activity [F(4,45)=4.8; p<0.003] (Figure 11) since reduced center
time and distance was only observed at doses where horizontal activity was also reduced.
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Discussion
The studies described here characterize indiplon as an effective sedative hypnotic in
rodents, with the pharmacological profile expected for an allosteric potentiator of
GABAA receptor function which has preference for α1 subunit-containing GABAA
receptors.
The in vivo pharmacology of indiplon is entirely consistent with in vitro studies that show
this compound to be a high affinity allosteric potentiator of GABAA receptors. Indiplon
has low nanomolar affinity for [3H] Ro 15-1788 and [3H] Ro 15-4513 binding sites in rat
brain membranes, and the binding of [3H]indiplon itself shows a pharmacology and
regional distribution of binding sites in rat brain consistent with that for α1 subunit-
containing GABAA receptors (Sullivan et al., 2004). Indiplon is also a fully efficacious
potentiator of GABAA receptor-mediated responses in neurons (Sullivan et al., 2004).
The predominantly sedative activity of indiplon in rats and mice, and reversal of this by
the benzodiazepine site antagonist, flumazenil, is in good correspondence with this in
vitro receptor profile, as is the fact that indiplon is active in the passive avoidance and
Vogel tests, and in blocking pentylenetetrazole-induced convulsions (unpublished
observations), activities consistent with an allosteric potentiator of GABAA receptors.
The sedative activity of indiplon was apparent in multiple assays in both rats and mice.
Inhibition of locomotor activity in both species, reduced ability to maintain balance on
the rotarod, reduced accuracy, increased omission errors and general response
suppression in the vigilance assays along with inhibition of activity in the open field test
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all suggested a consistent sedative profile. Indeed, the activity of indiplon in the passive
avoidance paradigm in mice, and the delayed non-match to sample paradigm in rats, both
widely used as measures of learning and memory, may have resulted from the sedative
activity of the compound, rather than a direct influence on the neuronal substrates of
learning and memory. This follows from the observations that the doses of indiplon that
were active in these paradigms closely matched the doses that produce sedation in mice
and rats (see Table 1). In the passive avoidance paradigm, the temporal profile of
indiplon’s action also matched that of its sedative effects. Further, indiplon did not alter
passive avoidance retention if it was given immediately after training, suggesting that it
did not have effects on memory consolidation (Cherkin, 1969; McGaugh, 1972). In the
delayed non-match to sample paradigm, short-term (4-32 sec delay intervals) memory
deficits were observed at a dose of indiplon that clearly produced a reduction in overall
responding, indicated by a decrease in total reinforcements obtained. Further, there was
no evidence of retrograde amnestic effects as indicated by a lack of effect in the delayed
non-match to sample paradigm 10 hours after dosing. Interestingly, there were also no
residual sedative effects 10 hours after dosing, as measured by the 5-choice serial
reaction time test of vigilance. The above findings suggest that the indiplon-induced
memory deficits were a function of generalized response suppression. Taken together, the
delayed non-match to sample and passive avoidance data do not provide strong evidence
that indiplon has specific effects on short-term memory processes or on memory
consolidation, although this must be more fully evaluated in future studies.
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The activity of indiplon in the Vogel test is consistent with that observed for both
benzodiazepine and non-benzodiazepine ligands in this, and other, conflict paradigms
(Gardner and Piper, 1982; Martin et al., 1993; Nazar et al., 1997; Griebel et al., 1998) and
may indicate that the compound has anxiolytic activity. However, in the mouse open field
test, where the anxiolytic effects of benzodiazepine ligands are also apparent (Crawley,
1985), indiplon showed no increase in center time, but a decrease in this parameter and in
total horizontal activity. This suggests that the potential anxiolytic effects of indiplon
were overshadowed in this test by the compound’s sedative effects, and the dose range
over which this occurred is consistent with the inhibition of locomotor activity in the
mouse observed previously. Interestingly, the dose range over which indiplon is active in
the Vogel test also coincides with the sedative dose range in the rat. One explanation for
these observations could be that both the anxiolytic and sedative activities of the
compound occur over the same dose range and that the sedative effects of the compound
can be overridden in a conflict paradigm, where animals have a considerable drive to
respond, whereas in the more “natural” setting of the open field test, sedation dominates.
Similar observations have been made previously in conflict versus ethologically-derived
tests comparing different benzodiazepine site ligands. Thus, “non-benzodiazepine”
agonists with selectivity towards GABAA receptors containing α1 subunits, including
zolpidem and zaleplon, have a different profile to benzodiazepines such as diazepam and
clorazepate (Griebel et al., 1996c; Griebel et al., 1996a; Griebel et al., 1996b; Nazar et
al., 1997; Griebel et al., 1998). Consistently, zolpidem and zaleplon show “anxiolytic”
effects in conflict procedures and anxiolytic activity is weak or absent in measures such
as the open field test, light-dark box and elevated plus maze. In addition, the effects of
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zolpidem and zaleplon in anxiety models overlays their sedative activity. Therefore, the
profile of indiplon with similar dose ranges for activity in assays for sedation, learning
and memory and anxiolytic activity (Table 1) is consistent with that for allosteric
potentiators of GABAA receptor function which have preference for α1 subunit-
containing GABAA receptors.
Our understanding of the roles played by GABAA receptor subtypes in benzodiazepine
ligand pharmacology has recently been confirmed and extended in an elegant series of
experiments where the affinity of the benzodiazepine site has been manipulated by point
mutations in the different α subunits with a subsequent “knock-in” approach to create
mutant mice that have lost benzodiazepine sensitivity in GABAA receptor subtypes
containing specific α subunits ((Mohler et al., 2002; Möhler et al., 2002). This work has
shown that certain pharmacological effects of benzodiazepine ligands are a result of an
interaction with particular α subunits, for example sedation with α1, anxiolysis with α2,
and memory effects with both α1 and α5 (Rudolph et al., 1999; Low et al., 2000;
McKernan et al., 2000; Crestani et al., 2002). As an efficacious allosteric potentiator with
preference for α1 subunit-containing GABAA receptors, indiplon’s in vivo profile is
entirely consistent with this work since it produces a predominantly sedative profile in
animals.
A major aim of the present study was to make a side-by-side comparison between the in
vivo sedative effects of indiplon, zaleplon, zolpidem and triazolam, and this has yielded
some interesting results. Table 1 compares the relative activities of these compounds
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across the in vivo measures taken and also includes a summary of in vitro receptor
affinity (from (Sullivan et al., 2004)). First, when assessed over all of the assays, indiplon
emerges as the most effective sedative versus zolpidem and zaleplon, consistent with its
higher affinity in the receptor binding assays. When the rank order is compared, the
relationship between receptor affinity and efficacy in the mouse and rat locomotor
activity, rat rotorod and mouse passive avoidance assays holds true between triazolam,
indiplon and zolpidem, with zaleplon being an outlier. Consequently, zaleplon appears to
be more effective in vivo than would be predicted from its receptor affinity. Interestingly,
dose-response curves for zaleplon appeared to be atypical compared to the other
benzodiazepine site ligands. In the mouse locomotor activity assay, inhibition by zaleplon
appeared to reach a lower maximum inhibition with a more shallow dose-response curve
(Figure 3B); similarly, the dose-response function for zaleplon in the passive avoidance
assay also appeared to be shallow compared with the other drugs tested. These
differences are not explained by the allosteric potentiation by this compound of the
GABAA receptor, since all of these ligands appear to be fully efficacious (Sullivan et al.,
2004). The reportedly high oral bioavailability of zaleplon in the rat of 80% (Beer,
1997)(cf oral bioavailability of zolpidem in the rat is 27% (Garrigou-Gadenne et al.,
1989)) may contribute to the effectiveness of zaleplon in vivo, although its blood-brain
barrier penetration is similar to zolpidem (brain:plasma ratio of approx. 0.5 (Garrigou-
Gadenne et al., 1989; Gaudreault et al., 1995)) and inferior to that for indiplon
(brain:plasma ratio = 1.7; vide supra). One possibility could be the existence of an active
metabolite for zaleplon, although the major metabolites are apparently weak or inactive
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displacers of benzodiazepine binding (Vanover, 1994). Another possibility may be an
additional pharmacological activity of the compound itself, or a metabolite.
In conclusion, the in vivo pharmacological profile of indiplon in rats and mice shows this
compound to be an effective sedative/hypnotic, consistent with its in vitro profile as a
high-affinity, allosteric potentiator of GABAA responses, with preference for α1 subunit-
containing GABAA receptors. The combination of high affinity, rapid onset and short
duration of action, as determined in both pharmacodynamic and pharmacokinetic assays,
should make this compound an attractive candidate as an improved treatment for
insomnia, a concept which is currently being tested in multiple clinical studies.
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Figure Legends
Figure 1. Chemical structures of indiplon, desmethyl-indiplon, zaleplon, zolpidem and
triazolam
Figure 2. Pharmacokinetic profile of indiplon after oral gavage. (A) plasma (diamonds)
and brain (circles) levels in the mouse (4 mg/kg; 2 mice per time point) and (B) plasma
levels (diamonds) in the rat (5mg/kg; 4 rats per time point).
Figure 3. Inhibition of non-habituated locomotor activity in the mouse. (A) Dose-
dependent reduction of locomotor activity by indiplon. Measurements were begun 30
minutes after oral gavage. (B) Dose-response curves for indiplon, zaleplon, zolpidem,
triazolam and desmethyl-indiplon. Locomotor activity counts were collected during the
30-40 minute post-dose interval and expressed as percent inhibition versus vehicle
controls. Number of mice/ group were: indiplon: 14-15; zaleplon: 15; zolpidem: 5-15;
triazolam: 3-20; desmethyl-indiplon: 14-15. (C) Duration of inhibition of locomotor
activity in the mouse by indiplon. Mice were placed into the locomotor test cages
immediately after oral gavage. There were 10-14 mice/group. * p<0.02 versus vehicle.
(D) Reversal of locomotor activity inhibition in the mouse by co-administration of
flumazenil (i.p.). There were 9-13 mice/group. * p<0.001 versus vehicle; + p< 0.01
versus indiplon-treated.
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Figure 4. Passive avoidance retention in the mouse. Mice were trained 30 minutes after
oral gavage. (A) Dose-dependent effect of indiplon. * p<0.0001 versus vehicle. There
were 5-15 mice /group. (B) Dose-dependent effect of zaleplon. * p<0.0001 versus
vehicle. There were 4-15 mice /group. (C) Dose-dependent effect of zolpidem. *
p<0.0001 versus vehicle. There were 4-15 mice /group. (D) Dose-dependent effect of
triazolam. * p<0.0001 versus vehicle. There were 4-18 mice /group.
Figure 5. Effect of indiplon on passive avoidance retention in the mouse. Impact of
interval between drug treatment and training (5-21 mice/group) * p<0.03 versus vehicle.
Figure 6. Inhibition of locomotor activity in the rat. Measurements began 30 minutes
after oral gavage. (A) Dose-dependent reduction of locomotor activity by indiplon. (B)
Dose-response curves for indiplon, zaleplon, zolpidem and triazolam. There were 8
rats/group. * p< 0.05; ** p<0.01 versus vehicle.
Figure 7. Rotarod activity in the rat 30 minutes after treatment. Dose-dependent effects of
(A) indiplon, (B) zaleplon, (C) zolpidem and (D) triazolam. There were 8-10 rats/group.
* p<0.01 versus vehicle.
Figure 8. Vigilance analysis in the rat using the 5-choice paradigm. Testing began 30
minutes after oral gavage. (A) Total reinforcements for indiplon (5-18 rats/group),
zaleplon (5-15 rats/group) and zolpidem (3-11 rats/group). *p<0.04 versus vehicle. (B)
Omission errors for indiplon (5-18 rats/group), zaleplon (5-15 rats/group) and zolpidem
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(3-11 rats/group). *p<0.03 versus vehicle. (C) Choice accuracy as measured by percent
correct responses for indiplon (5-18 rats/group), zaleplon (5-15 rats/group) and zolpidem
(3-11 rats/group). *p<0.02 versus vehicle.(D) Latency to respond for indiplon (5-18
rats/group), zaleplon (5-15 rats/group) and zolpidem (3-11 rats/group). *p<0.0005 versus
vehicle.
Figure 9. Delayed non-match to sample task in the rat. (A and B) Effect of indiplon 30
minutes after oral gavage (8-18 rats/group) on (A) percent correct choices and (B)
number of reinforcements obtained for each delay. (C and D) Effect of indiplon 10 hours
after oral gavage (4-5 rats/group) on (C) percent correct choices and (D) number of
reinforcements obtained for each delay. *p<0.006 versus vehicle.
Figure 10. Effect of indiplon in the Vogel test in the rat 30 minutes after oral gavage as
measured by the number of shocks taken during a session. There were 8-11 rats/group.
*p<0.016 versus vehicle.
Figure 11. Effect of indiplon in the open field test in the mouse 30 minutes after oral
gavage. (A) Percent time spent in the center of the open field, (B) horizontal activity.
There were 10 mice/group. *p<0.04 versus vehicle.
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Table 1: Comparison of the affinities and effective doses of indiplon, zaleplon, zolpidem
and triazolam in rodent assays. *In vitro data from (Sullivan et al., 2004).
Assay Indiplon Zolpidem Zaleplon Triazolam
Flumazenil Binding*, Rat
Cortex membranes (Ki, nM)
1.8 11.2 68.5 0.55
Potentiation of GABA
currents* Rat cultured
neurons (EC50, nM)
11.6 152 630 26.5
Inhibition of locomotor
activity Mouse (ED50, mg/kg,
p.o.)
2.7 24.6 6.1 0.04
Passive Avoidance Mouse
(MED, mg/kg, p.o.)
1 30 3 0.1
Inhibition of locomotor
activity Rat (ED50, mg/kg,
p.o.)
2.5 2.4 0.8 1.4
Rotarod impairment Rat
(ED50, mg/kg; p.o.)
5.9 14.8 5.4 4.8
Vigilance impairment Rat
(MED, mg/kg, p.o.)
3 10 3 ND
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Indiplon Zaleplon Zolpidem
Triazolam
Figure 1
Desmethyl-Indiplon
N
N N
OS
N
O
N
N N
N
O
CN
N
N
NO
N
NN
N
Cl
Cl
N
N N
OS
HN
O
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Figure 2A
B
ng/g
(ng
/mL)
ng/m
L
2000
1750
1500
1250
1000
750
500
250
00.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0
250
300
200
150
100
50
00 0.5 1 1.5 2 2.5 3 3.5 4 4.5 5 5.5 6 6.5
Time (hr)
Time (hr)
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Figure 3A
Time (min) after oral gavage40 50 60 70 80 90
Lo
com
oto
rA
ctiv
ity
0
500
1000
1500
2000
Vehicle 0.03 mg/kg 0.1 mg/kg
0.3 mg/kg 1.0 mg/kg 3.0 mg/kg 10 mg/kg 30 mg/kg 100 mg/kg
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Zolpidem
Indiplon
Zaleplon
Triazolam
Desmethyl-indiplon
0.0001 0.001 0.01 0.1 1 10 100
-10
10
30
50
70
90
110
Dose (mg/kg;p.o.)
%In
hib
itio
n o
f L
oco
mo
tor
Act
ivit
y
Figure 3B
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Figure 3C
Time after oral gavage (min)
20 40 60 80 100 120 140 160 180
To
talL
oco
mo
tor
Act
ivit
y
200
400
600
800
1000
1200
1400
1600
1800
2000Vehicle Indiplon (2 mg/kg)
**
* **
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*
+
To
talL
oco
mo
tor
Act
ivit
y
0
20
40
60
80
100
120
140
160
Vehicle Indiplon1.5 mg/kg Flumazenil
30 mg/kgFlumazenil45 mg/kg
Flumazenil10 mg/kg
Figure 3D
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Figure 4A
Indiplon (mg/kg;p.o.)
Ret
enti
on
Lat
ency
0
20
40
60
80
100
120
140
160
180
0.03 0.1 0.3 0.6 1 1.5
*
*
Vehicle
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Zaleplon (mg/kg;p.o.)
Vehicle 0.03 0.1 0.3 1 3 10 30 100
Ret
enti
on
Lat
ency
0
20
40
60
80
100
120
140
160
180
**
* *
Figure 4B
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Zolpidem (mg/kg;p.o.)
Vehicle 0.03 0.1 0.3 1 3 10 30 100
Ret
enti
on
Lat
ency
0
20
40
60
80
100
120
140
160
180
*
*
Figure 4C
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Triazolam (mg/kg;p.o.)
Vehicle 0.003 0.01 0.03 0.1 0.3 1 3 10
Ret
enti
on
Lat
ency
0
20
40
60
80
100
120
140
160
180
** * * *
Figure 4D
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Figure 5
Interval between treatment and training (min)
Vehicle
Indiplon
0 -30 -60 -120
Ret
enti
on
Lat
ency
0
20
40
60
80
100
120
140
160
180
*
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Figure 6A
To
talL
oco
mo
tor
Act
ivit
y
Time (min) after oral gavage
40 50 600
1000
2000
3000
4000
5000Vehicle 1 mg/kg 3 mg/kg 10 mg/kg
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Figure 6BIndiplon
0500
10001500
2000250030003500
Vehicle 1 3 10
Lo
com
oto
rA
ctiv
ity
Dose (mg/kg)
***
**
Zaleplon
0
1000
2000
3000
4000
5000
Vehicle 1 3 10
Lo
com
oto
rA
ctiv
ity
Dose (mg/kg)
**
** **
Zolpidem
0
1000
2000
3000
4000
Vehicle 3 10 30
Lo
com
oto
rA
ctiv
ity
Dose (mg/kg)
*
** **
Triazolam
0
1000
2000
3000
4000
Vehicle 1 3 10
Lo
com
oto
rA
ctiv
ity
0.04
Dose (mg/kg)
****
**
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Figure 7
Indiplon
Vehicle 1.0 3.0 10.0
Tim
e (s
eco
nd
s)
0
10
20
30
40
50
60
70
**
Dose (mg/kg)
A
Triazolam
Vehicle 0.3 1.0 3.0 10.0
Tim
e (s
eco
nd
s)
0
10
20
30
40
50
60
*
*
Dose (mg/kg)
DZolpidem
Vehicle 3.0 10.0 30.0
Tim
e (s
eco
nd
s)
0
10
20
30
40
50
60
*
*
Dose (mg/kg)
C
Zaleplon
Vehicle 1.0 3.0 10.0
Tim
e (s
eco
nd
s)
0
10
20
30
40
50
60
*
*
Dose (mg/kg)
B
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Figure 8A
Zolpidem
0
20
40
60
Vehicle 0.3 1 3 10 30
To
tal R
ein
forc
emen
ts
Dose (mg/kg)
Zaleplon
0
20
40
60
Vehicle 0.3 1 3 10 30
To
tal R
ein
forc
emen
ts
Dose (mg/kg)
Indiplon
0
20
40
60
Vehicle 1 3 10 30
To
tal R
ein
forc
emen
ts
Dose (mg/kg)
*** *
*
*
*
*
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Figure 8B
Indiplon
Vehicle 1.0 3.0 10.0 30.0
Om
issi
on
err
ors
0
5
10
15
20
25
30
35
40
45
50
Zolpidem
Vehicle 0.3 1.0 3.0 10.0 30.0
Om
issi
on
err
ors
0
5
10
15
20
25
30
35
40
45
50
Dose (mg/kg)
Dose (mg/kg)
*
*
*
*
*
Zaleplon
Vehicle 0.3 1.0 3.0
Om
issi
on
err
ors
0
5
10
15
20
25
30
35
40
45
50
Dose (mg/kg)
*
10.0
*
30.0
*
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Figure 8C
Vehicle 1.0 3.0 10.0 30.0
%C
orr
ect
Res
po
nse
s
0
20
40
60
80
100
Zaleplon
Vehicle 0.3 1.0 3.0 10.0
%C
orr
ect
Res
po
nse
s
0
20
40
60
80
100
Zolpidem
Vehicle 0.3 1.0 3.0 10.0 30.0
%C
orr
ect
resp
on
ses
0
20
40
60
80
100
Indiplon
Dose (mg/kg) Dose (mg/kg)
Dose (mg/kg)
A
C
B
** *
**
*
*
30.0
*
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Figure 8D
Lat
ency
to
res
po
nd
Zolpidem
Lat
ency
to
res
po
nd
Zaleplon
Lat
ency
to
res
po
nd
Dose (mg/kg)
Dose (mg/kg)
Indiplon
Vehicle 1.0 3.0 10.0 30.0
0.0
0.5
1.0
1.5
2.0
2.5
3.0
Dose (mg/kg)
Vehicle 0.3 1.0 3.0 10.0 30.0
0.0
0.5
1.0
1.5
2.0
2.5
3.0
* *
Vehicle 0.3 1.0 3.0
0.0
0.5
1.0
1.5
2.0
2.5
3.0
10.0
*
30.0
*
*
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Figure 9A
Delay Interval (sec)
%C
orr
ect
Ch
oic
es
1 sec 4 sec 16 sec 32 sec
50
60
70
80
90
100
110 Vehicle
*
*
Indiplon
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Figure 9B
Delay Interval (sec)
1 sec 4 sec 16 sec 32 sec
Nu
mb
er o
f R
ein
forc
emen
ts O
bta
ined
12
14
16
18
20
22
24
26
28
30
32
34
36VehicleIndiplon
* *
**
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Figure 9C
Delay Interval (sec)
1 sec 4 sec 16 sec 32 sec
%C
orr
ect
Ch
oic
es
70
75
80
85
90
95
100
105 Vehicle
Indiplon
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Delay Interval (sec)
1 sec 4 sec 16 sec 32 sec
Nu
mb
er o
f R
ein
forc
emen
ts O
bta
ined
12
14
16
18
20
22
24
26
28
30
32
34
36 VehicleIndiplon
Figure 9D
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Figure 10
Dose of Indiplon (mg/kg)
Vehicle 1.0 3.0 10.0 30.0
Nu
mb
er o
f sh
ock
s ta
ken
0
20
40
60
80
100
120
140
** *
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Figure 11
Vehicle 0.03 0.1 0.3 1.0
Per
cen
t ti
me
in c
ente
r
0
2
4
6
8
10
12
14
16
**
Vehicle 0.03 0.1 0.3 1.0
Ho
rizo
nta
l Act
ivit
y
0
500
1000
1500
2000
2500
3000
3500
**
Dose of Indiplon (mg/kg)
Dose of Indiplon (mg/kg)
A
B
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