in vivo pharmacological characterization of indiplon, a novel … · 2004. 7. 15. · jpet #63487 3...

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.

This article has not been copyedited and formatted. The final version may differ from this version.JPET Fast Forward. Published on July 15, 2004 as DOI: 10.1124/jpet.103.063487

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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 [email protected]

* 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

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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

This article has not been copyedited and form

atted. The final version m

ay differ from this version.

JPET

Fast Forward. Published on July 15, 2004 as D

OI: 10.1124/jpet.103.063487

at ASPET Journals on May 17, 2021 jpet.aspetjournals.org Downloaded from


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



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



%C

orr

ect

Ch

oic

es

70

75

80

85

90

95

100

105 Vehicle

Indiplon




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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

**



A

B




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in vivo pharmacological characterization of indiplon, a novel … · 2004. 7. 15. · jpet #63487 3...

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