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THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS
Copyright © 1998 by The American Society for Pharmacology and Experimental Therapeutics
JPET 285:41–53, 1998
Vol. 285, No. 1
Printed in U.S.A.
Zolpidem Physical Dependence Assessed Across Increasing
Doses Under a Once-Daily Dosing Regimen in Baboons1
ELISE M. WEERTS, NANCY A. ATOR, DOREEN M. GRECH2 and ROLAND R. GRIFFITHS
Behavioral Biology Research Center, Department of Psychiatry and Behavioral Sciences, Johns Hopkins University School of Medicine,
Baltimore, Maryland
Accepted for publication December 9, 1997
This paper is available online at http://www.jpet.org
Zolpidem is an imidazopyridine hypnotic, which exerts its
effects via the benzodiazepine (Bz or v, Langer and Arbilla,
1988) modulatory site on the GABAA receptor-chloride ionophore complex (Biggio et al., 1989; Lloyd and Zivkovic, 1988).
The distribution of zolpidem binding sites in rodent brain
parallels that of the classic Bz-Type 1-selective triazolopyridazine CL 218, 872 (Benavides et al., 1988, 1992; Langer and
Arbilla, 1988). Zolpidem preferentially bound GABAA receptor isoforms containing the alpha-1 subunit (combined with
beta-2, gamma-2) and showed lower affinity for receptor isoforms in which alpha-2, alpha-3 and alpha-5 subunits reReceived for publication July 23, 1997.
1
This research was supported in part by National Institute on Drug Abuse
Grant RO1 DA01147. Portions of these data were presented at the meeting of
the College on Problems of Drug Dependence, June 14 –19, 1997, Nashville,
TN.
2
Current address: Doreen M. Grech, Ph.D., Cato Research Ltd., 4364 S.
Alston Ave, Suite 200, Durham, NC 27713-2280.
The signs of flumazenil-precipitated withdrawal were summarized on a 9-point scale. Scores ranged from 1 to 5 in the 1.0
mg/kg condition, from 2 to 5 in the 3.2 and 10.0 mg/kg conditions and from 4 to 6 in the 32.0 mg/kg condition. Signs that
were considered intermediate in severity were observed. Specifically, tremor, jerk and/or rigidly braced posture was observed in one baboon at 1.0 mg/kg, two baboons at the next
two doses and all three baboons at 32.0 mg/kg. Vomit and/or
retch also occurred in two baboons at dose conditions above
1.0 mg/kg. Discontinuation of zolpidem dosing after 78 to 79
days resulted in mild withdrawal signs (e.g., number of pellets
obtained were lower and number of 1-min intervals increased in
which eyes were closed, or in which lying down, head lower
than torso posture and/or withdrawn posture were observed)
on the first day in two baboons. The peak withdrawal scores
were 4 or 5 on days 5 to 10; two baboons vomited and/or
retched and all three baboons showed tremor, jerk and/or
rigidly braced posture. Thus, zolpidem produced physical dependence under once-daily dosing conditions, and the severity
of the withdrawal syndrome can be characterized as intermediate.
placed alpha-1 (Pritchett et al., 1989; Pritchett and Seeburg,
1990). In contrast, classical Bz agonists bind less selectively
to GABAA isoforms containing alpha-1–alpha-5 subunits
(Pritchett et al., 1989; reviews in Möhler et al., 1992; Sanger
et al., 1994). It has been suggested that alpha-1-containing
GABAA receptors (e.g., combinations of alpha-1, beta-2, gamma-2) may be coextensive with those termed Bz1 (or v1)
(Benavides et al., 1992).
The pharmacological activity of zolpidem has been reported to differ from that of classical Bz agonists in several
preclinical tests in rodents. For example, zolpidem was less
effective than midazolam, triazolam and flunitrazepam in
producing myorelaxant effects and in protecting against seizures; but zolpidem was more potent in producing sedative
and hypnotic effects (Depoortere et al., 1986; Perrault et al.,
1990). Consummatory behaviors, which typically are increased by Bz agonists, were not altered reliably by zolpidem.
ABBREVIATIONS: Bz, benzodiazepine; GABA, g-aminobutyric acid; i.g., intragastric; p.o., per os.
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ABSTRACT
The current study examined behavioral effects and possible
development of physical dependence after once-daily doses of
zolpidem (0, 1.0, 3.2, 10.0, 32.0 mg/kg intragastrically [i.g.]) in
three baboons. Each dose was administered for 17 days and
then the dose was increased; the 32.0 mg/kg dose was administered for 27 days. Baboons had access to food pellets for 20
hr/day beginning 15 min after dosing. Each day, baboons were
presented with a fine motor task. Observation sessions were
conducted 1 hr after dosing on days 1, 10, 12 and 14 of each
dose condition and after termination of drug dosing. On days
10 and 14 of each dose condition, vehicle and flumazenil (5
mg/kg i.m.) were administered, respectively. Zolpidem increased the number of pellets obtained by two of three baboons. Vomit and/or retch and grimace (signs believed to be
indicative of abdominal discomfort) were observed in one or
two baboons during all zolpidem dose conditions (1.0 –32.0
mg/kg). Time to complete the fine motor task increased dosedependently in all three baboons, and incoordination was observed during the task in two baboons at 10.0 and 32.0 mg/kg.
Analysis of blood plasma showed that measurable levels of
zolpidem were present 24 hr after dosing in all drug conditions.
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Weerts et al.
dem after 2 weeks of i.v. administration in baboons produced
a decrease in the number of pellets delivered per day (Griffiths et al., 1992; Kaminski et al., 1993) and produced other
elements of a mild withdrawal syndrome (Kaminski et al.,
1993), which indicated that the baboons may have become
physically dependent on zolpidem. Mild to intermediate
withdrawal symptoms (including tremor, jerk, nausea and
abdominal pain) have been reported in patients treated for
insomnia with high doses of zolpidem (Cavallaro et al., 1993).
Thus, the effects of zolpidem appear to be more similar to
those of classic Bz agonists in nonhuman primates and humans than they are in the rat.
The present study was designed to further characterize the
behavioral effects and possible physical-dependence-producing effects of zolpidem in baboons. The effects of a range of
zolpidem doses (1.0 –32.0 mg/kg), delivered via the i.g. route,
were compared with a vehicle condition, using a once-daily
dosing regimen. The initial dose of zolpidem used in the
current experiments was the lowest dose to occasion druglever-appropriate responding ($80%) in most baboons in a
drug discrimination procedure (Griffiths et al., 1992). Specifically, three of five baboons trained to discriminate lorazepam (1.8 mg/kg p.o.) from the no-drug condition responded
on the lorazepam-appropriate lever after receiving 1.0 mg/kg
p.o. zolpidem. A dose of 3.2 mg/kg p.o. zolpidem occasioned
lorazepam-appropriate responding in four of five lorazepamtrained baboons. Thus, it was presumed that the lowest
zolpidem dose, 1.0 mg/kg i.g., chosen for the current experiment would be behaviorally active.
The dose was increased by a 0.5 log10 unit approximately
every 17 days. The Bz antagonist, flumazenil, was administered after 2 weeks of each dosing condition to determine
whether a precipitated withdrawal syndrome would occur. To
aid in evaluation of the dependence-producing effects of oncedaily dosing with zolpidem, blood plasma determinations of
zolpidem were made with blood samples obtained 24 hr after
dosing on days 15, 16 or 17 of each dose condition. After the
highest dose condition was completed, the nature and the
time course of spontaneous drug withdrawal was evaluated
after discontinuation of zolpidem dosing.
Methods
Subjects
Three adult male baboons (Papio cynocephalus, baboon GZ, and
Papio anubis, baboons DI and DS, obtained from Primate Imports,
New York, NY) served as subjects. All baboons had previous experience under operant conditioning procedures with food pellet reinforcement. Baboons GZ and DS had served in a study that investigated the physical dependence potential of i.g. triazolam and
zaleplon. Baboon GZ also had a 3-year history of i.v. self-administration of various nonsedative drugs (e.g., cocaine, imipramine). Baboon DI had a 9-year history of i.v. self-administration that included
sedative/anxiolytics (Griffiths et al., 1991). No experimental drug
conditions had been conducted with any of the baboons for 3 to 11
months before the current experiments and feeding generally had
been unrestricted. The baboons received one piece of fresh produce
and a multi-vitamin daily (generally at about 11:00 A.M.), and had
access to 1-g banana-flavored pellets (BIO-Serv, Frenchtown, NJ, for
baboons DS and DI, and P. J. Noyes, Lancaster, NH for baboon GZ)
for 20 hr each day (see below). Tap water was continuously available,
and the amount consumed was recorded daily. Body weights for the
three baboons ranged from 24.5 to 38.5 kg before the first experimental condition began.
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Zolpidem did not increase the intake of palatable food in
satiated rats (Yerbury and Cooper, 1989), standard food (i.e.,
rat chow) in food-restricted rats (Sanger and Zivkovic, 1988)
or the consumption of hypertonic saline solutions in rehydrating rats (Cooper and Desa, 1988). However, zolpidem did
increase consumption of palatable fluid in water-deprived
rats, and the increases were similar to those produced by
diazepam (Stanhope et al., 1993). The discriminative stimulus effects of zolpidem were distinguishable from those of
classic Bz agonists. Rats trained to discriminate chlordiazepoxide from saline did not generalize fully to zolpidem, even
when tested at doses that suppressed responding (Depoortere
et al., 1986; Sanger et al., 1987); all rats generalized to other
Bzs and to some non-Bz ligands. Furthermore, not all rats
trained to discriminate zolpidem from saline generalized to
chlordiazepoxide or to most other Bzs, even when tested at
doses that suppressed responding (Sanger et al., 1987;
Sanger and Zivkovic, 1986). Not all lorazepam-trained rats
showed full generalization to zolpidem, whereas diazepamand pentobarbital-trained rats did (Ator and Griffiths,
1992a). However, Rowlett and Woolverton (1997) found that
pentobarbital-trained rats did not generalize to zolpidem.
Rats trained to discriminate a low and a high dose of midazolam from the no-drug condition generalized to zolpidem by
making the low- but not the high-dose response (Sannerud
and Ator, 1995). Cross-tolerance between zolpidem and Bz
agonists apparently does not develop in mice and rats (Cohen
and Sanger, 1994; Cox et al., 1988; Sanger and Zivkovic,
1987). Although tolerance generally develops to the sedative
and ataxic effects of Bz agonists, there was only a small
degree of tolerance to the rate-decreasing effects of zolpidem
on operant behavior after repeated dosing (Sanger and Zivkovic, 1987, 1992). Similarly, there was no evidence of physical dependence as measured by a lack of convulsant activity
after flumazenil administration or discontinuation of chronic
zolpidem administration in mice and rats (Perrault et al.,
1992; VonVoightlander and Lewis, 1991). Taken together,
the behavioral data in rodents suggest a unique pharmacological profile for zolpidem when compared with classic Bzs.
In studies in nonhuman primates, Rowlett and Woolverton
(1997) found that rhesus monkeys trained to discriminate
pentobarbital from saline generalized to zolpidem. Griffiths
et al. (1992) found that four of five baboons trained to discriminate lorazepam from the no-drug condition and three of
three baboons trained to discriminate pentobarbital from the
no-drug condition generalized to zolpidem. Similarly, normal
human subjects trained to discriminate pentobarbital from
placebo also generalized to zolpidem (Rush et al., 1997). Zolpidem, triazolam and temazepam produced similar behavioral and subject-rated effects in normal human subjects in a
double-blind study (Rush and Griffiths, 1996). In other studies in human subjects, zolpidem and triazolam produced similar effects on most measures (e.g., behavioral performance
and subject-rated effects) (Evans et al., 1990; Mintzer et al.,
in press). However, there are differences between zolpidem
and triazolam in some performance measures in normal subjects (Mintzer et al., in press) and in subjective effects in
subjects with histories of drug abuse (Evans et al., 1990). In
baboons, behavioral signs of sedation and myorelaxation decreased with repeated administration of zolpidem, which
suggests the development of tolerance (Griffiths et al., 1992;
Kaminski et al., 1993). In addition, discontinuation of zolpi-
Vol. 285
1998
Intragastric catheters for baboons GZ and DS had been implanted
10 and 17 months, respectively, before the current experiments and
were still patent. The i.g. catheter for baboon DI was implanted 5
weeks before the current experiments. Procedures for the i.g. surgery
and description of the vest and tether system have been described
previously (Lukas et al., 1982). The catheter was implanted surgically into the stomach. The catheter exited in the midscapular region
of the back and was protected by a vest and tether system that
permitted the baboon free movement inside the cage. Baboons were
recovered fully from the surgery and showed normal dietary intake
before experimental conditions were initiated. Once the experimental dosing regimen began, the baboons were anesthetized with ketamine HCl (150 –300 mg) generally every 15 to 17 days (i.e., in the 3
days after the flumazenil challenge described below) to permit
weighing, medical examinations, exit site maintenance and cage
washing. Catheter blockage or equipment problems occasionally necessitated unplanned ketamine administration as noted below.
Apparatus
Procedures
Food reinforcement procedure. During all dosing conditions,
the baboons had access to an unlimited number of food pellets each
day by completing a fixed number of responses on the Lindsley
operandum for each pellet delivery (i.e., a fixed-ratio, FR, schedule of
reinforcement). The FR value was adjusted before beginning the
experimental conditions so that 1) the number of pellets delivered
per day was stable (i.e., no increasing or decreasing trends) for at
least 14 days, 2) virtually all pellets delivered per day were consumed (i.e., no more than two to three pellets were found in the pans)
and 3) the number of pellets delivered per day was sufficient to
maintain body weights in adult baboons of their size and activity
level. The pans under the baboons cages were inspected daily for
43
pellets to confirm that the pellets obtained were consumed. The final
FR value was 10 responses for each pellet delivery for all baboons.
Stable performance under an FR 10 schedule of food reinforcement
was established for at least a month before the vehicle base line (see
below) began. Pellet availability was correlated with illumination of
the jewel light above the Lindsley operandum. During i.g. dosing
conditions, pellet availability began 15 min after the i.g. injection
and continued for 20 hr; pellet access was terminated 4 hr before
drug dosing to facilitate drug absorption.
Once-a-day dosing conditions and flumazenil administration procedures. Injections were administered daily via the catheter at approximately 9:00 or 9:30 A.M. (615 min), depending on the
baboon. The successive dosing conditions were: vehicle, 1.0, 3.2, 10.0
and 32.0 mg/kg zolpidem. Each dose condition lasted 17 days, except
that the 32.0 mg/kg condition continued an additional 10 days after
the flumazenil challenge before drug dosing was terminated (as
described below). On days 10 and 14 of each dose condition, flumazenil vehicle and 5 mg/kg of flumazenil, respectively, were administered i.m. to assess whether a precipitated withdrawal syndrome
would occur. The high dose of flumazenil (5 mg/kg) was chosen
because 1) it has reliably precipitated withdrawal in baboons treated
with chronic high Bz doses (Ator and Griffiths, 1992b; Lamb and
Griffiths, 1984, 1985; Lukas and Griffiths, 1984) and 2) it facilitates
comparison of the present results with previous studies on Bz dependence in the baboon. Dosing with zolpidem continued for an
additional 3 days after flumazenil to permit restabilization and to
provide a window of opportunity for conducting the baboon weighing
and physical examination (as described above) before the next dosing
condition began. After 78 to 79 days of total dosing with zolpidem,
drug dosing was terminated.
Observations
Observations were conducted with use of a list of behavioral signs
and postures that had been established previously as reliable indicators of the Bz withdrawal syndrome, and provided a sensitive
assessment of activity levels, sedation and motor coordination (Ator
and Griffiths, 1992b; Lukas and Griffiths, 1982; Sannerud et al.,
1992). These behavioral signs and postures are defined in table 1.
The observer (EMW, DMG and research technician MFC) sat at a
small table with a laptop computer in front of each baboon’s cage.
The list of possible behavioral signs and postures that could be
scored were displayed on the computer screen. Frequency counts of
behavioral signs were collected in consecutive, 1-min intervals. A
computer-generated tone sounded at the beginning of each interval
prompting the observer to record each baboon’s posture. In addition,
observers could type comments at any time, which were saved in
1-min bins across the observation period; all observers were instructed to include in the comments whether vomiting, or just retching, occurred. They also noted if food-maintained responding, defecation and/or diarrhea occurred during each interval.
Before the experiment began, the behavioral definitions were
memorized by all observers and training observations were conducted by pairs of observers. During this period, the baboons were
habituated to the observers and observation procedures, and differences among observers in interpretations of the behavioral definitions and other aspects of recording behavior were resolved. The
criterion for agreement on occurrence and nonoccurrence of all behaviors and postures between all possible pairs of observers was 90%
or greater before experimental conditions were initiated. During the
experiment itself, independent observations were conducted by two
observers during randomly selected 30-min and 60-min observation
sessions; observations included all possible pairs of observers (i.e.,
EMW and DMG, DMG and MFC, MFC and EMW). The percent
reliability was calculated as follows: The occurrence or nonoccurrence of each behavior and posture recorded during each 1-min
interval of the observation session was counted for each observer.
The agreement between observers for the occurrence of each behavior and posture and the agreement between observers for the non-
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Baboons were housed individually in standard primate cages,
which also served as the experimental chambers, in the laboratory.
The tether was connected to a customized liquid swivel (IITC Life
Science, Woodland Hills, CA), which was attached to the top of the
cage. To maintain catheter patency, distilled water was infused
slowly (approximately 550 ml/24 hr) into the catheter using a peristaltic pump (model 1201, Harvard Apparatus, South Natick, MA).
Cages were equipped with a bench, which ran along one of the side
walls, a stainless steel water spout, which was attached to the front
of the cage, and an aluminum intelligence panel (45.7 3 63.5 cm),
which was mounted on the rear wall. A Lindsley operandum (Gerbrands Corp., Arlington, MA) was mounted in the lower left quadrant of the panel. A pull of the operandum sufficient to operate a
microswitch was required for the response. A green, 1.5-cm jewel
light was mounted above the operandum. A hopper, for delivery of
food pellets, was located in the center of the panel. The back wall of
the food hopper was a white Plexiglas panel (5 3 5 cm), which was
transilluminated during pellet delivery. The pellet feeder (BRS/LVE,
Inc., Laurel, MD or Gerbrands Corp., Arlington, MA), the water
bottle and the peristatic pump were on a grating above the cage.
Room ceiling lights were brightly illuminated for 13 hr/day (6:00 A.M.
to 7:00 P.M.) and dimly illuminated for the remaining 11 hr/day.
The number of pellets delivered per 30-min interval was collected
with IBM compatible personal computers with Med-PC software and
instrumentation (Med Associates, Inc., East Fairfield, VT). Behavioral observation data were collected using IBM compatible laptop
computers, programmed to provide a screen format and prompts for
entering each behavioral sign and posture; the raw data were saved
to diskettes. The raisin retrieval task used clear Plexiglas trays onto
which six 3-cm cups with 1-cm-high rims were mounted. The distance between the cups permitted each cup to be easily accessible
between the bars of the cage when the tray was placed against them
at the front of the baboon cage. The raisin retrieval task was timed
using a stopwatch.
Zolpidem Dependence
44
Weerts et al.
Vol. 285
TABLE 1
Definitions of behavioral signs and postures scored in observation sessions
Behavioral signs
Aggression
Ataxiaa
Bruxism
Eyes closed
Grooming
Jerka
Lip droop
Lip smack
Locomotion
Masturbation
Seizurea
Tremora
Vomit/retch
Wet dog shake
Yawn
Postures
Normal
Rigidly braced
Lying down
Head lower than torso
Withdrawn
Convulsing
a
Sitting upright; may be holding lightly onto bars; may have feet up.
Appears to brace itself by holding onto the cage bars while showing obviously enhanced muscle tension, faciculations,
and tremors.
Lying down on either the bench or cage floor, appears lethargic, and is unresponsive to normal verbal or physical
stimuli.
Standing on all four limbs with the head held below the waist (but not if level with or higher than the waist); may be
accompanied by vomit and/or retch, but can occur independently; may take the form of the back legs on the bench
and front legs on the cage floor; animal typically remains in posture for prolonged periods; not scored if also
reaching for objects or foraging in cage pan.
Sitting on either the floor or bench with chin on chest and unresponsive to normal stimuli.
Posture recorded during clonic or tonic-clonic seizure (see above).
cf. Adams and Victor (1993).
occurrence of each behavior and posture was divided by the total
number of intervals for that observation session. The overall interrater reliability for all behaviors and postures during the experiment
was greater than 97%.
All observation sessions were conducted after completion of the
raisin retrieval task (as described below) approximately 60 min after
the daily injection. The observers were not blind to the condition in
effect. On days 1 and 12 of each dosing condition, 30-min observation
sessions were conducted to assess the direct effects of zolpidem
during once-daily dosing. To determine whether a precipitated withdrawal syndrome would occur, observation sessions were conducted
immediately after i.m. administration of flumazenil vehicle and 5
mg/kg flumazenil, respectively, on days 10 and 14 of each dose
condition; these observations were extended to 60 min to encompass
the precipitated withdrawal syndrome. The first 30 min of the 60min observation on day 10 (when flumazenil vehicle was administered) were also included in the analysis of the direct effects of
zolpidem. Occasionally, the days of observations were shifted because of equipment problems or catheter blockage in baboon GZ.
[during the suspending agent vehicle condition, the flumazenil vehicle observation was on day 12; during the 10.0 mg/kg zolpidem
condition, observations were on days 11 (flumazenil vehicle), 13 and
15 (flumazenil)].
To assess whether a spontaneous withdrawal syndrome would
occur, a series of 30-min observation sessions was initiated 24 hr
after the last injection of 32.0 mg/kg zolpidem. Observations were
conducted daily for the first 15 days; on days 16 to 39, observations
were conducted every other day. Observations during drug withdrawal were conducted at approximately the same time of day as
previous observations. The variability in number of signs across days
and in whether certain behaviors are observed on certain days is to
some extent a function of the fact that the 30-min observation represents a relatively small sample of behavior that occurs each day.
Thus, the absence of a score for several behaviors should not be
interpreted to mean that the behavior was entirely absent, only that
it was not observed within the 30-min observation period.
Raisin Retrieval Task
The direct effects of zolpidem on fine motor coordination were
assessed daily with a raisin retrieval task; one raisin was placed in
each of the six equally spaced cups, and the tray was placed against
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Nose rub
Nose wipe
Scratch
Behavior directed at another animal or human consisting of barking, yawning with canines exposed, cage shaking,
raised eyebrows and staring.
Incoordination that is characterized by slow, uncertain movements (for example, when reaching, may reach for object
slowly and with jerking movement or may overshoot its mark) and tremor (action and postural); can also include
swaying or falling over during sitting and locomotion.
Gritting or grinding the teeth.
Eyelids closed over the eyeballs for at least 2 sec, as in sleeping.
Rubbing hair in search of objects (parasites, dirt) and removal of found objects.
Muscle contraction and/or relaxation leading to a quick jerking movement or twitch; jerk may be rhythmic and
repetitive or shock-like (e.g., clonic or myoclonic jerks); can be independent of seizure.
Loss of facial muscle tone evidenced by a characteristic droop of the lower lip.
Rapid movement of the tongue and lips resulting in a smacking sound that usually is directed at either another
animal or a human.
Gross movement from one location to another with use of the limbs.
Either manipulation of the flaccid penis until it is erect or manipulation of the erect penis, usually characterized by a
stroking movement.
Substantial displacement of the nasus externus from midline by either a limb or cage bars.
Quick, wiping movement across the nasus externus with a limb.
Rubbing or scraping slightly, as with the fingernails. Note: changes from one spot on the body to another defines the
beginning of a new scratching bout.
Consists of several components: 1) “Tonic spasm” that is characterized by contraction of muscle groups; usually takes
the form of extension of first, the back and neck and second, the arms and legs. Breathing may be suspended. 2)
“Clonic” jerks that are characterized by a mild, repetitive tremor with progression to brief, violent, rhythmic flexor
spasms that involve the entire body. After tonic and clonic phases, animal may lie still for about 5 min.
A more or less regular, rhythmic movement of the limbs, head, or trunk produced by synchronous contractions of
antagonistic muscles. Rhythmic quality distinguishes it from other involuntary movements. Its biphasic character
distinguishes it from clonic jerks. It frequently occurs during periods of demand on the muscles (action and
postural tremors) such as when the animal is reaching for an object.
Making an effort to vomit (often accompanied by a “gagging” sound) or disgorging the stomach contents through the
mouth; may not expel the vomitus; in such cases, the presence of vomit may often be assumed if the animal
subsequently engages in chewing and swallowing.
Rapid, side-to-side movement of the head and upper body, resembling the action of a wet dog shaking the water off of
its fur.
Opening the mouth wide with a prolonged, deep inhalation of air.
1998
the front of the cage. The tray was presented by a research technician (SRW or CDE) for 120 sec or until all six raisins were retrieved,
whichever occurred first. Technicians recorded the time (seconds) to
retrieve all six raisins, or if less than six raisins were retrieved, the
maximum time was recorded. Research technicians also completed a
checklist indicating the occurrence or non-occurrence of tremor and
incoordination (as defined in table 1), the number of raisins retrieved, the number of raisins dropped and specific comments about
behaviors observed during the task (e.g., “severe limb tremors” and
“fell off bench while doing task”). Before beginning experimental
conditions, all baboons were given repeated trials on the task, and
generally retrieved all six raisins in less than 15 sec. The task was
presented 1 hr after dosing each day; on days when observations
occurred (e.g., days 1, 10, 12 and 14), the task was conducted immediately before the observation session. On days when flumazenil or
its vehicle was administered, the raisin retrieval task was repeated
immediately after the 60-min observations (i.e., 2 hr after dosing
with vehicle and zolpidem) by the person that conducted the observation.
On days 15, 16 or 17 of each dosing condition, blood samples were
obtained when baboons were anesthetized with ketamine as described above. All blood was drawn at about 9:00 A.M., approximately
24 hr after the injection; the dose for that day was given after blood
was drawn and when the baboon was awake from the anesthesia.
Blood samples (approximately 10 ml) were injected immediately into
a lithium-heparinized tube and centrifuged at 3200 rpm for 12 min.
The plasma then was withdrawn and frozen in a polypropylene tube
until analysis at Pharmacia and Upjohn, Inc. (Kalamazoo, MI).
Plasma zolpidem levels were assayed according to the method of
Salva and Costa (1995) with some modifications. Plasma was thawed
and centrifuged to a clear particulate matter. A 2-ml sample of
plasma was transferred to a conical tube; 5 ng of an internal standard and 20 ml of 10 N sodium hydroxide were added. The internal
standard was a zolpidem analog in which the dimethyl amide functionality was replaced by dimethyl piperazine amide. A 4-ml sample
of ethyl acetate was added and the mixture was vortex-mixed. After
the phases separated upon standing, the organic layer was removed
and transferred to another conical tube. Another 4 ml of ethyl acetate
was added and the mixture was vortex-mixed again. After the phases
separated upon standing, the organic layer was removed and the two
organic layers were combined and dried with a stream of nitrogen. A
150-ml sample of mobile phase was added to the dried material and
vortex-mixed. The solution was transferred to a 1.5-ml Eppendorf
centrifuge tube and spun at 14,000 rpm for 5 min. A 50-ml sample of
the clear supernatant was injected onto an high-performance liquid
chromatograph Water’s Symmetry C18 column. The high-performance liquid chromatography flow rate was 0.5 ml/min. A Water’s
470 fluorometer detector was used to detect zolpidem and the internal standard at 254 l ex and 390 l em. Water’s Maxima software was
used to integrate peaks that co-eluted with zolpidem and the internal
standards. Plasma concentrations of zolpidem were calculated knowing the amount of internal standard added to plasma (5 ng), the
integrated areas of relevant peaks and the relative response factors
determined for equal amounts of zolpidem and internal standard
added to and extracted from plasma from baboons treated with
vehicle.
Drugs
The present study used zolpidem tartrate, and then zolpidem base
(Research Biochemicals International, Natick, MA). The amount of
zolpidem base weighed for each dose was adjusted by use of the 1.24
salt/base ratio (Durand et al., 1992) so that the amount of base used
(0.807, 2.58, 8.07, 25.8 mg/kg, respectively) would be equivalent to
that in the 1.0, 3.2, 10.0, 32.0 mg/kg doses that were calculated based
on the salt. Zolpidem base was used for baboon DI at doses of 1.0, 3.2,
45
10.0 mg/kg; for baboon DS at 3.2 and 10.0 mg/kg; and for baboon GZ
at 32.0 mg/kg. All other doses were zolpidem tartrate. The weight of
the baboon at the end of each condition was used to calculate the dose
for the next condition. Zolpidem stock solutions were mixed with
BIO-Serv Agent K (Frenchtown, NJ), 2 g/l blended in distilled water,
with a blender. The stock solution was kept in a brown glass bottle
under refrigeration for up to 3 days. After mixing on a magnetic
stirrer, an appropriate quantity of the stock was drawn up for each
baboon’s dose each day. Higher injection volumes were used at
higher doses because of problems with catheter blockage after drug
injections. The injection volumes were as follows: 10 ml for 1.0 and
3.2 mg/kg, 50 ml for 10 mg/kg and 200 to 500 ml (depending on the
weight of the baboon) for 32 mg/kg zolpidem. In the 32 mg/kg condition, the dose for each individual baboon was mixed in a electric
blender immediately before administration. Injections were followed
by a 10- to 20-ml flush of the BIO-Serv suspending agent suspension.
Flumazenil (5 mg/kg, Hoffmann-LaRoche, Basel, Switzerland) was
prepared in a vehicle of propylene glycol/ethanol (95%)/sterile water
(40:10:50 v/v). The mixture was injected i.m. at a volume of 2 ml
immediately after mixing.
Data Analysis
A single-subject design was used in which each baboon served as
his own control. The direct effects of dosing with zolpidem on pellets
per day and on all behaviors recorded during the 30-min observational sessions on days 1 and 12 and in the first 30 min of the 60-min
observation on day 10 (when flumazenil vehicle was administered)
were judged to be significant based vehicle control z-scores. Control
observations were conducted before and after drug dosing; these
included days 1, 10 and 12 during the original vehicle base line and
the last 3 days of postwithdrawal vehicle observations (days 31, 33,
35, 37 or 39, depending on the baboon). For behaviors that were
predicted to increase (e.g., lip droop, ataxia, withdrawn chin on chest
posture, lying down, eyes closed), significance was determined if the
observed frequency of that behavior or posture exceeded the vehicle
control z-score, above which 5% of the scores would be expected to fall
for a one-tailed test. For all other behaviors that were predicted to
increase or decrease, the z-scores delineated the top 2.5% and the
bottom 2.5% for a two-tailed test.
A constellation of behaviors considered to be indicative of a Bztype withdrawal syndrome in the baboon was used to derive a “withdrawal score” for both flumazenil-precipitated withdrawal and spontaneous withdrawal. In deriving the withdrawal score, 1 point was
assigned in each of nine categories of behaviors or postures; the
maximum possible score was 9. The nine categories of behavioral and
postural differences used to determine the withdrawal score were as
follows: 1) pellets per day decreased; 2) the time to complete the
second raisin retrieval task increased; 3) the number of 1-min intervals increased in which eyes were closed or in which lying down,
head-lower-than-torso posture and/or a withdrawn chin-on-chest
posture were observed; 4) locomotion increased or decreased; 5) selfdirected behaviors (nose rub, nose wipe, scratch and wet dog shake)
increased; 6) aggressive threat, bruxism or yawn increased; 7)
tremor (limb/body), jerk and/or rigidly braced posture increased; 8)
vomit and/or retch increased; 9) seizures increased. It is important to
note that this is a nonweighted scale; a higher score does not necessarily denote a more severe withdrawal.
Behaviors during the 60-min flumazenil-challenge observations
were judged to be significantly increased if they were higher than the
frequencies recorded during the following three observation sessions:
1) the flumazenil vehicle during the original suspending agent vehicle condition, 2) flumazenil during the original suspending agent
vehicle condition and 3) flumazenil vehicle during the same dosing
condition. These three control observations accounted for the effects
of the suspending agent vehicle, of the flumazenil alone and of
chronic drug on the behavior.
The incidence of spontaneous withdrawal behaviors scored during
a 30-min observation session after drug dosing ended was judged as
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Blood Plasma Drug Level Determinations
Zolpidem Dependence
46
Weerts et al.
Vol. 285
Results
The Effects of Zolpidem During Once-a-Day Dosing
Observations. All three baboons showed increases in one
or more behavioral signs associated with sedation during
zolpidem dosing, particularly at the higher doses (10.0, 32.0
mg/kg). Among the specific behaviors listed that could be
scored, the sedative/myorelaxant effects of a drug would be
detected by increases in ataxia, eyes closed, lip droop, decreases in locomotion and increases in the frequency of resting postures (e.g., lying down and/or the withdrawn chin-onchest posture). Locomotion was not decreased in any baboon
compared with vehicle. Lip droop and ataxia were not observed during vehicle control conditions. Lip droop was observed in two baboons at 3.2 and 10.0 mg/kg, and in all three
baboons at 32.0 mg/kg zolpidem. Ataxia was observed in two
baboons at the two lower zolpidem doses (1.0, 3.2 mg/kg), and
more frequently in all three baboons at the two higher doses
(10.0, 32.0 mg/kg). During observation sessions, ataxia was
typically recorded during operation of the food lever and
during position changes. Severe ataxia was noted during the
raisin retrieval task in two baboons; comments written by the
observers indicated that baboon GZ was “falling off of bench”
during raisin retrieval task on day 22 of 32.0 mg/kg zolpidem,
and that baboon DI “fell off the bench twice” while doing the
raisin retrieval task on day 11 of 10.0 mg/kg zolpidem. Baboon DS showed less ataxia, but showed an increased incidence of resting postures (i.e., lying down and/or withdrawn
chin-on-chest posture) relative to vehicle.
In addition to the behavioral signs of sedation recorded
during the observation sessions, chronic zolpidem produced
some signs that may be associated with abdominal discomfort (i.e., head-lower-than-torso posture, vomit and/or retch
and grimace) in two baboons. Vomit and/or retch and grimace
were not observed during the initial vehicle base-line observations or during the postwithdrawal observations (i.e., baseline levels were recovered). During observation sessions, baboon DI showed grimacing and/or retching at 1.0, 3.2 and
32.0 mg/kg zolpidem, and baboon DS vomited at 32.0 mg/kg
zolpidem. Grimacing and retching typically were accompanied by head-lower-than-torso posture in baboon DI. Vomiting was observed outside of the observation period in baboon
DS; DS vomited on day 16 of 3.2 mg/kg zolpidem, and on days
9, 21 and 22 of 32.0 mg/kg zolpidem.
The frequency of locomotion, aggression, bruxism, lip
smacks, nose wipes, nose rubs, wet-dog shakes, grooming,
tremor, jerk and yawns were not systematically altered in
the three baboons by zolpidem administration. Rigidly
braced posture or convulsing were never observed during
zolpidem dosing.
Raisin retrieval task. Figure 1 shows the effects of zolpidem on the time (seconds) to complete the raisin retrieval
task for the first 14 days of each dose. The task was conducted approximately 1 hr after dosing with vehicle or zolpidem. During the vehicle base line, the time to complete the
task (i.e., retrieve all six raisins) was relatively stable for
each baboon. The time to complete the raisin retrieval task
increased in a dose-dependent manner. As dose increased,
the time to complete the task also became more variable for
each baboon. Time to complete the task clearly increased
above vehicle in all three baboons at the 10.0 and 32.0 mg/kg
doses.
In addition to taking longer to complete the raisin retrieval
task, baboons showed incoordination and tremors during the
task (data not shown). Incoordination was variable across
days and dosing conditions, but tended to increase dosedependently in two of three baboons. At the lowest zolpidem
dose (1.0 mg/kg), incoordination was observed for 7, 2 and 0
of 16 total test days in GZ, DS and DI, respectively. Incoordination was observed during the 10.0 mg/kg condition for 11
of 14 total test days in baboon GZ (The task was not conducted on days 2, 3 and 9 because baboon GZ had to be
anesthetized to unblock the catheter.) and for 11 and 2 of 16
total test days in baboons DS and DI, respectively. During
the 32.0 mg/kg condition, incoordination was observed for 23
of 23 total test days in baboon GZ (Days 8 and 9 were lost
because of the need to unblock the catheter) and 3 and 19 of
25 total test days in baboons DS and DI, respectively. No
instances of tremor or incoordination were observed during
the initial vehicle condition. Tremor occasionally was observed during dosing: Baboon DI showed tremor at 10.0 and
32.0 mg/kg zolpidem, and baboon DS showed tremor on days
5 and 11 at 32.0 mg/kg.
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significantly increased or decreased as follows: For behaviors that
were predicted to increase during withdrawal, points were assigned
only if a) the observed frequency of that behavior or posture exceeded
the vehicle control z-score, above which 5% of the scores would be
expected to fall for a one-tailed test. Because locomotion was predicted to increase or decrease during withdrawal (increased activity
has been reported during withdrawal from Bzs; Rickels et al., 1990),
the z-scores for this behavior delineated the top 2.5% and the bottom
2.5% for a two-tailed test. The vehicle control z-scores were based on
the frequencies recorded during a total of six observation sessions;
these included the three observation sessions (i.e., days 1, 10, 12)
during original suspending agent vehicle base line, and the last 3
days of observations during the postwithdrawal period (i.e., days 31,
33, 35, 37 or 39, depending on the baboon). An additional criterion for
inclusion in the withdrawal score was that b) the observed frequency
of behavior also had to exceed the frequencies obtained during the
three observations during the 32.0 mg/kg condition which preceded
termination of zolpidem dosing. This criterion accounted for both
time and/or drug-produced changes in the behaviors.
Yanagita and Takahashi (1970) used the presence of or change in
specific behaviors to characterize the severity of withdrawal as
“mild,” “intermediate” or “severe” in barbiturate-dependent rhesus
monkeys, and later to grade withdrawal from different Bzs including
chlordiazepoxide, diazepam and triazolam (reviews in Woods et al.,
1987). To allow comparison across studies, classifications of withdrawal as mild, intermediate and severe, which were based on those
used by Yanagita and colleagues, also were used in the present
study. Specifically, behavioral signs that would result in a mild
classification included aggression, mild tremor and reduction in pellets delivered per day in the current study. Additional behaviors that
have been associated with mild Bz withdrawal in the baboon include
increased self-directed behaviors (e.g., nose rubbing, nose wiping,
scratching), withdrawn posture (i.e., sitting with chin on chest) and
postures that may be associated with abdominal discomfort and
nausea (e.g., head-lower-than-torso posture). Signs that resulted in
an intermediate classification included aggravated tremor, rigidly
braced postures (i.e., muscle rigidity), impaired motor activities and
vomit and/or retch. Signs that resulted in a severe classification were
the occurrence of seizures and convulsions. Thus, withdrawal would
be characterized as intermediate if vomit, retch, tremors, jerks
and/or rigidly braced posture was observed and severe if seizure
activity was observed regardless of the total number of signs observed.
1998
Food-maintained behavior. Figure 2 shows the number
of pellets delivered per day during the first 14 days of each
dosing condition. Evaluation of the 30-min bins of food-maintained responding showed that during vehicle conditions,
pellets were consumed during multiple feeding bouts
throughout the day; the total pellets per day ranged between
108 and 277. During zolpidem dosing, the number of pellets
per day gradually increased in a dose-dependent manner in
two (GZ and DS) of the three baboons; by the end of the 32.0
mg/kg dosing condition, body weights had increased from
31.3 to 32.7 kg in baboon GZ and from 38.4 to 41.8 kg in
baboon DS. The pellet data in 30-min bins indicated that
baboons GZ and DS began responding immediately after
pellets became available and that most of the pellets per day
were obtained in the first 2 to 4 hr of the 20-hr period of pellet
47
Fig. 2. The number of pellets delivered per day during once-daily dosing
with zolpidem in baboons GZ, DS and DI. Pellets were available under an
FR10 schedule for 20 hr beginning 15 min after dosing. Data shown are
for the first 14 days of each dose condition. On days 10 and 14 of each dose
condition, baboons received i.m. injections of flumazenil vehicle and 5
mg/kg flumazenil, respectively, approximately 1 hr after the dose of
zolpidem. Days on which GZ received ketamine to unblock the catheter
have been omitted and adjacent points are not connected. The shaded
area in each panel represents the range for the vehicle control base line
for each baboon.
availability. For baboon DI, there were relatively small
changes in the number and distribution of pellets obtained
during the 20-hr period of pellet availability during zolpidem
dosing conditions; at low doses (1.0, 3.2 mg/kg) pellets were
slightly increased above vehicle on some days and body
weight was increased by 0.5 kg. However, when compared
with the 3.2 mg/kg condition, pellet intake during the 10.0
and 32.0 mg/kg conditions returned to lower levels (but generally within the vehicle base-line range); weight for baboon
DI decreased from 24.5 kg in the 3.2 mg/kg condition to 23.4
kg by the end of the 32.0 mg/kg condition. Daily inspection of
the pans and food hoppers indicated that virtually all pellets
delivered were consumed (i.e., no more than two to three
pellets were found in the pan under the cage).
Flumazenil-Precipitated Withdrawal
The effects of the Bz antagonist flumazenil on behaviors
relevant to a Bz withdrawal syndrome are summarized with
withdrawal scores (fig. 3). Flumazenil administration after 2
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Fig. 1. Time taken to complete the fine motor task during once-daily
dosing with zolpidem in baboons GZ, DS and DI across consecutive days.
Data shown are the time (seconds) to retrieve six raisins from a tray
presented at the front of the cage on days 1 to 14 of each dose condition.
The maximum time allowed for retrieving all six raisins was 120 sec.
These data are from the task that was conducted approximately 1 hr after
bolus dosing. Days on which baboon GZ received ketamine to unblock the
catheter have been omitted (the raisin retrieval task was not conducted)
and adjacent points are not connected. The shaded area in each panel
represents the range for the vehicle control base line for each baboon.
Zolpidem Dependence
48
Weerts et al.
weeks of daily administration of the lowest dose (1.0 mg/kg)
of zolpidem increased one or more signs when compared with
control observations (see “Data Analysis”); withdrawal scores
ranged from 1 to 5. In the 3.2 and 10.0 mg/kg conditions, peak
withdrawal scores ranged from 2 to 5. The highest withdrawal scores were at the 32.0 mg/kg condition; peak withdrawal scores were 6, 6 and 4 for baboons GZ, DS and DI,
respectively.
Flumazenil did not reduce the number of pellets delivered
for that day below the range of pellets for the control conditions (see also last day shown in each condition in fig. 2). The
raisin retrieval task that was presented 1 hr after flumazenil
administration was refused by baboon DS at doses of 3.2
mg/kg zolpidem and higher, but there were no effects in this
task after flumazenil for the other two baboons. The number
of baboons showing increases in the number of 1-min intervals spent with eyes closed, or in certain postures (i.e. lying
down, withdrawn with chin on chest posture, and/or in the
head-lower-than-torso posture, which is often associated
with nausea and vomiting) were dose-dependently increased:
one of three baboons showed an increase in these postures at
1.0 mg/kg zolpidem, as did two of three baboons at 3.2 and
three of three at 10.0 and 32.0 mg/kg. Aggressive behaviors
(i.e., aggressive threats, yawning and bruxism) increased in
baboon GZ in all dose conditions, and in all three baboons at
the highest zolpidem dose condition (32.0 mg/kg). All three
baboons showed changes in locomotion, which were unsystematically related to dose. Two baboons (DS, DI) showed
higher self-directed behaviors (i.e., nose rubbing, nose wiping, scratching and wet dog shakes) after flumazenil in all
zolpidem dose conditions. The third baboon (GZ) showed
increased self-directed behaviors at 1.0 and 32.0 mg/kg zolpidem, but not at other doses. Baboons GZ and DI showed
increased tremor, jerk and/or rigidly braced posture after
flumazenil in three of the four zolpidem dose conditions, but
baboon DS did so only at the highest dose (32.0 mg/kg).
Seizures were not observed in any baboon. Vomit and/or
retch was observed after flumazenil in baboon GZ in the 3.2
and 32.0 mg/kg zolpidem conditions and in baboon DS at 32.0
mg/kg. Although not included in the withdrawal score, observers recorded defecation in written comments during the
observation sessions: all three baboons showed increased
defecation within 5 to 15 min after flumazenil administration
at 32.0 mg/kg zolpidem, and diarrhea was observed in baboons GZ and DS.
In summary, intermediate withdrawal signs (i.e., tremor,
jerk and/or rigidly braced posture) were observed in only 1
baboon after flumazenil at the 1.0 mg/kg zolpidem condition.
The other two baboons showed mild signs of withdrawal (e.g.,
increased self-directed behaviors). At the 3.2 mg/kg zolpidem
condition, intermediate withdrawal signs were observed in
two of three baboons; tremor, jerk and/or rigidly braced posture was increased for both baboons, and vomit and/or retch
was also increased in the baboon that had showed tremor
and/or jerk at the previous dose condition. At 10.0 mg/kg
zolpidem, mild to intermediate withdrawal signs were also
observed; one baboon each showed increased self-directed
behavior, aggression and tremor, jerk and/or rigidly braced
posture. At 32.0 mg/kg zolpidem, intermediate withdrawal
signs were observed in all three baboons; vomit and/or retch
was increased in two baboons, and tremor, jerk and/or rigidly
braced posture was increased in all three baboons. Thus, the
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Fig. 3. Withdrawal scores after administration of flumazenil (5 mg/kg
i.m.) during each zolpidem dosing condition in baboons GZ, DS and DI.
Sixty-minute observation sessions were conducted after injection of vehicle and flumazenil. The maximum possible score was 9: 1 point was
assigned for each of seven categories of behavior recorded during the
observation session; 1 point for increased duration or refusal of the raisin
retrieval task conducted at the end of the observational session (i.e., 1 hr
after flumazenil vehicle or flumazenil administration and 2 hr after
dosing); and 1 point was assigned if the number of pellets delivered that
day was decreased. Points were assigned if the particular measure was
decreased (pellets, locomotion only) or increased (all other behaviors)
when compared with all the control observations (i.e., the suspending
agent vehicle base line, the flumazenil during the suspending agent
vehicle condition and the flumazenil vehicle during each drug dose condition).
Vol. 285
1998
Zolpidem Dependence
49
number of signs and the severity of the signs observed during
flumazenil-precipitated withdrawal tended to increase across
the zolpidem dosing conditions.
Spontaneous Withdrawal
Levels of Drug in Blood Plasma
Figure 6 presents plasma levels for zolpidem 24 hr after
dosing with each dose of zolpidem. Zolpidem was detected 24
hr after drug administration in all three baboons at even the
lowest doses. Zolpidem levels in plasma generally increased
dose-dependently. Plasma levels ranged from 0.66 to 2.87
ng/ml at 1.0 mg/kg zolpidem and from 1.36 to 4.09 ng/ml at
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Dosing with zolpidem ended after the 32.0 mg/kg dosing
condition. At the time drug was discontinued, the total number of days of dosing with zolpidem (1.0 –32.0 mg/kg) was 78
days for baboons DS and DI and 79 days for baboon GZ. The
effects of termination of chronic zolpidem dosing on signs
during observation sessions, food-maintained behavior, and
time to complete the raisin retrieval task are summarized by
withdrawal scores. Figure 4 shows the spontaneous withdrawal scores across the first 15 days after zolpidem dosing
ended. Baboons GZ and DS had scores of 2 on the first day,
but baboon DI first showed withdrawal signs on day 2 (score
of 1). The peak scores ranged from 4 to 5 across the three
baboons, and the peak score first occurred by 5 to 10 days
after dosing ended. By the 15th day of this condition, scores
ranged from 3 to 4.
Withdrawal from zolpidem did not disrupt performance of
the raisin retrieval task. In fact, the duration to complete the
task rapidly returned to vehicle control levels in all three
baboons after the discontinuation of zolpidem dosing. In addition, baboons generally completed the task without signs of
tremor or incoordination. During the observation sessions,
there was an increased number of 1-min intervals with postural changes (i.e., lying down, head-lower-than-torso posture, withdrawn chin-on-chest posture) and/or eyes closed.
Locomotion was relatively unaffected, but all baboons
showed increased frequency of self-directed behaviors (i.e.,
nose wipe, nose rub, scratch and/or wet-dog shakes), and
aggressive behaviors (i.e., aggressive threats, yawns, bruxism). The number of times these contributed to the withdrawal score varied across baboons, and did not show a
pattern across days. All three baboons showed tremor, jerk
and/or rigidly braced posture; but for baboons GZ and DS, it
was on 5 and 7, respectively of the first 15 days and for DI, it
was only on 4 of those days. Vomit and/or retch was scored on
2 or 3 of the first 10 days of withdrawal for baboons DS and
DI. Seizures were not observed in any of the baboons.
As also shown in figure 5, pellets/day decreased below
base-line vehicle control levels for at least the first 15 days in
two baboons (GZ, DS), and then increased toward the vehicle
control levels. Weight loss was also evident after termination
of zolpidem dosing in baboon GZ (from 32.7 to 30.5 kg), and
DS (from 41.8 to 39.2 kg) when compared with weights during the 32.0 mg/kg zolpidem condition that preceded the
withdrawal condition; these body weights were similar to
those obtained during the initial vehicle base line. The number of pellets delivered per day and body weight for baboon
DI after termination of zolpidem dosing were similar to those
obtained during vehicle control and during the 32.0 mg/kg
zolpidem dosing conditions.
Fig. 4. Daily withdrawal scores for the first 15 days after discontinuation of zolpidem dosing in baboons GZ, DS and DI. Thirty-minute observations were conducted at the same time each day. Points were assigned
if the particular measure exceeded the z-score that was calculated from
the nine control observations (see “Data Analysis”). The raisin retrieval
task used for this evaluation was conducted each day, 1 hr after dosing
with the suspending agent vehicle or zolpidem. Other details are the
same as for figure 3.
3.2 mg/kg zolpidem. Plasma levels were increased substantially in two baboons (DS and DI, 19.4 and 17.4 ng/ml, respectively), and were slightly increased in the third baboon
(GZ, 4.23 ng/ml) at 10.0 mg/kg zolpidem. At 32.0 mg/kg
zolpidem, plasma levels were higher for baboon GZ (11.2
ng/ml), similar to the 10.0 mg/kg dose for baboon DS (19.6
ng/ml) and reduced in baboon DI (3.41 ng/ml). Chromatograms showing the relative peak areas for each dose indicated a lack of proportionality for zolpidem for baboon DI for
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Weerts et al.
Vol. 285
Fig. 6. Plasma levels of zolpidem 24 hr after once-daily dosing. Blood
samples were collected on day 15, 16 or 17 of each dose condition. Each
data point represents a single determination in baboons GZ (open
square), DS (open circle) and DI (open triangle). The line represents the
group mean.
the 10.0 and 32.0 mg/kg doses. Comparison with chromatograms of the control plasma extracts indicated, however, that
there was proportionality for a major metabolite of zolpidem,
which suggests that the lower plasma concentration of zolpidem at 32.0 mg/kg may have been due to more rapid metabolism of zolpidem by baboon DI during this dose condition.
Discussion
The current results demonstrated that zolpidem produced
behavioral signs of sedation, myorelaxation and motor impairment under once-daily dosing conditions in baboons. Zolpidem has produced motor impairment in rodents (Sanger et
al., 1987, 1996) and humans (Dingemanse et al., 1995). Signs
of sedation, including ataxia and/or decreased arousal, have
been observed in baboons after the repeated administration
and self-injection of zolpidem (Griffiths et al., 1992; Kaminski
et al., 1993) and of high doses of Bz agonists (e.g., chlordiazepoxide, diazepam, lorazepam, midazolam and triazolam)
(Ator et al., 1996; Griffiths et al., 1981, 1991; Sannerud et al.,
1989). Thus, the sedative effects of zolpidem in the current
study were similar to the sedative effects of Bz agonists and
were consistent with the sedative-hypnotic profile of zolpidem in rodents (Depoortere et al., 1986; Sanger et al., 1987;
Sanger and Zivkovic, 1988) and humans (Balkin et al., 1992;
Langtry and Benfield, 1990).
Benzodiazepine agonists typically increase consummatory
behaviors in rats and nonhuman primates, including the
baboon (Cooper and Moores, 1985; Foltin et al., 1989; Randall
et al., 1960; Weerts et al., 1993). In the current study, zolpidem increased food-maintained behavior; and this increase
was progressive in two of the three baboons across the weeks
in which the i.g. dose was increased every 17 days. Because
virtually all pellets delivered were consumed by the baboons,
this increase also represents an increase in food intake. Similar effects on food intake were observed with the triazolobenzodiazepine hypnotic triazolam (Ator et al., 1996) and the
pyrazolopyrimidine zaleplon in baboons with the same oncedaily dosing procedure (Ator NA, Weerts EM, Kaminski BJ,
Kautz MA and Griffiths RR, unpublished observations). The
finding that zolpidem increased food-maintained behavior
and food intake in the current experiment is consistent with
previous studies in baboons in which zolpidem was delivered
via the i.v. route (Griffiths et al., 1992; Kaminski et al., 1993),
but contrasts with studies in rats that showed that zolpidem
did not alter consummatory behaviors (Cooper and Desa,
1988; Sanger and Zivkovic, 1988; Yerbury and Cooper, 1989;
cf., Stanhope et al., 1993, who found increases in drinking
palatable fluids in water-deprived rats).
Although zolpidem increased food-maintained behavior in
baboons, zolpidem also produced signs suggestive of abdominal discomfort, including vomiting, in two of three baboons.
These signs were not observed during vehicle control conditions and have not been observed during dosing with other
sedative-hypnotics (e.g., triazolam) using this dosing procedure in baboons (Ator et al., 1996). Evans et al. (1990) reported that zolpidem (15.0, 30.0 and 45.0 mg) (but not triazolam, 0.25, 0.5, 0.75 mg) increased human subject ratings of
“queasy/sick to stomach,” and also produced emesis (5 of 15
subjects vomited after the highest zolpidem dose, and 4 of 15
subjects vomited after the two lower doses.) In a post-marketing survey in insomniac patients (n 5 1972) treated with
zolpidem (Ganzoni et al., 1995), there were 33 reports (1.7%)
of gastrointestinal distress that included nausea, vomiting,
abdominal pain and/or diarrhea.
There was little evidence of tolerance to the behavioral
effects of zolpidem during once-daily dosing. In fact, the
hyperphagic effects and motor deficits produced by zolpidem
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Fig. 5. The number of pellets delivered per day after discontinuation of zolpidem dosing in baboons GZ, DS and DI. Data shown are consecutive days
after end of dosing with zolpidem (open circles). Pellets were available under an FR10 schedule for 20 hr beginning 15 min after dosing with vehicle.
Days on which ketamine was administered have been omitted, and adjacent points are not connected. The shaded area in each panel represents the
range for the original vehicle control base line for each baboon. Filled circles are the mean pellets delivered per day for each baboon during the 32.0
mg/kg zolpidem condition that preceded discontinuation of zolpidem dosing; vertical lines through the means represent the range.
1998
51
characterized in the baboon (Ator and Griffiths, 1992b; Lukas and Griffiths, 1982; Lamb and Griffiths, 1984; Sannerud
et al., 1989). The current study graded withdrawal as mild,
intermediate or severe based on the characterization of barbiturate and Bz withdrawal in rhesus monkeys by Yanagita
and colleagues (Yanagita and Takahashi, 1970, Woods et al.,
1987, see “Methods”). Administration of the Bz antagonist
flumazenil during once-daily dosing with zolpidem precipitated a mild-to-intermediate withdrawal syndrome (as evidenced by increased vomit and/or retch, tremor, jerks and/or
rigidly braced posture), which peaked at the highest dose of
zolpidem (32.0 mg/kg). As few as 2 weeks of zolpidem administration were sufficient to produce some signs of physical
dependence, and the number of signs observed and apparent
severity of physical dependence increased in a dose- and
time-related manner. Severe withdrawal signs (e.g., seizures)
were not observed in any baboon. The current findings are
consistent with those of Schoch and colleagues (as reviewed
in Stephens and Sanger, 1996) in which the partial inverse
agonist sarmazenil precipitated a withdrawal syndrome in
squirrel monkeys treated with high doses of zolpidem. The
mild-to-intermediate precipitated withdrawal syndrome was
similar to that observed in studies with other Bz agonists
given p.o. or i.g. in baboons and in which agonist dose and/or
duration of dosing increased (Ator and Griffiths, 1992b; Lukas and Griffiths, 1984; Sannerud et al., 1989).
A mild-to-intermediate withdrawal syndrome also was observed in all baboons when chronic zolpidem dosing was
discontinued abruptly. Mild signs of withdrawal (e.g., reduced food maintained behavior, increased postural changes,
aggressive behaviors and self-directed behaviors) were apparent within the first few days after zolpidem was discontinued, followed by intermediate withdrawal signs (e.g.,
tremor, jerk and/or rigidly braced posture and vomit and/or
retch) within 5 to 7 days after zolpidem was discontinued.
Seizure, the most severe sign generally seen during Bz withdrawal, was not observed in any baboon. The time-limited
suppression of food-maintained behavior after discontinuation of zolpidem dosing in the present study is similar to that
reported previously (Griffiths et al., 1992; Kaminski et al.,
1993), and is consistent with that observed in baboons during
withdrawal from classic Bz agonists and the b-carboline abecarnil (Lamb and Griffiths, 1984; Lukas and Griffiths, 1982;
Sannerud et al., 1992). In humans, signs of withdrawal after
abrupt discontinuation of Bzs typically include nausea, vomiting and loss of appetite (Rickels et al., 1990). It is interesting that the one baboon for which pellets per day did not
decrease during withdrawal was the one baboon for which
pellets per day had not increased progressively across the
entire period of dosing. Research to investigate this phenomenon is in progress.
Chronic Bz administration reliably produces physical dependence in a variety of species (for review see Woods et al.,
1987, 1992). Previous studies in rodents concluded that there
was no evidence of zolpidem physical dependence after
chronic administration (Perrault et al., 1992; VonVoightlander and Lewis, 1991). The development of physical dependence in the present study differs with the findings of those
studies, but is consistent with the observations that zolpidem
physical dependence appeared to develop after high doses of
zolpidem in squirrel monkeys (Schoch and colleagues as cited
in Stephens and Sanger, 1996) and under conditions of re-
Downloaded from jpet.aspetjournals.org at ASPET Journals on October 12, 2016
progressively increased across the dosing conditions until
drug was discontinued. Tolerance to the sedative and ataxic
effects of Bz agonists in baboons typically occurs within the
first few days of repeated dosing (Lamb and Griffiths, 1985;
Lukas and Griffiths, 1982; Sannerud et al., 1989). However,
the ataxic effects of daily i.v. zolpidem (3.2 or 5.6 mg/kg) in
baboons did diminish with repeated dosing (Griffiths et al.,
1992; Kaminski et al., 1993). Aside from route of administration, there were many dosing parameters that could account
for the apparent lack of tolerance in the present study. In
particular, progressively increasing the zolpidem dose every
17 days in the present study may have masked the development of tolerance. A lack of tolerance to effects of triazolam
(Ator et al., 1996) and zaleplon (Ator NA, Weerts EM, Kaminski BJ, Kautz MA and Griffiths RR, unpublished observations) also was found using a similar dosing regimen and
set of behavioral assessments in baboons. It is possible that
tolerance may have developed if longer cycles (i.e., more than
17 days) of dosing conditions were used. However, tolerance
was not evident even after 27 days of the 32.0 mg/kg dose
condition. The lack of tolerance to the sedative effects of
zolpidem in the current study is consistent with the results of
studies in rats (Perrault et al., 1992; Sanger and Zivkovic,
1987, 1992; Sanger et al., 1994) and with the finding from
clinical studies that no tolerance to the hypnotic effect of
zolpidem was found in humans with insomnia (review in
Salva and Costa, 1995).
The mean elimination half-life of zolpidem in humans after
single oral doses (5–20 mg p.o.) has ranged across studies
from 1.5 to 3.2 hr (review in Salva and Costa, 1995). Although elimination half-lives in rats and cynomolgus monkeys have been shorter than in humans (Durand et al., 1992),
it is reasonable to assume that the elimination half-life in
adult male baboons might be more similar to that in humans
(Fridman and Popova, 1988). If the elimination half-life in
baboons is assumed to be within the range of those reported
above for humans it seems likely that, given the large total
doses administered (e.g., approximately 30 mg at the 1.0
mg/kg dose), some drug still would be present 24 hr after
dosing. In human volunteers, mean peak plasma concentrations of 200 ng/ml were reached by 2.3 hr on day 15 of
repeated zolpidem dosing (20 mg/kg/day p.o., for 15 days),
which declined to about 10 ng/ml after 12 hr and to less than
5 ng/ml after 24 hr (Durand et al., 1992; Thénot et al., 1988).
Similar low concentrations of zolpidem in blood plasma were
obtained 24 hr after dosing with 1.0 and 3.2 mg/kg in the
current study in baboons. In the current study, levels of
zolpidem in plasma were dose dependent across the 1.0 to the
10.0 mg/kg condition, but there was some indication that
baboons may have metabolized the 32.0 mg/kg dose differently (J. Althaus, personal communication). Previous studies
have not reported an accumulation of zolpidem in plasma
with chronic dosing in rats (5.0 mg/kg21 i.p., once daily, for 7
or 28 days) (Trenque et al., 1994) or human volunteers (20
mg/day p.o. for 15 days) (Thénot et al., 1988); and the results
of the study in humans suggested that zolpidem neither
induced nor inhibited its own metabolism.
The behavioral signs that are characteristic of Bz withdrawal are similar to the behavioral signs that are characteristic of withdrawal from barbiturates in humans and laboratory animals (Henningfield and Ator, 1986; Woods et al.,
1987). The behavioral signs of Bz withdrawal have been well
Zolpidem Dependence
52
Weerts et al.
Acknowledgments
The excellent technical assistance of Michael Cichocki, Christine
Ebaugh and Samuel Womack is gratefully acknowledged. We also
thank Dr. Barbara J. Kaminski for her helpful contributions to the
successful completion of this study and John S. Althaus of Pharmacia & Upjohn, Inc. for carrying out the blood plasma analysis and
supplying the description of it. We thank Pharmacia and UpJohn
Company for a grant to defray the cost of durg supplies.
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