Anxiolytic and Side-Effect Profile of LY354740: A Potent, Highly Selective, Orally Active Agonist for Group II Metabotropic Glutamate Receptors
←
→
Page content transcription
If your browser does not render page correctly, please read the page content below
0022-3565/98/2842-0651$03.00/0
THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS Vol. 284, No. 2
Copyright © 1998 by The American Society for Pharmacology and Experimental Therapeutics Printed in U.S.A.
JPET 284:651–660, 1998
Anxiolytic and Side-Effect Profile of LY354740: A Potent,
Highly Selective, Orally Active Agonist for Group II
Metabotropic Glutamate Receptors
DAVID R. HELTON,1 JOSEPH P. TIZZANO, JAMES A. MONN, DARRYLE D. SCHOEPP and MARY JEANNE KALLMAN
Toxicology Research (D.R.H., J.P.T., M.J.K.) and Neuroscience Research Division (J.A.M., D.D.S.), Eli Lilly and Company, Greenfield, Indiana
Accepted for publication October 27, 1997 This paper is available online at http://www.jpet.org
Downloaded from jpet.aspetjournals.org at ASPET Journals on August 30, 2015
ABSTRACT
LY354740 is a conformationally constrained analog of gluta- diazepam, acute administration of LY354740 did not produce
mate which is a potent agonist for group II cAMP coupled sedation, cause deficits in neuromuscular coordination, interact
metabotropic glutamate receptors (mGluRs). The discovery of with central nervous system depressants, produce memory
this novel pharmacological agent has allowed the exploration of impairment or change convulsive thresholds at doses 100- to
the functional consequences of activating group II mGluRs in 1000-fold the efficacious doses in animal models of anxiety.
vivo. In an effort to evaluate the clinical utility of LY354740 as an Thus, LY354740 has anxiolytic activity in animal models that
anxiolytic, we examined its effects in the fear potentiated startle are sensitive to benzodiazepines such as diazepam. However,
and elevated plus maze models of anxiety and compared the at anxiolytic doses in these models, LY354740 produced none
results with the clinically prescribed anxiolytic diazepam. In the of the unwanted secondary pharmacology associated with di-
fear potentiated startle and elevated plus maze models, both azepam. These data indicate a functional role for group II
LY354740 and diazepam produced significant anxiolytic activ- mGluRs in fear/anxiety responses in animals and suggest that
ity (ED50 values of 0.3 and 0.4 mg/kg p.o. for fear potentiated compounds in this class may be beneficial in the treatment of
startle and 0.2 and 0.5 mg/kg for the elevated plus maze, anxiety-related disorders in humans without the side effects
respectively). The duration of pharmacological effect for seen with currently prescribed medications.
LY354740 in the efficacy models was 4 to 8 hr. In contrast to
Glutamate is the major excitatory neurotransmitter in the mechanisms (Sladeczek et al., 1985; Nicoletti et al., 1986;
mammalian CNS, contributing excitatory input to virtually Sugiyama et al., 1987; Schoepp and Johnson, 1988). These
all neurons of the brain and spinal cord. The ubiquitous metabotropic glutamate receptors (mGluRs) represent a het-
nature of glutamate excitation supports a role for glutama- erogeneous family of proteins that are coupled to multiple
tergic neurotransmission in most physiological processes of second messenger systems (Schoepp and Conn, 1993; Nakan-
the CNS including sensory and motor functions, central reg- ishi and Masu, 1994; Pin and Duvoisin, 1995) and are divided
ulation of respiratory and cardiovascular centers, cognition into three groups with similar pharmacology, molecular
and regulation of perception and emotion. Similarly, altered structure and second messenger coupling.
glutamatergic neuronal transmission has also been hypoth- Group I mGluRs include mGluR1 and mGluR5, group II
esized to have a central role in many neurological and psy- receptors include mGluR2 and mGluR3 and group III
chiatric disorders, including neurodegenerative diseases, ep- mGluRs consist of mGluR4, mGluR6, mGluR7 and mGluR8.
ilepsy, schizophrenia and anxiety (Danysz et al., 1995). When expressed in nonneuronal cells, group I mGluRs couple
Until recently, the effects of glutamate were thought to be to the activation of phosphoinositide hydrolysis, although
exclusively mediated by ion channel-mediated ionotropic re- group II and group III mGluRs are negatively linked to cAMP
ceptors (iGluRs), which include NMDA, AMPA and kainate formation. Each mGluR subtype is uniquely and differen-
receptor subtypes (Hollmann and Heinemann, 1994). How- tially distributed in the CNS. Group II mGluRs (mGluR2 and
ever, it is now known that glutamate can also couple to mGluR3) are highly localized within forebrain regions in the
second messenger pathways through G-protein-dependent
rat suggesting specific roles in modulation of glutamatergic
transmission in those regions (Ohishi et al., 1993a; Ohishi et
Received for publication May 23, 1997.
1
Current address: Quintiles BRL, 1300 N. 17th Street, Suite 300, Arling- al., 1993b; Schoepp, 1994; Pin and Duvoisin, 1995). Group II
ton, VA 22209. mGluRs are thought to exist at presynaptic sites where they
ABBREVIATIONS: CNS, central nervous system; iGluR, ionotropic glutamate receptor; NMDA, N-methyl-D-aspartic acid; AMPA, 2-amino-3(3-
hydroxy-5 methylisoxazol-4-yl)propionic acid; mGluR, metabotropic glutamate receptor; HSD, Tukey’s studentized range.
651652 Helton et al. Vol. 284
function as negative feedback autoreceptors to inhibit the cally available anxiolytics have limited clinical efficacy be-
release of glutamate (possibly through effects on voltage- cause of their adverse side effect profile, dependence produc-
sensitive calcium channels), or postsynaptically where they ing properties (Dantzer, 1977; Greenblatt and Shader, 1978;
can modulate cell function via alterations in A-kinase activ- Woods et al., 1987, 1992; Treit, 1991). Therefore, a nonaddic-
ity (ultimately by changing protein phosphorylation) and/or tive, highly efficacious anxiolytic devoid of benzodiazepine-
direct modulation of various ion channels (i.e., K1, Ca11) like side effects would be of great clinical value for the treat-
(Schoepp, 1994; Pin and Duvoisin, 1995). Specifically, fore- ment of anxiety and related disorders.
brain areas that express group II mGluRs include limbic In an effort to evaluate the role of group II mGluRs in
structures that participate in the control of emotionality and anxiety, we examined the effects of LY354740 [or its racemic
anxiety states. A compound acting on mGluRs in these brain form, (6)LY314582] in two animal models that detect anxio-
areas would be hypothesized to modulate behavioral states in lytic activity, the fear potentiated startle model and elevated
animals associated with these brain areas. plus maze. We also profiled the secondary (potential side-
LY354740 [(1S,2S,5R,6S-2-aminobicyclo[3.1.0]hexane-2,6- effect) pharmacology of LY354740 in comparison to diaze-
dicarboxylate monohydrate], a conformationally constrained pam, including the effects of both agents on spontaneous
analog of glutamate, is a nanomolar potent, highly selective activity, neuromuscular coordination, interaction with a CNS
and orally active agonist at group II/cAMP coupled mGluRs depressant, electro-convulsant threshold and learning and
(mGluR2 and mGluR3) (see fig. 1) (Monn et al., 1997; Schoepp memory.
et al., 1997). Until the recent discovery of LY354740, there
Downloaded from jpet.aspetjournals.org at ASPET Journals on August 30, 2015
were no known group II mGluR agonists that were nanomo-
lar potent, highly selective and systemically active. Thus, the Materials and Methods
discovery of this novel pharmacological agent has allowed Animals. All experiments were performed in accordance with Eli
exploration of the functional consequences of activating Lilly and Company Animal Care and Use Policies. Male Long Evans
group II mGluRs in vivo. rats (180 – 400 g) and male NIH Swiss mice (18 –35 g) were obtained
The fear potentiated startle procedure, a model based on from Harlan Sprague-Dawley, Cumberland, IN. Male CD-1 mice
fear which is reportedly selective for the identification of (18 –35 g) were obtained from Charles Rivers Laboratories, Portage,
anxiolytic drugs, has been described by Davis (1979, 1986). In MI. All animals were acclimated at least 3 days before testing.
Animals were housed at 23 6 2°C (relative humidity 30 –70%) and
this procedure, a neutral stimulus such as a light (condi-
given Purina Certified Rodent Chow (PMI International, Inc., St.
tioned stimulus) is repetitively paired with an aversive stim- Louis, MO) and water ad libitum. The photo period was 12 hr of light
ulus such as shock (unconditioned stimulus). After condition- and 12 hr of dark, with dark onset at approximately 1800 hr.
ing, when the animals are presented with loud acoustic Drugs. (1)LY354740 monohydrate [(1S,2S,5R,6S-2-aminobicyclo
stimuli, enhanced startle responses are elicited when the [3.1.0]hexane-2,6-dicarboxylate monohydrate], (2)LY366563 mono-
startle stimulus is preceded by the light. The augmentation hydrate or racemic (6)LY314582 (Monn et al., 1997) were dissolved
of startle responses engendered in this model are reported to in a vehicle of purified water and neutralized with 5 N NaOH to a pH
result from a classically conditioned increase in fear (Davis, of approximately 7 to 8. Diazepam (Sigma Chemical Co., St. Louis,
1979). MO) was suspended in purified water by the drop-wise addition of
A second model of anxiety, the elevated plus maze, is a Tween 80. Hexobarbital (Sigma) was solubilized in purified water by
the addition of 0.2 N sodium hydroxide. Hydrochloric acid (0.1 N)
widely used test based on the natural aversion of rodents to
was subsequently added to adjust the pH prior to dilution to the final
heights and open spaces, which has been validated for both volume with purified water. NMDA (Research Biochemicals Inter-
rats and mice (Lister, 1987; Dawson and Trinklebank, 1995; national, Natick, MA) was solubilized in 0.9% physiological saline.
Helton et al., 1995), and, as with fear potentiated startle, is Control animals received the respective vehicle. Food was removed
sensitive to both anxiolytic and anxiogenic drugs. at least 1 hr before compound administration.
Clinically proven anxiolytics such as diazepam (Valium) Fear potentiated startle. SL-LAB (San Diego Instruments, San
and buspirone (Buspar) are effective in reducing the fear Diego, CA) chambers were used for conditioning sessions and for
(increased startle responding) associated with the presenta- producing and recording startle responses. A respondent condition-
tion of the light in the potentiated startle model (Davis, 1979; ing procedure was used to produce potentiation of startle responses.
Kehne et al., 1988) and at increasing open-arm activity in the Briefly, on days 1 and 2, rats were placed into dark startle chambers
in which shock grids were installed. After a 5-min acclimation pe-
elevated plus maze (Helton et al., 1995). However, all clini-
riod, each rat received a 1 mA electric shock (500 msec) preceeded by
a 5-sec presentation of light (15 W) which remained on for the
duration of the shock. Ten presentations of the light and shock were
given in each conditioning session with an inter-trial interval of 10
sec. Startle responding was measured through transducers located
in the center of a platform located under the animal. Recorded values
represent a maximum change in voltage from baseline and are re-
corded and presented as a peak maximum voltage (Vmax). Forty-eight
hours after the second conditioning session, rats were administered
LY354740, diazepam, or water, and startle testing sessions were
conducted. A block of 10 consecutive presentations of acoustic startle
stimuli (110 dB, non-light-paired) was presented at the beginning of
the session to minimize the influence of the initial rapid phase of
habituation to the stimulus. This was followed by 20 alternating
trials of the noise (110 dB white noise, 50 msec in duration) alone or
Fig. 1. Chemical structure of LY354740 (1S,2S,5R,6S)-2- noise preceeded by the light. All intertrial intervals were eight sec-
aminobicyclo[3.1.0]hexane-2,6-dicarboxylate monohydrate. onds in duration. Excluding the initial trial block, startle response1998 Anxiolytic Effect of LY354740 653
amplitudes for each trial type (noise-alone vs. light 1 noise) were Twenty min after i.p. administration of diazepam (0.3–3 mg/kg),
averaged for each rat across the entire test session. Data were NMDA (25 mg/kg) or LY354740 (administered as the racemate
presented as the difference between noise-alone and light 1 noise. (6)LY314852) (50 –200 mg/kg), saline was placed on each cornea
Diazepam (0.03–1 mg/kg, i.p.) or LY354740 (0.01–10 mg/kg, oral) immediately before placement of the eyes in corneal electrodes. Cur-
was administered 30 or 60 min, respectively, before evaluation in rent was then delivered with shock intensities ranging from 4 to 9
potentiated startle. For oral duration of action testing, LY354740 (3 mA (0.2-sec shock duration). The types of convulsions (clonic, tonic or
mg/kg, oral) was administered 2, 4, 6 or 8 hr before testing. tonic-extensor) observed within 10 sec were recorded.
Elevated plus maze. Construction of the elevated plus maze was Learning and memory— conditioned avoidance respond-
based on a design validated for mice by Lister (1987). The entire ing. Rats were anesthetized with isoflurane gas and surgically im-
maze was made of clear Plexiglas. The maze was comprised of two
planted with Alzet osmotic pumps (Alza Corporation, Vacaville, CA)
open arms (30 3 5 3 0.25 cm) and two enclosed arms (30 3 5 3 15
filled with water or LY354740 delivering 3 mg/kg/day. All rats were
cm). The floor of each maze arm was corrugated to provide texture.
implanted on the same day and testing was conducted in three
The arms extended from a central platform and angled at 90° from
different groups according to the following schedule: rats receiving 3
each other. The maze was elevated to a height of 45 cm above the
table and illuminated by a red light. Individual infrared photocells mg/kg/day LY354740 or vehicle were tested on days 2 through 6.
were mounted along each arm of the maze. For closed and open arms Separate rats receiving 3 mg/kg/day LY354740 or vehicle were tested
three photocells were positioned at equal intervals along the axis of on days 7 through 11. Separate rats with 3 mg/kg/day LY354740 or
each arm. For nosepoke activity photobeams were 0.5 cm from the vehicle exposure for 12 days were tested on days 1 through 5 after
entry of the open and closed arms. NIH Swiss mice were individually pump removal. An Omnitech Shuttle System (Columbus, OH) was
placed in the closed arm of the maze and the number of closed, open used to collect behavioral data. The avoidance procedure was carried
Downloaded from jpet.aspetjournals.org at ASPET Journals on August 30, 2015
and nosepoke counts were recorded and used as a measure of arm out in a two-compartment shuttle box with a grid floor through
entries and time spent on various sections of the maze over a 5-min which electric current was delivered. Each test session consisted of
test period. LY354740 (0 or 3 mg/kg, oral) was administered to mice 20 trials and each animal was tested for 5 consecutive days. The
at 1, 4 or 6 hr before evaluation in the maze. intertrial interval for each test session was variable (30 6 10 sec). A
Spontaneous activity. Spontaneous activity of individual rats combination of light (6 W) and acoustic (90 dB tone) stimuli was
was recorded using Multi-Varimax Activity Monitors (Columbus In- used, with a signal duration of 5 sec. Shock intensity was set at 0.5
struments, Columbus, OH). Interruptions of three individual infra- mA and the shock interval was 5 sec. To avoid the shock, rats learned
red photocells were recorded by computer. Activity counts were cu- to move from one side of the chamber to the other side at the cue
mulated after administration of diazepam (1–10 mg/kg, oral) or (avoidance) or move when the shock was administered (escape). Data
LY354740 (10 –300 mg/kg, oral) at 15-min intervals for 1 hr. Ambu- were tabulated as mean percent escape or mean percent avoidance
latory (locomotor or horizontal movement) counts represent the sum responses.
of individual photocell beam breaks (n 5 3 beams/cage) within a
Learning and memory—passive avoidance responding.
representative time interval. Nonambulatory (stereotyped or nonlo-
Male CD-1 mice were tested in a 2-day passive avoidance task.
comotor movement) counts represent the sum of the repetitive break-
Diazepam (3–30 mg/kg, oral) or LY354740 (3–30 mg/kg, oral) was
ing of individual photocells.
administered on test day one 60 min before acquisition training.
Sensorimotor reactivity—auditory startle. The auditory star-
Passive avoidance performance was evaluated using the Omnitech
tle response of each rat was recorded using San Diego Instruments
startle chambers (San Diego, CA). Startle was assessed across 10 Shuttle Control System (Omnitech Electronics), which consisted of
trials with a 5-min adaptation period at a background noise level of eight, two-compartment shuttle chambers and a constant current
70 6 3 dBA, followed by 10 presentations of auditory (120 6 2 dBA shock generator. Mice were individually placed in the shuttle cham-
white noise, 50 msec in duration) stimuli presented at 8-sec inter- ber, and the session was initiated with a one min acclimation period.
vals. Startle was measured through transducers located in center of Day 1 acquisition testing consisted of three, 180-sec training trials
a platform located under the animal. Recorded values represent a which were signaled by illumination of a light (6 W) on the side the
maximum change in voltage from baseline and are recorded and animal was on in the chamber. During each trial, a foot shock of 0.5
presented as a peak maximum voltage (Vmax). During testing, rats mA was delivered through the grid floor if the mouse crossed to the
were administered LY354740 (1–10 mg/kg) orally and evaluated for other compartment which was kept dark. When the animal crossed
sensorimotor responsiveness after a 60-min pretreatment period. into the dark compartment a footshock was administered continu-
Neuromuscular coordination—rotarod. Rats were placed on a ously until the animal returned to the illuminated compartment.
rotarod (model V EE/85, Columbus Instruments) accelerating from 1 Trials were separated by 1-min intervals of total darkness. On day 2,
to 80 revolutions/4 min. All rats were given two control trials at least 24-hr (approximately) retention testing consisted of a 1-min acclima-
12 hr apart before evaluation of diazepam or LY354740. Rats were tion period followed by a single, 180-sec retention trial in which no
tested on the rotarod 60 min after administration of diazepam (1–10 foot shock to mice was delivered upon crossing in any group. Com-
mg/kg, oral) or LY354740 (30 –300 mg/kg, oral). The number of puter-recorded data for each animal for each trial are reported as the
seconds each rat remained on the rotarod was recorded. mean latency to first crossing.
Interaction with CNS depressants— hexobarbital-induced
Statistical evaluations. Potentiated startle, elevated plus maze,
sleep times. Male CD-1 mice were given a single oral dose of
spontaneous activity, sensorimotor reactivity, neuromuscular coor-
diazepam (1–10 mg/kg) or LY354740 (1–10 mg/kg) 60 min before
dination, hexobarbital-induced sleep time and learning and memory
challenge with hexobarbital (100 mg/kg, i.p.). Observation of the
assessments were evaluated by an analysis of variance. When sig-
mice began immediately after hexobarbital injection. Once the
mouse was asleep, it was transferred to a prenumbered chamber and nificant treatment effects were obtained, post hoc comparisons were
placed on its back. Mice were considered awake when they could made using a Tukey’s studentized range (HSD) test. In all analyses,
successfully right themselves (all four feet in contact with the sur- two-tailed statistical tests were used and the level of significance was
face) or until 240 min had elapsed. Once a mouse righted itself, it was set at P # .05. Respective SI50 values [shock intensity (mA) inducing
placed on its back once more and allowed to right a second time for tonic-extensor convulsions in 50% of the mice] for electroshock were
confirmation. Sleep duration was recorded for each mouse. calculated by probit analysis (log10) and compared to respective SI50
Convulsive liability— electroshock-induced convulsions. values obtained in the presence of LY354740 at the 95% confidence
Electroshock was conducted in male CD-1 mice according to the limits. Statistical analyses were performed using Statistical Analy-
method of Swinyard et al. (1952) with the following modifications. sis Systems (SAS Institute, Inc., Cary, NC).654 Helton et al. Vol. 284
Results
Fear potentiated startle. Administration of diazepam
(fig. 2) resulted in a dose-related attenuation of fear potenti-
ated startle responding with significant attenuation at 0.1,
0.3 and 1 mg/kg diazepam (ED50 5 0.4 mg/kg) (Tukey’s HSD,
P # .05). Oral administration of LY354740 (60-min pretreat-
ment) produced a dose-dependent attenuation of fear poten-
tiated startle responding in rats with significant reductions
at 0.1, 1, 3 and 10 mg/kg (ED50 5 0.3 mg/kg LY354740) (fig.
3) (Tukey’s HSD, P # .05). Baseline startle responding dur-
ing fear potentiated startle was not altered at any dose of
diazepam or LY354740 examined (data not shown; see fig. 7
for LY354740 acute startle results). The inactive isomer of
LY354740, (2)LY366563 (Monn et al., 1997), did not atten-
uate the fear potentiated startle response at oral doses up to
10 mg/kg (fig. 3). Oral administration of 3 mg/kg LY354740,
a dose that produced a significant reduction in fear potenti-
Downloaded from jpet.aspetjournals.org at ASPET Journals on August 30, 2015
ated startle, produced a significant attenuation of fear poten-
tiated startle responding for at least 6 hr after administra-
tion (fig. 4) (Tukey’s HSD, P # .05).
Elevated plus maze. Oral administration of 3 mg/kg
LY354740 produced a significant increase in nosepoke activ-
ity at 1, 4 and 6 hr after administration (fig. 5) (Tukey’s HSD,
P # .05). Significant increases in open-arm activity was
found at 1 and 4, but not 6, hr postdosing (fig. 5). Closed-arm
activity counts were not significantly altered at any time-
point (fig. 5). The inactive isomer, (2)LY366563, did not alter
open, nosepoke or closed-arm activity at oral doses up to 10 Fig. 3. Effect of orally administered (1) LY354740 (above left) or orally
mg/kg (data not shown). administered (2)LY366563 (the inactive isomer) (above right) on fear
potentiated startle responding in male Long Evans rats. Values represent
Spontaneous activity. Diazepam administration signifi- the mean (6S.E.) startle amplitude (Vmax) expressed as the difference
cantly reduced spontaneous ambulatory (fig. 6) and nonam- between noise-alone and light 1 noise. LY354740 or LY366563 was
bulatory (data not shown) activity levels in rats at 10 mg/kg administered orally 60 min before evaluation of startle responding. * Sig-
nificantly different from control P # .05.
(oral) from 30 through 60 min postdosing (Tukey’s HSD, P #
.05). LY354740 did not significantly alter spontaneous am-
bulatory (fig. 6) or nonambulatory (data not shown) activity
levels at oral doses up to 300 mg/kg.
Sensorimotor reactivity—auditory startle. There
were no significant changes in auditory startle responding in
rats at oral doses of LY354740 as high as 10 mg/kg (fig. 7), a
dose that completely suppresses fear potentiated startle re-
sponding (see fig. 5). In addition, no significant changes were
seen in startle habituation at doses up to 10 mg/kg (data not
shown).
Neuromuscular coordination—rotarod. Orally admin-
istered diazepam significantly reduced the number of sec rats
spent on the rotarod at doses of 3 and 10 mg/kg given 60 min
before testing (fig. 8) (Tukey’s HSD, P # .05). LY354740
administration (30 min pretreatment) did not produce any
significant changes in neuromuscular coordination as evalu-
ated on the rotarod at oral doses up to 300 mg/kg (fig. 8).
Interaction with CNS depressants— hexobarbital-in-
duced sleep times. Orally administered diazepam produced
dose-dependent increases in hexobarbital-induced sleep
times in CD-1 mice with significant effects at 3 and 10 mg/kg
(169 and 231% increases with respect to control, respectively)
Fig. 2. Effect of intraperitoneally administered diazepam on fear poten- (fig. 9) (Tukey’s HSD, P # .05). There were no significant
tiated startle responding in male Long Evans rats (n 5 8 rats/treatment increases in sleep time after oral administration of LY354740
group). Values represent the mean (61 S.E.) startle amplitude (Vmax) (60 min pretreatment) at doses up to 10 mg/kg (fig. 9).
expressed as the difference between noise-alone and light 1 noise. Diaz-
epam was administered 30 min before evaluation of startle responding. Convulsive liability— electroconvulsive shock. Diaz-
* Significantly different from control P # .05. epam (i.p.) effectively attenuated all electroshock-induced1998 Anxiolytic Effect of LY354740 655
Downloaded from jpet.aspetjournals.org at ASPET Journals on August 30, 2015
Fig. 5. Effect of LY354740 (3 mg/kg) on automated elevated plus maze in
Fig. 4. Effect of LY354740 (3 mg/kg) on fear potentiated startle respond-
male NIH Swiss mice after oral pretreatment times of 1, 4 and 6 hr.
ing in male Long Evans rats after oral pretreatment times of 2, 4, 6 or 8
Nosepoke counts represent incomplete movements (head only) into the
hr (circles). The black bar represents the effect of LY354740 at 1 hr (data
open arm from the closed arm. Open counts represent activity in the open
taken from fig. 3). Values represent the mean (6S.E.) startle amplitude
(sideless) arms of the maze. Closed counts represent activity in the
(Vmax) expressed as the difference between noise-alone and light 1 noise.
enclosed arms of the maze. Scores represent mean 6 S.E. of activity
* Significantly different from water control P # .05 (square).
counts in each zone during a 5-min test period. * Significantly different
from control P # .05.
tonic convulsions at doses of 1 and 3 mg/kg (fig. 10). In
contrast, the ionotropic glutamate receptor agonist, NMDA,
produced a significant increase in the number of electro- or ionotropic (NMDA, AMPA or kainate) glutamate receptors
shock-induced convulsions when tested at a single i.p. dose of (Monn et al., 1997; Schoepp et al., 1997). The high potency
25 mg/kg (fig. 10). No significant changes were observed in and selectivity of LY354740 for group II mGluRs make
the current intensity producing 50% tonic convulsions follow- LY354740 a novel pharmacological tool for investigating the
ing administration of LY354740 (administered as the race- role of group II mGluRs in physiological processes and ex-
mate (6)LY314582, i.p.) at doses up to 200 mg/kg (fig. 10). ploring the potential therapeutic applications of such agents.
Learning and memory— conditioned avoidance re- Benzodiazepines such as diazepam (Valium) are the most
sponding. Continuous administration of LY354740 (3 mg/ widely prescribed medications for the treatment of anxiety
kg/day, s.c. infusion) to rats did not impair the acquisition of and related disorders. The 5HT1a agonist, buspirone (Bus-
avoidance responding on days 2 to 6 (fig. 11) or days 7 to 11 par), is also available, but is limited in use due to marginal
of exposure (data not shown). Cessation of LY354740 expo- efficacy and delayed onset of action (Riblet et al., 1982).
sure did not disrupt the acquisition of avoidance responding Benzodiazepines act at a specific site on the GABAA receptor
during the first 5 days of withdrawal (fig. 11). to facilitate fast-inhibitory synaptic transmission throughout
Learning and memory—passive avoidance. Oral diaz- the CNS. This likely accounts for their widespread therapeu-
epam (0, 3, 10 or 30 mg/kg) did not alter the acquisition phase tic applications including use as anticonvulsants, anxiolytics,
(acquisition trials 1, 2, 3—learning day 1) of passive avoid- muscle relaxants, sedative-hypnotics and anesthetic agents.
ance responding in CD-1 mice at any dose examined. How- However, the widespread distribution of benzodiazepine re-
ever, diazepam produced a dose-dependent disruption of the ceptors also produces side-effects and other liabilities such as
retention phase (retention trial—memory day 2) of passive CNS depression, abuse potential, physical dependence and
avoidance with significant deficits in responding at 10 and 30 withdrawal reactions (Woods et al., 1992). In most synapses,
mg/kg (fig. 12) (Tukey’s HSD, P # .05). LY354740, at oral the inhibitory effects of GABA are opposed by glutamate
doses up to 30 mg/kg, did not disrupt the acquisition phase or excitation and theoretically, agents that block the postsyn-
the retention phase of passive avoidance responding (fig. 12). aptic excitatory effects of glutamate produce pharmacology
similar to benzodiazepine pharmacology (Coyle, 1987). Con-
sistent with this theory, antagonists for NMDA and/or AMPA
Discussion receptors have been shown to produce anxiolytic effects in
LY354740, a conformationally constrained analog of gluta- the fear-potentiated startle (Kim et al., 1993; Anthony and
mate, is a highly potent and selective group II (mGluR2 and Nevins, 1993) or elevated plus maze models (Wiley et al.,
mGluR3) receptor agonist with no appreciable agonist/antag- 1995) of anxiety. However, in contrast to NMDA or AMPA
onist activity at metabotropic group I and group III mGluRs receptors that are postsynaptic and participate directly in656 Helton et al. Vol. 284
Fig. 6. Effect of orally administered diaz-
epam (left panel) or orally administered
LY354740 (right panel) on spontaneous
ambulatory activity levels in male Long
Evans rats accumulated at 15-min inter-
vals for 60 min. Activity counts represent
the sum of individual photocell beam
breaks averaged at 15 min intervals
(mean 6 S.E.). * Significantly different
from control P # .05.
Downloaded from jpet.aspetjournals.org at ASPET Journals on August 30, 2015
sion within the amygdaloid complex has also been linked to
anxiolytic responses in animals (Campeau and Davis, 1995).
For example, intraamygdala injections of NMDA receptor
antagonists prevent the acquisition of fear-potentiated star-
tle responses in rats, although having no effects on the ex-
pression of fear responses in conditioned animals (Miseren-
dino et al., 1990; Anthony and Nevins, 1993). However, the
intraamygdala injection of the AMPA/kainate receptor an-
tagonist 6-cyano-7-nitroquinoxaline-2,3-dione blocks the ex-
pression of fear-potentiated startle responding in animals
(Kim et al., 1993). Within the amygdaloid complex there is
evidence that tonic excitation through glutamate pathways
and evoked excitatory postsynaptic potentials can be blocked
by antagonists for NMDA and AMPA/kainate ionotropic re-
ceptors that act at postsynaptic sites (Rainnie et al., 1991), or
metabotropic agonists (group II and III) that act presynapti-
cally to diminish postsynaptic AMPA receptor mediated fast-
excitatory postsynaptic potentials (Rainnie and Shinnick-
Gallagher, 1992). Interestingly, the oral administration of
LY354740 produced a dose-dependent attenuation of fear
potentiated startle responding with significant reductions at
0.1, 1, 3 and 10 mg/kg (ED50 5 0.3 mg/kg LY354740) (fig. 3).
Fig. 7. Effect of orally administered LY354740 on sensorimotor reactivity
as evaluated using auditory startle responding in Long Evans rats.
Baseline startle responding was not altered at any dose of
LY354740 was administered by oral gavage 60 min before testing. Values LY354740 examined (fig. 7). The effects observed were ste-
represent the mean (6S.E.) peak response (maximum startle amplitude) reoselective because the inactive enantiomer of
averaged over 10 trials (n 5 10 rats/treatment group). (1)LY354740, (2)LY366563, was without effect in these
models at oral doses up to 10 mg/kg (fig. 3). Additionally, oral
fast-excitatory synaptic transmission, group II mGluRs have administration of 3 mg/kg LY354740, a dose that produced a
unique pre- and postsynaptic localizations (Petralia et al., significant reduction in fear potentiated startle, produced a
1996) which can alter second messengers to indirectly mod- significant attenuation of fear potentiated startle responding
ulate fast-synaptic transmission (Schoepp and Conn, 1993). during the first 6 hr after administration (fig. 4). Thus, in the
To evaluate the potential clinical utility of LY354740 as an fear potentiated startle model, LY354740 and diazepam pro-
anxiolytic, we examined its effects in the fear potentiated duced significant anxiolytic activities with very similar po-
startle and elevated plus maze animal models of anxiety. tencies (ED50 values of 0.3 and 0.4, respectively, see table 1).
The potentiated startle procedure is a model based on fear We have previously shown that in the elevated plus maze,
which is selective for the identification of anxiolytic drugs oral administration of LY354740 produces dose-dependent
(Davis, 1979; 1986; see above). The amygdala has been im- increases in open arm activity with no effect on closed-arm
plicated as an important brain region for acquisition, reten- activity (Monn et al., 1997). In this study, we further exam-
tion and expression of conditioned fear and the actions of ined the time course of activity for LY354740 after oral ad-
anxiolytic drugs (Davis, 1992; Davis et al., 1993, 1994). The ministration of 3 mg/kg, a dose that produced maximal in-
suppression of excitatory glutamatergic synaptic transmis- creases in open-arm activity when administered 30 min1998 Anxiolytic Effect of LY354740 657
Fig. 8. Effect of orally administered diaz-
epam (left panel) or orally administered
LY354740 (right panel) on neuromuscu-
lar coordination as evaluated on the ro-
tarod in male Long Evans rats (n 5 10
rats/dose group). Diazepam or LY354740
was administered by oral gavage 30 min
before testing. Values represent the mean
(6S.E.) number of sec rats remained on
the rotarod. * Significantly different from
control P # .05.
Downloaded from jpet.aspetjournals.org at ASPET Journals on August 30, 2015
Fig. 9. Effect of orally administered diaz-
epam (left panel) or orally administered
LY354740 (right panel) on hexobarbital-
induced sleep time in male CD-1 mice.
Diazepam or LY354740 was administered
by oral gavage 60 min before administra-
tion of hexobarbital (10 mg/kg, i.p.). Val-
ues represent mean (6S.E.) sleep time in
min (n 5 10 mice/treatment group). * Sig-
nificantly different from control P # .05.
Fig. 10. Effect of i.p. administered diaze-
pam (left panel) or i.p. administered
NMDA or LY354740 (administered as the
racemate (6)314582) (right panel) on
electroshock-induced convulsive patterns
in male CD-1 mice. Diazepam, NMDA or
LY354740 was administered i.p. 20 min
before testing. Values represent the num-
ber of animals (expressed as percent) dis-
playing tonic convulsions at each current
level (n 5 40 mice/dose-response).
before testing. LY354740 produced significant increases in In addition to anxiolytic activity, administration of diaze-
open-arm and nosepoke activity through the first 4 hr after pam and other benzodiazepines can result in sedative, ataxic
administration. Again, closed-arm activity counts were not and cognitive side-effects (Greenblatt and Shader, 1978;
significantly altered at any timepoint (data not shown). Dantzer, 1977) and with repeated exposure, tolerance and658 Helton et al. Vol. 284
Fig. 11. Effect of s.c. administered
LY354740 (3 mg/kg/day) on the acquisi-
tion of avoidance responding during treat-
ment (left panel) or after the withdrawal
of LY354740 (right panel) in male Long
Evans Rats. LY354740 was administered
for 12 consecutive days in osmotic pumps.
Values represent mean (6S.E.) percent
successful avoidance (n 5 8 rats/treat-
ment group).
Downloaded from jpet.aspetjournals.org at ASPET Journals on August 30, 2015
Fig. 12. Effect of p.o. adminis-
tered diazepam (left panel) or
p.o. administered LY354740
(right panel) on the acquisi-
tion (acquisition trials 1, 2,
3—learning day 1) and reten-
tion (retention trial—memory
day 2) of passive avoidance re-
sponding in male CD-1 mice.
Diazepam or LY354740 was
administered 60 min before
testing during the acquisition
phase. Values represent mean
(6S.E.) of latency to first
crossing (n 5 8 mice/treat-
ment group). * Significantly
different from control P # .05.
dependence. In our series of experiments, diazepam produced LY354740. However, although memory for the task was not
sedation with therapeutic ratios (based on efficacy ED50/ altered by LY354740, a significant disruption of retention
Rotarod ED50) of only 19.8- and 36-fold higher than those was noted for diazepam treated animals (fig. 12).
producing anxiolytic effects in fear potentiated startle and As a class of pharmaceutical compounds, benzodiazepines
the elevated plus maze, respectively (table 1). In contrast, consistently enhance the effects of other CNS depressants.
LY354740 did not produce changes in neuromuscular func- Potentiation of hexobarbital-induced sleep time was used to
tion at doses as high as 300 mg/kg providing therapeutic evaluate potential interactions of LY354740 with CNS de-
ratios of .1000 and .600, respectively (table 1). pressants. Hexobarbital is a short-to-intermediate acting
The difference in side-effect profile between diazepam and barbiturate that exerts its primary pharmacological effect on
LY354740 is also reflected in overt clinical signs and spon- the CNS by enhancing inhibition of GABA-mediated neuro-
taneous activity tests. In these tests, diazepam, but not transmission (Olsen, 1982). Test compounds may shorten or
LY354740, produced marked changes in coordination, and prolong hexobarbital-induced sleep time by acting directly on
activity (table 1). Additionally, LY354740 did not produce the CNS (excitation or depression) or indirectly via inhibition
cognitive impairment during the first 11 days of continuous of hepatic enzymes involved in hexobarbital metabolism. As
administration or during the first 5 days after withdrawal. expected, hexobarbital-induced sleep times were dose-depen-
Furthermore, the acquisition (learning) of a passive avoid- dently increased by diazepam supporting its known interac-
ance task was not disrupted by either diazepam or tive activity with sedative-hypnotics. However, LY3547401998 Anxiolytic Effect of LY354740 659
TABLE 1
Comparative doses (ED50) of diazepam (DIAZ) or LY354740 in efficacy and secondary pharmacology models
Efficacy/
ED50 ED50 Efficacy/Rotaroda
Rotaroda
Test Species
DIAZ LY354740 DIAZ LY354740
Potentiated startle (anxiety) Rats 0.4 0.3 19.8 .1000
Elevated plus maze (anxiety) Mice 0.2b 0.5c 36d .600
Spontaneous activity (sedation) Rats 6.9 .300
Rotarod (neuromuscular coordination) Rats 7.9 .300
(7.1 i.p.)
Hexobarbital-induced sleep (interaction with CNS depressants) Mice 1.4 .10
Electroconvulsive shock (CNS depression) Mice 0.5 (i.p.) .200 (i.p.)
Passive avoidance (learning and memory) Mice 3.1 .30
a
Therapeutic ratios are based on the efficacy ED50/rotarod ED50.
b
Helton et al., 1995.
c
Monn et al., 1997.
d
Ratio is based on i.p. data.
did not increase sleep time indicating that LY354740 may Danysz W, Parsons CG, Bresink I and Quack G (1995) Glutamate in CNS disorders.
Drug News and Perspectives 8:261–277.
Downloaded from jpet.aspetjournals.org at ASPET Journals on August 30, 2015
not produce CNS depression or interact with other CNS Davis M (1979) Diazepam and flurazepam: Effects on conditioned fear as measured
depressants at clinically relevant doses (fig. 9). with the potentiated startle paradigm. Psychopharmacology 62:1–7.
Davis M (1986) Pharmacological and anatomical analysis of fear conditioning using
Benzodiazepines are also used clinically as anticonvul- the fear-potentiated startle paradigm. Behav Neurosci 100:814 – 824.
sants. Changes in electroconvulsive shock thresholds after Davis M (1992) The role of the amygdala in fear-potentiated startle: Implications for
administration of diazepam or LY354740 were evaluated. animal models of anxiety. Trends Pharmacol Sci 13:35– 41.
Davis M, Falls WA, Campeau S and Kim M (1993) Fear-potentiated startle: A neural
Although diazepam increased the threshold for electroshock- and pharmacological analysis. Behav Brain Res 58:175–198.
induced convulsions, no significant increases in convulsive Davis M, Rainnie D and Cassell M (1994) Neurotransmission in the rat amygdala
related to fear and anxiety. Trends Neurosci 17:208 –214.
thresholds were seen with LY354740 (fig. 10). In addition, Dawson GR and Tricklebank MD (1995) Use of the elevated plus maze in the search
the proconvulsive ionotropic receptor agonist, NMDA, de- for novel anxiolytic agents. Trends Pharmacol Sci 16:33–36.
creased convulsive thresholds in electroshock (fig. 10). These Greenblatt D and Shader R (1978) Dependence, tolerance, and addiction to benzo-
diazepines: Clinical and pharmacokinetic considerations. Drug Metab Rev 8:13–
data suggest that LY354740 may not produce CNS depres- 28.
sion or proconvulsive activities in humans. Helton DR, Berger J, Czachura JF, Rasmussen K and Kallman MJ (1995) Central
nervous system characterization of the new cholecystokininB antagonist
In summary, LY354740 is a novel, nanomolar potent, LY288513. Pharmacol Biochem Behav 53:493–502.
highly selective and orally active agonist at group II/cAMP Hollmann M and Heinemann S (1994) Cloned glutamate receptors. Annu Rev Neu-
rosci 17:31–108.
coupled metabotropic glutamate receptors. These data indi- Kehne JH, Cassella JV and Davis M (1988) Anxiolytic effects of buspirone and
cate that activation of in vivo group II mGluRs with gepirone in the fear-potentiated startle paradigm. Psychopharmacology 94:8 –13.
LY354740 produces benzodiazepine-like efficacy in the fear- Kim M, Campeau S, Falls WA and Davis M (1993) Infusion of the non-NMDA
receptor antagonist CNQX into the amygdala blocks expression of fear-potentiated
potentiated startle and elevated plus maze models of anxiety. startle. Behav Neural Biol 59:5– 8.
However, unlike benzodiazepines, LY354740 produces no Lister RG (1987) The use of a plus-maze to measure anxiety in the mouse. Psycho-
pharmacology 92:180 –185.
CNS depression and other unwanted benzodiazepine-like ac- Miserendino MJD, Sananes CB, Melia KR and Davis M (1990) Blocking of acquisi-
tivities in animals. This atypical profile of anxiolytic activity tion but not expression of conditioned fear-potentiated startle by NMDA antago-
and large safety margin in animals for LY354740 may reflect nists in the amygdala. Nature 345:716 –718.
Monn JA, Valli MJ, Massey SM, Wright RA, Salhoff CR, Johnson BG, Howe T, Alt
the novel mechanism and regional selectivity produced by CA, Rhodes GA, Robey RL, Griffey KR, Tizzano JP, Kallman MJ, Helton DR and
this agent. These data support the functional role of group II Schoepp DD (1997) Design, synthesis and pharmacological characterization of
(1)-2-aminobicyclo[3.1.0]hexane-2,6-dicarboxylate: A potent, selective, systemi-
mGluRs in anxiety. Additionally, LY354740 may be a useful cally active group 2 metabotropic glutamate receptor agonist possessing anticon-
pharmacological tool for evaluating the therapeutic rele- vulsant and anxiolytic properties. J Med Chem 40:528 –537.
vance of group II receptor activation in anxiety and other Nakanishi S and Masu M (1994) Molecular diversity and functions of glutamate
receptors. Annu Rev Biophys Biomol Struct 23:319 –348.
CNS-related disorders in humans. Nicoletti F, Meek JL, Iadarola MJ, Chuang DM, Roth BL and Costa E (1986)
Coupling of inositol phospholipid metabolism with excitatory amino acid recogni-
tion sites in rat hippocampus. J Neurochem 46:40 – 46.
Ohishi H, Shigemoto R, Nakanishi S and Mizuno N (1993a) Distribution of the
Acknowledgments messenger RNA for a metabotropic glutamate receptor, mGluR2, in the central
nervous system of the rat. Neuroscience 53:1009 –1018.
The authors thank Vanessa Benagh, Angie Boring, Kenneth Fir- Ohishi H, Shigemoto R, Nakanishi S and Mizuno N (1993b) Distribution of the
man, Kelly Griffey, Allen Johnson and Deah Modlin for their fine mRNA for a metabotropic glutamate receptor (mGluR3) in the rat brain: an in situ
technical assistance in performing the behavioral studies. hybridization study. J Compar Neurol 335:252–266.
Olsen RW (1982) Drug interactions at the GABA receptor-ionphore complex. Annu
Rev Pharmacol Toxicol 22:245–277.
Petralia RS, Wang Y-X, Niedzielski AS and Wenthold RJ (1996) The metabotropic
References glutamate receptors, mGluR2 and mGluR3, show unique postsynaptic, presynap-
Anthony EW and Nevins ME (1993) Anxiolytic-like effects of N-methyl-D-aspartate- tic and glial localizations. Neuroscience 71:949 –976.
associated glycine receptor ligands in the rat potentiated startle test. Eur J Phar- Pin J-P and Duvoisin R (1995) The metabotropic glutamate receptors: Structure and
macol 250:317–324. functions. Neuropharmacology 34:1–26.
Campeau S and Davis M (1995) Involvement of the central nucleus and basolateral Rainnie DG, Asprodini EK and Shinnick-Gallagher P (1991) Excitatory transmission
complex of the amygdala in fear conditioning measured with fear-potentiated in the basolateral amygdala. J Neurophysiol 66:986 –998.
startle in rats trained concurrently with auditory and visual conditioned stimuli. Rainnie DG and Shinnick-Gallagher P (1992) Trans-ACPD and L-APB presynapti-
J Neurosci 15:2301–2311. cally inhibit excitatory glutamatergic transmission in the basolateral amygdala
Coyle JT (1987) Excitotoxins, in Psychopharmacology: The Third Generation of (BLA). Neurosci Lett 139:87–91.
Progress (Meltzer HY ed.) pp 333–340, Raven Press, NY. Riblet LA, Taylor DP, Eison MS and Stanton HC (1982) Pharmacology and neuro-
Dantzer, R (1977) Behavioral effects of benzodiazepines: A review. Biobehavioral Rev chemistry of buspirone. J Clin Psychiatry 43:11–16.
1:71– 86. Schoepp DD and Johnson BG (1988) Excitatory amino acid agonist-antagonist in-660 Helton et al. Vol. 284
teractions at 2-amino-4-phosphonobutyric acid-sensitive quisqualate receptors Treit T (1991) Anxiolytic effects of benzodiazepines and 5-HT1a agonists, in 5-HT1a
coupled to phosphoinositide hydrolysis in slices of rat hippocampus. J Neurochem Agonists, 5-HT-3 Antagonists and Benzodiazepines: Their Comparative Behavioral
50:1605–1613. Pharmacology (Rodgers RJ and Cooper SJ eds), pp 107–131, Wiley, Chichester.
Schoepp DD and Conn PJ (1993) Metabotropic glutamate receptors in brain function Wiley JL, Cristello AF and Balster RL (1995) Effects of site-selective NMDA receptor
and pathology. Trends Pharmacol Sci 14:13–20. antagonists in an elevated plus-maze model of anxiety in mice. Eur J Pharmacol
Schoepp DD (1994) Novel functions for subtypes of metabotropic glutamate recep- 294:101–107.
tors. Neurochem Int 24:439 – 449. Woods JH, Katz JL and Winger G (1987) Abuse liability of benzodiazepines. Phar-
Schoepp DD, Johnson BG, Wright RA, Salhoff CR, Mayne NG, Wu S, Cockerham SL, macol Rev 39:251– 414.
Burnett JP, Belagaje R, Bleakman D and Monn JA (1997) LY354740 is a potent Woods JH, Katz JL and Winger G (1992) Benzodiazepines: use, abuse, and conse-
and highly selective group II metabotropic glutamate receptor agonist in cells
quences. Pharmacol Rev 44:151–347.
expressing human glutamate receptors. Neuropharmacology 36:1–11.
Sladeczek F, Pin J-P, Recasens M, Bockaert J and Weiss S (1985) Glutamate
stimulates inositol phosphate formation in striatal neurones. Nature 317:717–719.
Sugiyama H, Ito I and Hirono C (1987) A new type of glutamate receptor linked to Send reprint requests to: Dr. Darryle D. Schoepp, Neuroscience Research
inositol phospholipid metabolism. Nature 325:531–533. Division, Lilly Corporate Center, drop 0510, Eli Lilly and Company, Indianap-
Swinyard EA, Brown WC and Goodman LS (1952) Comparative assays of antiepi- olis, IN 46285.
leptic drugs in mice and rats. J Pharmacol Exp Ther 106:319 –330.
Downloaded from jpet.aspetjournals.org at ASPET Journals on August 30, 2015You can also read
