0022-3565/93/2662-1O18$03O0/O

THE JOURNAL

Copyright

0? PHARMACOLOGY

AND

0 1993 by The American

EXPERIMENTAL

Society

Vol. 266, No. 2

in U.S.A.

THERAPEUTICS

Pharmacology

for

and Experimental

Therapeutics

Printed

Imidazenhl: A New Partial Positive

Aminobutyric
Acid (GABA) Action
PIETRO
ERMINIO

GIUSTI,
COSTA

IVICA

DUCIO,

GIULIA

PUIA,

ROBERTO

Allosteric Modulator of -yat GABAA Receptors1

ARBAN,

ARMIN

WALSER,

ALESSANDRO

GUIDOTTI

and

Fidia-Georgetown
Institute for the Neurosciences
(l.D., G.P., A.G., E.C.), Georgetown
University
Medical School, Washington,
District of Columbia;
Department
of Pharmacology
(PG., R.A.), University
of Padua, Padua, Italy; and Hoffmann-La
Roche, Ltd. (A.W.), Nutley, New Jersey

Accepted

for publication

April 1 2, 1993

ABSTRACT

Positive
allosteric
modulators
of -y-aminobutyric
acid (GABA)A
receptors,
including
benzodiazepines
and congeners,
can be
classified
into three categories:
1) full allostenc
modulators
(i.e.,
triazolam
and alprazolam)
that act with high potency and efficacy
at many GABAA receptors,
2) selective allostenc modulators
(i.e.,
diazepam)
that act with high potency
and high efficacy
at selected GABAA receptors;
and 3) partial allostenc
modulators
(i.e.,
bretazenil)
that act with high potency
but low efficacy
at many
GABAA receptors.
lmidazenil,
an imidazobenzodiazepine
carboxamide, has been characterized
as a novel representative
of the
partial allostenc
modulator
class. When tested on a broad spectrum (native and recombinant)
of GABAA receptors,
imidazenil
positively
modulates
the GABA-elicited
Cr currents
with a 4- to
5-fold higher potency
but an efficacy (30-50%) lower than that
of diazepam,
and it antagonizes
the effects of the latter drug.
lmidazenil
in vitro (K1 = 5 x 1
M) and in vivo (IDso
0.2 ,mol/
kg i.v.) displaces
[3H]fiumazenil
from its brain binding sites and
in vivo it possesses
a marked anticonflict
profile in the rat Vogel

Classical

BZDs

tranquilizers

and

and

a number

of chemically

anticonvulsants

produce

unrelated
their

minor

pharmacologi-

cal effects by allosterically

and positively
modulating
the GABA
action
at specific
ionotropic
receptors
(GABAA
receptors)
(for
review see Costa,
1991).
At least six different
genes encoding
for the atpha subunits
of GABAA receptors
are present
in the mammalian
brain.
In
addition,
the mammalian
brain expresses
other genes (3-beta,
3-gamma,
2-rho
and 1-detta),
encoding
for structurally
heterogeneous
GABAA receptor
subunit
families
(Pritchett
et at., 1989;
Olsen and Tobin,
1990; Burt and Kamatchi,
1991). Should
all
the mRNAs
expressed
in the brain from these genes be functional
in encoding
for GABAA
receptor
subunits
and should
Received
I

This

Grant

for publication
work

BPN-1

was
1 ROl

ABBREVIATIONS:

bicuculline-HCI;
system.
1018

supported

January
in part

ii,

1993.
by National

MH49486-O1.

BZD,

benzodiazepines;

PTZ, pentylenetetrazol;

Institute

on Medical

Health

conflict-punishment
test and is 1 0 times more potent than bretazenil and 1 00 times more potent than diazepam
or alprazolam
in antagonizing
bicucullineand pentylenetetrazol-induced
seizures. Unlike diazepam
and alprazolam,
which induce sedation
and ataxia and potentiate
the effects of ethanol and thiopental
at doses similar to those that produce
anticonflict
effects and
occupy
50% of brain flumazenil
binding sites, imidazenil
does
not produce
ataxia or sedation
in rats nor does it potentiate
the
effects of ethanol or thiopental
in doses 30- to 50-fold higher
than those required
for the anticonflict
effect and for 100%
occupancy
of brain flumazenil
binding sites. Furthermore,
when
administered
with diazepam,
imidazenil
blocks in a dose-related
fashion the sedative,
ataxic effects of this drug and thus acts on
these unwanted
responses
as an antagonist
(i.e. , like flumazenil).
In all tests, imidazenil
has the pharmacological
profile of a partial
allosteric
modulator,
but is more potent than bretazenil,
has a
longer biological
half-life and, in rodents,
is virtually
unable to
cause sedation,
ataxia or to potentiate
ethanol toxicity.

their cognate
tor structures,
100

subunits
be assembled
to form pentameric
recepone would reach a theoretical
value of more than

structurally

different

GABAA

receptor

subtypes

that

are

expressed
in vivo.
of heterooligomeric

Thus, GABAA receptors

appear
proteins,
probably
pentameric,

to be a class
which pos-

sess

structural

high-affinity

an

binding

be

amazing
for

located

subunits

BZDs

and

in the
of

GABAA

diversity.
other

putative
receptors

A specific

pharmacological

extracellular

congeners

domain

(Pritchett

and

could

of the
Seeburg,

apha
1991).

However,
the presence
of a gamma-2
receptor
subunit
is required
to maximize
the efficacy
of the allosteric
modulation
of
the GABA
action
at the GABAA
receptors
(Pritchett
et aL,
1988; Puia et aL, 1991).
The identification
of structurally
different
and neuron-specific
native
GABAA
receptors
(Fritschy
et aL, 1992) and the discovery
of chemically
different
positive
modulators
(i.e., zolpidem,
abecarnil
and alpidem)
that either
selectively

bind

to

specific

GABAA

receptor

subtypes

(Langer

GABA, -y-aminobutyric
acid; FAM, full allosteric modulators; SAM, selective allosteric modulators; BIC,
DMSO, dimethylsulfonic
acid; trifluoracetic acid; PAM, partial allosteric modulators;
CNS, central nervous

Partial Modulators

1993

et aL,

and

1990;

different
tor

Zivkovic

Zivkovic,

et aL,

1992;

efficacy

the

Massotti
et aL, 1991;
aL, 1990)
or modulate

1990;

Stephens

et

action

of GABA

at various

1992; Ducic
may find positive
allosteric
modulators
which
not only have different,
selective
profiles

therapeutic

effects.

In fact,

liability

of

but

it now

BZDs

in a predictable

certain

GABAA

efficacy
Sanger

on all GABA
and Zivkovic,

GABAA

recep-

with

tolerance

subt35

minor

and

pharmacological

with

side

congeners

either

their

or their

may

selectivity

for

indiscriminately
et at.,

low
1990;

Jenck
et at.,
of newly
emerging
allosteric
modulators

1993).
The development
low efficacy
positive

of information
and brain

requires
heterogeneity,

an understanding
of the
but also imposes
the

concerning
distribution

the stoichiometry
of specific
GABAA

and
recep-

acting
with high potency
and
GABAA
receptor
subtypes
(i.e.,
SAM, acting with high potency
of selected

GABA

receptor

dem); and 3) PAM,

efficacy
on a great

efficacy
at a great
variety
of
triazolam
and alprazolam);
2)
and efficacy
at a small number

subtypes

acting
variety

(i.e.,

diazepam

and

alpi-

with high potency,

but low intrinsic
of GABAA
receptor
subtypes
(i.e.,

bretazenil).
The PAM,
because
of their
high potency
and low
intrinsic
activity
on many
GABAA
receptor
subtypes,
may act
as antagonists
of compounds
of the FAM
and SAM
classes.
Thus,
PAM
slightly
enhance
the GABAergic
tone
at a great
number
of synapses
but because
of their
low efficacy
they leave
virtually
intact
the physiological
gating
of GABAA
receptors
by

neurally
released
The FAM have
elicit
a great
memory
and

nounced
cause
tion;

quanta
a high

ability

anticonvulsant

effects

and

The
liability

side effects
The
SAM

memory

ataxia
and
the rodents

and

ethanol
develop
dependence

may

of ethanol
and
et aL, 1990;
Facklam

barbiturate
et at., 1992;

and

including
loss of
have
a less pro-

psychotic

are endowed
fail to cause

a new

liability

disorders,

and barbiturate
a fast tolerance

PAM
and

(Haefely
In the present
study
profile
of bretazenil,
ative
profiles
more
those
peutic

of severe
symptoms.

to induce

tracted
treatment.
and dependence

imidazenil,

of GABA.
tolerance
and dependence

number
psychotic

sedation,
moreover,

potentiation

IMIDAZENIL

Fig.

1.

Structure

develop

but

potentiato their
upon

pro-

with low tolerance

sedation,
ataxia
or
depressant

effects

Jenck
et at., 1992).
the pharmacological
PAM class, to that of

we have compared
a compound
of the

imidazobenzodiazepine

carboxylic

acid

deny-

(fig. 1) which belongs

to the PAM. The pharmacological
of the two drugs appear
to be similar
but imidazenil
is
potent,
longer
lasting
and has a higher
selectivity
for
pharmacological
actions
indicative
of a potential
theraanticonvulsant
and anxiolytic
value.

temperature
access

and Methods

Male
Park,

Sprague-Dawley

PA).

Animals

rats

were

were

housed

from

in groups

Zivic-Miller
of five per

(Allison

cage

at a

water.

and

a 12-hr

light-dark

All the experiments

cycle

were

and

with

free

between

performed
Co.

(Kala-

a gift

from

Roche (Nutley, NJ). Thiopental,

BIC, PTZ and GABA
from Sigma Chemical
Co. (St. Louis, MO). Imidazenil

Hoffmann-La

were purchased

1,5-a] [1,4]benzodiazepine-3and [4C]imidazenil

were synthesized
at Hoffmann-La
Roche.
Imidazenil
was 99% pure when
analyzed
by reverse
phase
high-performance
liquid chromatography.
Drugs were injected
i.v. in a volume
of 1 ml/kg
or per p.o. gavage
in a volume
of 6 ml/kg,
and the doses were expressed
in micromoles
per kilogram.
6-(2-bromophenyl)-8-fluoro-4H-imidazo[
(fig.

Binding

1)

Studies
in vitro.
BZD binding studies were conducted
with minor
of a previously
published
method
(Massotti
et aL, 1991).
cortices
and spinal
cords
obtained
from
250- to 300-g
male

Binding
modifications

Cerebella,

were homogenized

rats
50

volumes

with Bninkmann

ice-cold

50

mM

Polytron

Tris-citrate

[model]

buffer,

pH

for
7.1,

30 sec
and

in

were

centrifuged
for 10 mm at 48,000 x g at 0*C. The pellets were washed
5
times by resuspension
and recentnifugation
in the same
volume
of icecold buffer
and stored
at -20C
for at least 18 hr. After thawing,
aliquots
of the membrane
suspensions
were pelleted
and resuspended
in 50 volumes
by

BZD

of the

binding

membrane

same

site

(81
or

absence

different
pH 7.1.

buffer.

ligands

protein

flumazenil

Inhibition
for

suspension

Ci/mmol,

presence

of

New

of [3H]flumazenil

investigated

was

60

mm

England

binding

by incubating
at

4C

Nuclear,

200 g

with

1 nM

Boston,

MA)

of
[3H]

in the

diazepam
or various
concentrations
volume of 1 ml of 50 mM Tnis-citrate

10 iM

were

ligands,

of

in a total
buffer,
Imidazenil
and other
drugs
to be tested
were
dissolved
in
DMSO and diluted
in the incubation
medium
immediately
before
the
assay.
The maximal
DMSO concentration
in the final incubation
medium
was 1% (v/v),
this concentration
failed to influence
the binding
of [3H)flumazenil
to brain
membranes.
At the end of the incubation
period
the samples
were filtered
under vacuum
through
Whatman
GF/
B filters and washed twice with 5 ml ofcold buffer. Parallel
exper.iments
performed

in the

is in absence

and

described

1992).

The

GABAA

presence

of 50 M

[3H]PK

have

11195
possibility

W8.8

studied
protocols:

or in the

bicuculline).

binding

in detail

to established

studies

previously

of 30 zM

GABA

[3H]-4Cl

(that

diazepam

were

conducted
with methods
we
et aL, 1991; Romeo et aL,
binds to receptors
other than
synaptic
membranes
according

(Massotti

that imidazenil
in crude rat brain
[3H]glycine

absence

[3H)GABAA,

or [3H]dibenzocyclohepteneimine

(Danysz et at., 1990); [3H]spiperone

(Apud and Ito, 1991); a-[3H]amino3-hydroxy-5-methyl-4-isoxazolepropionic
acid (Nielsen
et aL, 1990);
[3Hjketansenin
(Apud and Ito, 1991); [3H]naloxone
(Bylund
and Yamamura, 1990); t-[S]butylcyclophosphorothionate
(Gallo et aL, 1985);
[3H]baclofen
(Bowery et aL, 1985);
(3-hydroxylphenyl)-[3HJN-(1-pro-

(Devane

obtained

6-(2-bromophenyl)-8-fluoro-4-H-imi-

2:00 and 5:00 P.M. Alprazolam

was a gift from the Upjohn
mazoo,
MI). Diazepam
and bretazenil
(Ro 16-6028)
were

1986);

Animals and Drug Administration

imidazenil

of 22 0.5#{176}Cwith

to food

pyl)pipenidine

Materials

of

dazo[1 ,5-aJ[1.-4]benzodiazepine-3-carboxamide.

carboxamide

by using
a broad
spectrum
of recombinant
GABAA
et at. (1991)
and
Ducic
et a!. (1993)
have
that
tranquilizing
and antiepileptic
drugs
which
are
allosteric
modulators
of GABA
action
at GABAA
can be classified
into
three
major
groups:
1) FAM,

reported
positive
receptors

NH2

dependence

receptor
subtypes
(Haefely
1992; Stephens
et aL, 1990;

only

A1IIIIIII

of GABAA
receptors
pharmacological
and

Puia

receptors,

with

GABAA

associated

that

their

receptors
not
receptor
structural

acquisition
the abundance
tor subtypes.
Recently,

are

manner

receptor

Ducic
et at.,
of generalized

of GABAA

also

appears

and

change

1992;
classes

Sanger

(Haefely
et at., 1990; Pi#{241}a
et at., 1991; Jenck
et at.,
et aL, 1993)
has given
rise to the hope
that
one

subtypes

1019

of GABAA Receptors

(Koe

et aL, 1989);
[3H]L-365,260
(Bylund
and Yamamura,
1990);
et aL, 1988). The competition
experiments

[I]pindolol

(Chang
and
Lotti,
and [3H]CP-55,940
were run in tnipli-

cats with six different

concentrations
of competing
S.E.M. values for the displacement
of [3Hjflumazenil
by the

nonlinear

curve-fittingprogram

based

on LIGAND

ligand. IC
were determined
(McPherson,

1020

Giustl et al

1987).

Statistical

Vol. 266

comparisons

of the

estimated

parameters

were

made

by Students
t test
for unpaired
values
or by analyses
of variance
followed
by Dunnetts
test. In all cases P < .01 was considered
statistically significant.
The GABA ratio was calculated
by dividing
the ICo
of the ligand in the presence
of BIC with the ICo of the same ligand
in presence
of 30 M GABA.
Binding
in vivo.
The method
used
for these
studies
is similar
to
that described
by Facklam
et al. (1992).
Imidazenil,
alprazolam,
bretazenil
or diazepani
were
administered
i.v.

at

before
3 mm
and

variouS

doses

to

groups

of

five

male

(200-250

rats

g)

10

mm

sacrifice.
[3H]Flumazenil
(50 Ci/kg
i.v., 1 mlfkg) was injected
before sacrifice.
The animals
were decapitated,
their forebrains
cerebella were excised rapidly and homogenized
in a fixed volume

(1/10

w/v)

of Tnis-HC1

homogenate

buffer

(50

mM,

pH

7.4).

One

milliliter

of the

filtered
immediately
through
Whatman
were washed
3 times with 3 ml ofcold buffer.

GF/B filters
and the filters
Nonspecific
binding
was defmed as radioactivity
bound
to membrane
when
the rats
were given 20 imol/kg
i.v. of clonazepam
10 mm before sacrifice.
No
major
[3Hjflumazenil
metabolites
were found in the rat brain
3 mm
after the i.v. injection
of this
radioactive
compound
(similar
results
are
reported
in the literature).
The ICo S.E.M.
values for each experiment
were calculated
by computer-assisted
curve
fitting
program
(EBDA) (McPherson,
1987).
was

The fractional

BZD binding
Fractional

site occupancy

occupancy

where

b = LD, dose inhibiting

(micromoles
per kilogram);
x

(%)

specific

was

calculated

as follows:

[3H]flumazenil

binding

by 50%

(micromoles
per kilogram)
and C
by Facklam
et aL (1992).
Statistical
comparisons
for the estimated
parameters
were made by
analysis
of variance
followed
by the Dunnetts
test.
Electrophysiological
recordIngs.
Experiments
were performed
on primary
cultures
of rat cortical
neurons
and
transformed
human
=

slope factor

embryonic

calculated

kidney

dose

as described

293

cells

transfected

with

plasmids

containing

cDNAs encoding for different

subunits
of GABAA
receptor.
rat cortical neurons were prepared as described previously

Neonatal

(Alho et a!.,

1988).
Transformed
Culture

human

Collection

Medium

embryonic

No.

CRL

kidney

1573)

were

293
grown

cells
in

(America

Minimal

Type
Essential

Grand Island, NY) supplemented

with 10% fetal
penicillin
(100 U/mI) (Gibco) and streptomycin
(100 UI
ml) (Gibco)
in 6% CO2 incubator.
Exponentially
growing
cells were
dispersed
with trypsin
and seeded at 2 x 10 cells per 35-mm dish in 2
ml of culture
medium.
Transfection
was performed
with
the calcium
phosphate
precipitation
technique
(Puia et aL, 1991). Alpha-i,
a4,ha-2,
apha-3,
a4,Iia-5,
beta-i, gamma-i
and gamma-2
GABAA
receptor
subunit cDNAs inserted
singly into the eukaryotic
expression
vector pCIS2
were used to transfect
the 293 cells. Cells were incubated
with calcium
bovine

(GIBCO,

serum,

phosphate
in the presence of supercoiled
plasmids (3 zg ofeach
per 35-mm dish) for 12 to 16 hr at 37C under 3% CO2. The
was then removed
and the cells were rinsed twice with culture
and

in fresh

incubated

electrophysiological

medium

studies

cr

GABA-activated

for 24 hr at 37CC under

(Puia

currents

et a!.,

plasmid
medium

medium

6% CO2 before

1991).

were measured

as described

previously

single-electrode
voltage
clamp technology
in
the whole-cell configuration
(Puia et aL, 1991; Ducic et a!., 1993). The
recording
pipette
contained
145 mM CsCl,
1 mM MgCl2,
11 mM
ethylene
glycol bis($-aminoethyl
ether)-N,N-tetraacetic
acid and 10
at

room

mM
7.2).

with

temperature

4-(2-hydroxyethyl)-1-piperazineethanesulfonic
Cells

were

bathed

in 145

mM

NaCl,

5 mM

KC1,

in proximity
of the cell body, and pressure pulses lasting 5 sec
were applied
between
two successive
GABA pulses delivered
at a
frequency
of 6/mm.
Ion currents
were amplified
by a patch
clamp
amplifier,
filtered at 1500 Hz and recorded
on a chart recorder
for offline analysis.
Dose-response
curves
were analyzed
by the computer
program
of Tallanida
and
Murray
(1987)
equipped
with
a statistical
package to calculate
the ECo.
Activfty

Antiseizure

seizure
tests.
(+)-BIC-HCI
and PTZ
to a concentration
of 0.27 mol/ml
and infused
into the tail vein of rats
300 g) at a constant
rate
of0.42 ml/min.
The time ofthe
jerk and of full tonic-clonic
convulsion
was observed.
The BIC and PTZ dose required
to elicit
these
BIC and PTZ
in isotonic
saline
imol/ml
for PTZ

were dissolved
for BIC and 5
(weighing
250first myoclonic
responses

was

expressed
as micromoles
per kilogram.
With this infusion procedure
rats of 250 to 300 g reached full tonic-clonic
convulsions
in approximately 3 mm after receiving
1.26 0.11 ,mol/kg
of BIC or 253 19
of PFZ. In rats that failed to convulse
because
of treatment
with BDZ uganda the infusion
was stopped
after
20 mm.
The mean threshold
dose of BIC or PTZ needed
to induce seizures
in control
rats was subtracted
from
the dose
of BIC or PTZ required
to induce seizures
in rats treated
i.v. 10 mm earlier
with
different
doses
of BZD ligands.
Dose-response
curves were analyzed
by the computer
program
of Tallarida
and Murray
(1987)
equipped
with statistical
zmol/kg

to calculate
the EDo.
Chemical
kindling
induced
by PTZ.
duced in rats by i.p. injection of 20 mol/kg

package

+(b/X)C

placed

Kindled
of PTZ

seizures were inevery 2nd day for

for 1 hr after
each

9 weeks (Giorgi
et at., 1991). Rats were observed
PTZ injection
and seizures
were recorded according
to the following
scale:
0 = no response;
1 = ear and facial twitching;
2 = myoclonic
body jerks;
3 = clonic
forelimb
convulsions;
4 = generalized
clonic
convulsions
with rearing
and falling down episodes;
and 5 = generalized
convulsions
with tonic extension
episodes
or status
epilepticus.
The
mean value of three seizure
scores recorded
for each rat in each week
of treatment
was used for statistical
analysis.
Animals
that had a
seizure
score of 4 to 5 after three
consecutive
injections
of PTZ were
defined as kindled and their treatment
was discontinued.
Fifteen
days
after, they were treated
by p.o. gavage with various
doses of imidazenil
and diazepam
and were challenged
30 mm later with 20 imol/kg
i.p. of
PTZ. Rats were observed
for 1 hr after the challenging
PTZ injection
to determine
the incidence
of various
types of convulsions.
The dose of
imidazenil
and diazepam
that reduces by 50% the number
of convulsing
rats (ED)
was calculated
by probit analysis according to the method
of Finney
(1971).

Drug

on Motllfty

Actions

Locomotor

activity.

experimental

room

Animals

for at least

(200-250

g b.wt.)

were

kept

in

the

the experiment.
Groups
of
eight animals
received
different
i.v. doses
of the drug
under study 10
min before
the test. The locomotor
activity
(number
of photobeam
interruptions)
was measured
for 20 min by a Digiscan
Animal
Activity
Monitor
(Omnitech
Electronics,
Inc., Columbus,
OH). Dose-response
curves were analyzed
and ED values were estimated
by the computer
program
ofTallarida
and Murray (1987).
Rotarod
test. Animals
(200-250
g b.wt.) were placed on a horizontal
rod (diameter,
5 cm) rotating at the rate of 8 revolutions
per mm, 17

cm above

the bench

(Treadmill

Basile,

before

testing,

Comenio,

Italy).

Groups

acid-CsPH

(pH

2 mM

and

of the rats and was estimated

5 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfomc
acid-NaOH
(pH
7.4). GABA (0.5 M) was applied
by iontophoresis
with 30-msec
pulses
of positive
current
(25-50 nA).
Drugs
were
dissolved
in DMSO
and diluted
in bath
solution
so that
the final DMSO concentration
did not exceed 0.01%.
Drugs
were
delivered
by pressure
(2-4 psi) from 5- to 10-aim diameter
micropipettes

10 mm

7700,

the tested
drugs
i.v. In
the test phase, a rat was considered
ataxic if it fell 3 times during a 3rain period
test. The EDo value was the dose producing
ataxia in 50%

CaCl2

of six animals,

4 hr before

received

by the program

of Tallanida

and

Murray

(1987).

Barbiturate
Thiopental

into

and
sodium

the tail vein

administration

Ethanol

or ethanol

(diluted

1:1 with saline) were injected

(200-250
g) 10 mm after the i.v.
or the BDZ uganda. One minute after thiopen-

of unrestrained

of saline

Potentiation
rats

Partial Modulators

1993
tal or ethanol,
tested by gently
time

required

more

that

the righting
reflex
placing
the animals
for them

was assessed.
Righting
reflex was
on their backs and measuring
the
an upright
position.
Animals
requiring
areflexic.
No loss of righting
reflex

to regain

30 sec were considered

was observed when thiopental
(18.9 mol/kg)
were
given alone. Ten animals
were used
compounds.
The ED, values were calculated
ing

to Finney

or ethanol

(12

60 mm at a flow rate of 0.5 ml/min.

To identify
the retention
times of
the authentic
compounds
and of their
metabolites
all the elution
fractions
were counted.

mmol/kg)

Results

for each dose of the tested

by probit analysis
accord-

Binding

Loss of Righting

Table
1 shows
a comparison
of the potency
of
with that of diazepam,
bretazenil
and alprazolam
to
displace
from crude synaptic
membranes
the binding
of [3H]
flumazenil,
an antagonist
(Bonetti
et at., 1982)
of the positive
and negative
allostenic
modulators
of the GABA
action
on
In vitro.
imidazenil

Reflex

Rats were administered

imidazenil
(1.89, 3.78, 7.56, 15.12, 30.25, 60.5
and 121 imol/kg,
by p.o. gavage)
and 30 mm later a dose of diazepam
(35, 50, 70, 105 and 130 mol/kg
i.v.), so that for each dose of imidazenil
5 doses
of diazepam
were
tested.
One
minute
after
i.v. dia.zepam
the
righting

reflex

was

righting

assessed.

reflex

were

The

EDre

values

by probit

determined

for

inducing

analysis

loss

according

GABAA

Anticonflict

to Finney

the

and

experiments,

by Vogel

Briefly,

Each
chamber

punishment

behavioral

as modified

(180-240

experiment.

habituation

the

et a!. (1971)

animals

virtually

ing [3Hjflumazenil
at least
one order

Activity

these

receptors,

tivity.
In membranes
cerebella,
the potency

of the

(1971).

o_

Experiments

(1971).

Diazepam-Induced

For

1021

of GABAA Receptors

g) were

rat

was

by Giusti

deprived

allowed

(a chamber

devel-

et a!. (1991)

of water

to

identical

paradigm
for

become

to the

was used.

72

hr

familiar

testing

with

a
but

However,

displace

membranes

before

chamber

to

of

in cerebellum
aiprazolam

of intrinsic

from
(K

rat

modulatory

brain

the

potency

[3H]flumazenil

rat

cord

spinal

ofbretazenil
and
that
of diazepam

of imidazenil
from

the

and

crude

is considerably

and cortex
and similar
(see table
1). Furthermore,

ac-

cortices
and
M) in displac-

5.10_b

is slightly
higher
than
that
of magnitude
higher
than

alprazolam.

tazenil

devoid

prepared
of imidazenil

bre-

synaptic

lower

than

that

to that
of diazepam
and
it should
be emphasized

without
water) for 5 mm immediately
before the test in order to avoid
exploration-induced
delay of drinking.
After
this
period,
the rat was
transferred
to the testing
chamber
(28 x 20 x 20 cm with a stainless-

that in the cerebellum

and the cortex
the slope
(nH) of the [3H]
flumazenil
displacement
curve
by imidazenil
is close
to 1, but
in the spinal
cord this value
is 0.57. GABA
(50 M)
added
to

steel

cerebellar

membranes

imidazenil

(IC,o

grid

floor)

(Lafayette

Instruments,

Lafayette,

IN).

Water

was

provided
with a stainless-steel
drinking
tube (see Giusti et al., 1991 for
details).
Each rat was allowed to complete
a 10-sec licking period before
the start of a 3-mm test period.
The number
of licking
periods
(each

period

equal

to

3 sec

of cumulative

drinking)

was

recorded

in unpun-

ished rats and in rats punished

with an electric
shock (0.8 mA, 1 sec
duration)
delivered
through
the drinking
tube after
each drinking
period.
Programming
for the test session
was controlled
by a solidstate modular
programming
apparatus
(Atto Instruments,
Washington,
DC). Rats that failed to start drinking
after
being placed for 5 mm in
the test chamber
were excluded
from the experiments.
Ten animals
were used for each dose ofthe drugs under investigation.
Alprazolam,
diazepam,
bretazenil
and imidazenil,
suspended
(by 30-sec
sonication)

The

in saline,

anticonflict

were

effect

injected

i.v.

of a drug

protection

10 mm

before

was calculated
A-K
B -

the

test.

Where

A = average
number
of licking
periods
in a group
of drugrats receiving
punishment;
B = average
number
of licking
in a group of drug-treated
rats not receiving
punishment;
and
K = average
number of licking periods
in a group of saline-treated
rats
receiving
punishment.
In the absence
of punishment
(B ), the average
number
of licking periods
observed
in drug-treated
rats did not differ
significantly
from those in saline-treated
rats, at the doses tested.
The relative
drug
potency
(ED
dose of drug that gives 50%
protection)
in the conflict
test was derived
from the percentage
of
protection
data by probit analysis
according
to Finney
(1971). For all
these parameters
the fiducial
limits were referred
to P = .01.
treated
periods

Determination

of [H]FIumazenil

or [1C]lmidazenii

in

the

Brain

At different
times after i.v. injections,
[3H]flumazenil
or [4C]imiwere extracted
from the brain with chloroform-methanol
(2:1,
v/v) containing
1% acetic
acid.
The
chloroform-methanol
extract
was
dazenil

lyophilized,
resuspended
in H2O
Millipore
filter and injected
on a
x 20 cm) equilibrated
with H2O
and [4C]imidazenil
were separated
the column
with a gradient
from

1%

TFA,

filtered

through

the

rat

a 0.45-

5- Bondopack
C15 column
(250 mm
containing
1% TFA. [3H]Flumazenil
from their metabolites
by eluting
0 to 60 ml of acetonitrile,
1% TFA in

1.7-fold

of GABA

the

brain,

imidazenil

(1

M)

fails

to

glial

cells;

from

[3H]baclofen

dopamine

tansenin

from

and

from

GABAB

[3H]naloxone

or

vivo.

The

from
[3H]4Cl

recognition
receptors

sites
located

[3H]

[IJ

relationship

on
in

spiperone
[3H]ke-

[3H]glycine,

a-[3H]amino-3-hydroxy-5

from

glutamate

recep-

[3H]-3-hydroxyl-[phenyl)-N-(1-propyl)or sigma
receptors;
H]L-356,260

pipenidine
from opiate
cholecystokinin
receptors,
receptors
and [3H]CP-55,940
In

prepared

receptors;
or

nM,

presence

receptors;

acid

propionic

in the

receptors;

5-hydroxytryptamine2

of

0.050

displace

5-hydroxytryptamine1

[3H]dibenzochlorohepteneimine

potency

0.58

diazepam
or [3H]PK
11195 bound
to their
mitochondrial
diazepam
binding
inhibitor

tors;

100%

by

presence

n = 6) and by 4.6-fold
that of diazepam
(IC
of GABA = 8.3 1.4 nM, n = 6).
By using crude cortical
synaptic
membranes

methyl-4-isoxazole

as follows:

increases

in the

pindolol
from
from cannabinoid
between

the

from

adrenergic
receptors.

beta

fractional

occupancy

by [3H]flumazenil
of high affinity
recognition
sites for BZDs
located
on GABAA receptors
and the dose of alprazolam,
bretazenil,
diazepam
and imidazenil
injected
into rats 10 mm
before the measurement
of [3Hjflumazenil
binding
is shown
in
figure

and

2. The

the

mazenil

figure

2 insert

reports

the

binding

for the

displacement

nonspecific
in the

equipotent

in

same

rats.

Imidazenil

displacing

and

[3HJflumazenil

ID,

the

slope

factor

of [3H]flu-

bretazenil

are

from

virtually

forebrain

and

they are 10- and 20-fold

more potent
than diazepam
or alprazolam. The four drugs have the same rank order of potency
in
displacing
[3Hjflumazenil
from
the
cerebellum
(data
not
shown).
Efficacy

of Diazepam,

Imidazenil

and Bretazenil

on the

Positive Allosteric Modulation of GABA-Elicfted Cr Currents

in Native

and Recombinant

Imidazenil

fails

GABAA

to displace

Receptors

[3H]GABA

(t[rsS]butylcyclophosphorothionate)
aptic

membranes

of

cortex

(20 nM)

(5 nM)
or

cerebellum

(data

or [S]TPBS

from

crude

syn-

not

shown)

and

1022

Giusti

et al.

Vol. 266

TABLE 1
Inhibition
by lmidazenhl
and Congeners
of [3H]flumazenil
binding
to crude synaptosomal
membranes
of rat CNS structures
The concentration of [3HJflumazenhl in these experiments was 1 nM. AS binding experiments
were COndUCted in presence of 50 M bicuculline.
Cerebe1um

Cortex

IC50

IC,,

flH

nM

Diazepam
Alprazolam
Bretazenil
Imidazenil

r1

IC,

nM

38 2.3
21 1 .6

1.5
0.90

Spinal Cord

0.11
0.080***

1.0

nM

42 2.8
1 8 1 .9
2.6 0.36

1 .2 0.023
0.83 0.085
0.10

1.0 0.020

1.0

1 .1
1 .1

43

1 .8
65 33*.
97 4.2**
38 4#{149}5**

0.048
0.080
1.0 0.20
fl 0.010

0.23***

..

The lC values of bretazenil,

aiprazolam
and imidazenil
in the spinal cord are statistically
different
(P
for imidazenil are statistically
different (P < .001) from diazepam and aiprazolam.
but not from bretazenil.

< .01) from

0.90
0.67
0.60

0.15
0.090
0.25

0.57 0.22

that of cortex and cerebellum;

the

values

100

80
C)
C
U

Aiprazolam
Dazepam

0.

60

150

C)
C)

Bretazenil

(V

40

(U
C

Diazepam

C)
(U

100

C
Qi

20

50

1.

U1

0
0.01

0.1

Mmol/kg
Fig. 2. In vivo
imidazenil

displacement

from

10

-9

100

forebrain

of

[3H]flumazenil

by

and its congeners.

Parameter
lDmol/kg)

Slope factor

Diazepam

Alprazolam

1 .8 0.2
1 .6 0.21

2.2
0.93

Nonspecific

12

Bretazenil

0.82

lmidazenil

0.20

0.22 0.039

0.027
1 .3 0.24
10

0.33

8.9

-7

log

Lv.
rat

-8

1 .4

-6

-5

[ M

Fig. 3. Dose-response
curve for the positive modulation of GABA-gated
Cl currents by diazepam (S) and imidazenil (U) in cortical neurons. Each
value represents
the mean S.E.M. for six cells. The EC of diazepam
was approximately
50 nM and that of imidazenil
was approximately
10
nM.

0.18

12

(%)

binding

[3H]flumazenil
(50 MCi/kg, 0.6 nmol) and BZD ligands were injected
i.v. in rats 3 and 1 0 mm, respectively,
before sacrifice.
Results
are
expressed as mean S.D. , lD values were calculated as described in

test (see Materials

and Methodsfl).bNonspecific

total binding) was determined

of clonazepam.

binding (percentage

after administration

of a 20-zmol/kg

of
dose

z
fails

to elicit

cortical

cells

receptors
not

chloride
in primary

expressed

shown).

modulator

moral

action

neurons

cell

lines
the

GABAA

culture

receptors
(i.e.,

dose-response

tumor
acts

of GABA

GABAA

kidney

cortical

imidazenil

in primary

native

receptors

or in recombinant

in 293 human

neurons

recombinant

in

cultures

However,
of the

cortical

currents

on
(fig.

kidney
as

rat

3) or

expressed
in
DNA
transfection)
relationship

293

(data
of

rat

a variety

of

for

the

allo-

latory
efficacy
of a maximal
(i0
M) effective
dose of imidazenil
at alpha-1-beta-1-gamma-2;
atpha-1-beta-1-gamma-1;
a!pha-2-beta-1-gamma-2;
atpha-3-beta-1-gamma-2;
and alpha-5-

than

that

recombinant
GABAA
receptors
is uniformly
to that
of bretazenil
and is consistently
lower

of diazepam

(fig. 4).

be

human
tu(fig. 4). In

stenic
modulation
of GABA
evoked
Cl currents
by imidazenil
and diazepam
indicates
that
imidazenil
is 4 to 5 times
more
potent
in causing
a 50% positive
modulation
of GABA
action,
but less efficacious
than
diazepam
(fig. 3). The positive
modu-

beta-1-gamma-2
low and similar

I-

allostenic

receptors
on

GABAA

cell line

a positive

native

of

DL4ZEPAM

BRETAZENIL

IMIDAZENIL

Fig. 4. Efficacy of the positive allosteric modulators of GABA action on

native and recombinant
GABAA receptors.
Diazepam,
imidazenil
and
bretazenil

human

potentiate
kidney

GABA-gated

cr

current

with

different

efficacy

in

293 cell lines expressing

GABAA receptors
containing
the
and various molecular forms of alpha and gamma subunits

beta-i subunit
and in primary culture of rat cortical neurons.
Each bar represents
the
mean S.E.M. of 8 to 10 cells. Maximally
efficacious
doses (10
M) of
the drugs given by pressure application.
*)
< .05 when compared
to the

effect elicited by diazepam.

Partial Modulators

1993

When

cells

transiently

gamma-2
were
and diazepam

transfected

of GABA-evoked

Cl

to values
(fig. 5).

to those
elicited
inhibition
of

similar
similar

modulation
(fig.

with

treated
simultaneously
(i0
M), the efficacy

of GABA

currents

with
of the

elicited

action

can

atpha-1-beta-1-

imidazenil
positive

The

M)

(i0
modulation

by diazepam

was

ties

be

selected

with

potency

and

clonazepam

GABA

of imidazenil,

diazepam

at GABAA

quantifying
receptor
vulsions

efficacy

and

action

bretazenil

the

shift

in

in vivo

the

threshold

and

PTZ

Both
BIC and PTZ are blockers
fail to interact
directly
with

imidazenil,
bretazenil,
these
receptors.
BIC

diazepam
is an isostenic

sites
GABA

ing

of GABA

to the

channel

domain

allosteric
dose-related

increase

tonic-clonic

convulsions.

dose

does
of

in the

not

the

of GABA
to interact

imidazenil
action
on
with
the

the

receptors.

As shown

GABAA

allostenic

BIC
the
and

the

because

dose;

and

modulator
above

PTZ

modulator
clonazepam

tested
this

remains
are

value

of bretazenil,

clonazepam,

of

the

GABAA

receptor
binding

function
sites
for

dose of bretazenil.
imidazenil
action

Figure
fold

1991),

presumably

ionotropic

receptors

pam

(fig.

potent

dazenil

increase

in the

thresh-

comparable

to

alprazolam.

However,

or PTZ
6 and

threshold

when

higher

is

and

hand

against
that
are

a synergistic

potency

are
ofthe

seizures

coad-

different

(compare

and bretazenil
are
against
BIC-induced

figs.

significantly
seizures.

action
of imidazenil
(i.e., against
lasting
than that of a 10-fold larger

is also

kindled

more

seizures

in dosage,
than
that

potent

than

(no graded

imidazenil

the
of

diaze-

response).

is approximately

diazepam

in this

test.

fail

anticonflict

effects

at doses

to modify

potency

diazepam

sites on
positive

doses

of

after

20-

(data

dose

of bretazenil

not

is

clonazepam

imidazenil

and

behavioral

a larger
[3H]flumazenil

of the

GABAA
receptors
lasts
approximately

higher
2 hr

Bretazenil
neither

and

decreases

contrast,
diazepam
and induce
ataxia

diazepam.

El

only

on

of

reducing
in vivo,

imi-

conflict

diazepam,

doses

2) that,
The

punishment-induced

of bretazenil,
the

(table

behavior.

and

bretazenil
suppression

percentage
recognition

than
sites

(see table
5). In this test
4 hr, whereas
the action
is practically

undetectable

shown).

Locomotor
Activity, Ataxia, Potentiation
of Thiopental-,
Ethanol-Induced
and Antagonism
of Diazepam-lnduced
Loss of Righting Reflex

bretazenil
convulsions

drinking

reducing

that

located
action

of a 10-fold
of

in

or alprazolam

that
are
imidazenil

effecdose

unpunished

that
are 50%
efficacious
in
caused
by punishment
occupy,

alprazolam.
Importantly,
against
BICand
PTZthat elicited
by diazepam
3- to 10-fold
higher
than

alprazolam

se,

per

allostenic
(figs. 3 and

binding
7, each

efficacy
of imidazenil,
from
BIC and PTZ

This

maximal

convulsant

positive
cause

increasing

are

Figure

alprazolam

ofthe

that

shows

to elicit

and congeners
GABAA
receptors

other

we
major

Test

Imidazenil

dose
with

brain.

the

and

the

(Bar-

threshold

action.

the

no

On

Imidazenil

that

after

interval

or PTZ,

despite
the difference
at least
2 hr longer

11 demonstrates
more

Anticonflict

even

10).

mm

time

BIC

PTZ-induced

in the PTZ-induced

all the
tested

GABA

and

10

efficacy
of cliazepam
low efficacy
values

imidazenil
PTZthan

In fact,
persists

with
receptor

indefinitely

BIC

on BIC-

in a dose-

the
the

diazepam

However,
the anticonvulsant
BIC) is considerably
longer

con-

whereas

is lower
than
that
of diazepam
and
the
maximal
efficacy
of imidazenil
induced
seizures
is only 30 to 40% of
and alprazolam,
but its potency
is
that

7) reveals
that
potent
against

a characteristic

the

unchanged

given.
The
in protecting

tested

6 and
more

et a!.,

in figures

has

drugs

at

in

PTZ
proper-

0.1 to 2.5 mol/

constant
with

9). A comparison

of

by bind-

of

modulators
4), but fail

expected

(fig.

by

(Puia
action,

this

to rise

ministered

when

and

increases

detectable

of imidazenil.

is operative

the

convulsions
are

BIC

from

Moreover,

was

shifts
toward

evaluated

tonic-clonic

1 AM.

which
the

inhibit
pharmacokinetic

of imidazenil

metabolites

action

to

of imidazenil

shows
that
imidazenil
the BIC convulsions

bretazenil

modulators

to

to trigger

and
aiprazolam
located
on
antagonist
of GABA
at the

or PTZ

However,

continue

positive

BIC

0.05

probability

dose

content

doses

injection,

the

to induce

1983).
6 and 7 show that pretreatments
modulators
of the GABAA

Figures

tive

from

imidazenil
to its peculiar

brain

(for

characteristic

alprazo-

of
due

were

of GABAA
the specific

GABAA receptor
recognition
PTZ inhibits
allostenically
ken et at.,

bretazenil,

in increasing

receptors

antagonists
BIC
(graded
response).

doses

i.v.)

imidazenil
imidazenil

The

old

the

manner

kg

In Wvo Efficacy
and Potency of Positive Allostenc
Modulators
of GABAA Receptors
in Inhibiting
BIC- or P12Induced Seizures

that

because

by imidazenil
applied
alone
diazepam
positive
allostenic
obtained

efficacy
is not

related

reduced

5).

lam,

low

convulsions

1023

of GABAA Receptors

Diazepam

10 pM

Imidaxenil

10 pM

injected

locomotor

activity

and alprazolam
for doses
as small

i.v.
nor

up

to

60

produce

reduce
locomotor
as 2 to 3 mol/kg

zmol/kg
ataxia.

In

activity
(table

Diazepam
+

10 pM
Imidazenil
10 pM

imidazenil

Bretazenil

10 pM

Diazepam
+ Bretixenil

10 pM
10 pM

Fig. 5. Antagonism
imally

efficacious

GABA-activated

by imidazenil and bretazenil at maxmodulatory

doses of diazepam
on
Cl currents in human kidney 293 cell

lines expressing
GABAA
receptors
containing
alpha-i
beta-i -gamma-2
subunits.
Each
bar represents
the
mean S.E.M. for five cells. P < .05 when compared
to diazepam-treated
cells; P < .01 when compared to
diazepam-treated
cells.

1024

Giusti

Vol. 266

et al.

Fig. 6. Increase in BIC seizure threshold by various positive allosteric

modulators
of GABAA receptors. In these
experiments the threshold dose of BIC needed to induce
tonic-clonic convulsions in control rats (1 .26 zmo1/kg i.v.)
was subtracted from the dose of BIC required to induce
convulsions in rats treated I 0 mm earlier with different i.v.
doses of the anticonvulsant
drug. Each value is the mean
S.E.M. of 8 to 10 rats. All values depicted
here are
significantly higher(P < .05)than controls. In rats that failed
to convulse because
of treatment with various positive
allosteric modulators
of GABAA receptors, the infusion was
stopped after 20 mm and the equivalent value of BIC
infused was used for the graphic
representation
of the
data.

16

.1

Imaz.nil

C
0.01

0.1

i#{243}o

10

pmol/Kg

1000

$00

Fig. 7. Increase
in PTZ seizure threshold by various
positive allostenc modulators of GABAA receptors. In
these experiments the threshold dose of PTZ needed
to induce tonic-clonic convulsions in control rats (253
zmol/kg iv.) was subtracted from the dose of PTZ
required to induce convulsions in rats treated 10 mm
earlier with different
i.v. doses
of the anticonvulsant
drug. Each values is the mean S.E.M. of 8 to 10
rats. All values depicted here are significantly higher
(P < .05) than controls. In rats that failed to convulse
because of the treatment, the infusion was stopped
after 20 mm and the equivalent
value of PTZ infused
was used for the graphic representation
of the data.

jooo

400

Im
Srs#{149}az.nil
C
0.01

0.1

100

10
i.v.

pmol/Kg

1$

V.hkl.

6
Imidaz.nil

0.3

lmidaz.nil

0.6

lmidaz.nil

1.2

I
.1

-I

00
Diozpom

10
(pmol/Kg

20

i.v.)

antagonizes the anti-BIC seizure action of diazepam.

Note that imidazenil (0.3, 0.6 or 1.2 ,mol/kg
i.v.) possesses
anti-BIC
seizure effects in the absence of diazepam. lmidazenil was infused i.v.
immediately before diazepam. BIC was injected 10 mm later. Each value
is the mean S.E.M. of 8 to 10 rats.

lo

Fig. 8. Imidazenil

AIpsazolam

(pmol/Kg

Lv.)

9. Diazepam
potentiates
the anti-BIC seizure action of alprazolam.
Diazepam
(1 .76 and 0.88 rno1/kg i.v.) was injected immediately before
aiprazolam. BIC was infused 10 mm later. Each value is the mean
Fig.

S.E.M. of 8 to 10 rats.

1993

Partial

2
of imidazenil
and ataxia
TABLE

3.5

and congeners

Effects

0
C
0
U

3.0

on conflict, locomotor

2.5

.0

CU

2.0

ED,,,

1.5

Anticonflict

Drugs

Locomotor

1.0

pincl/kg

C)

Diazepam

2.0

0.5

Alprazolam
30

Fig. 10. Time course

against

of 10 rats.

60

120

Time

(mm)

of the anticonvulsant

180

4.0

Clonazepam

240

of bretazenil

and

I0

4.

DIAZEPAM

administered

were

>60

>60

ED,,,

7.
0.1

58

Diazepam

LL.

10

Alprazolam

(48-70)
>60

Clonazepam

>60

Bretazenil
Imidazenil

Fig. 11. Comparison

of the potency of imidazenil and diazepam

on the
induced by PTZ. Imidazenil and diazepam
were administered by p.o. gavage 30 mm before challenge with 20 molfkg
i.p. of
PTZ in animals kindled
as described
under MateriaJs
and Methods.
Groups of 10 rats were used for each point so that each experimental
group consisted
of 40 rats. A group of control rats was administered
saline (1 mI/kg) per p.o. gavage
30 mm before challenge with PTZ. The
ED values and the CL (P = .01) were calculated from the percentage
of protection
data by probit analysis according
to Finney (1971).
kindling

1.9

and bretazenil,

potentiate
reflex

the
(table

unlike

diazepam

thiopental3). Moreover,

and

or ethanol-induced
rats pretreated

2.1

>60
>60

ED,,, for the various BZDS in inducing loss of righting reflex when given alone
with 12 mmol/kg Lv. of ethanol or 18.9 mel/kg iv. of thIOpentaI.

TABLE 4
Antagonism

by imidazenil

of diazepam-induced

of the

motor

impairment

or of the

potentiation

figure

limits in parentheses.

Confidence

Diazwn.lnduced
Loss of
Ri1thg Reflex (ED,,,)

,mof/kg

loss of
imida-

Aiprazolam,

one of the FAM,

in

doses
or

in

that
potentiatmg

are

and diazepam,
50%

efficacious

ethanol-

3 with

those
of
in vivo

absence

decreasing

thiopental-in-

(48-70)

1 .89
3.78
7.56

56
59
67

(48-67)
(51-69)
(50-88)

81

(45-1 50)

87 (73-100)
110 (97_130)**
1 15 (1 00-1 30)

lmidazenil was njected Lv. Immediately before the test.

Tested at five doses between
35 and 130 Mmol/kg

Methods).

in

and

of the

one of the SAM,

58

15.12

we have

binding

MJr1c/kg iv.

30.25
60.5
I 21

reflex,

2.

loss of righting

reflex

zenil p.o. and then given 58 imol/kg

of diazepam
i.v. fail to
exhibit
the characteristic
loss of righting
reflex
elicited
by
diazepam
alone (table 4). The imidazenil
antagonism
is dosedependent.
To facilitate
the understanding
of the correlation
existing
between
fractional
occupancy,
by various
BZDs, of the
BZD
recognition
sites located
on GABAA
receptors
and the
ethanoland thiopental-induced
loss of righting
assembled
in table 5 the data of tables
2 and
the percentage
of inhibition
of [3H]flumazenil

5.4
(3.5-7.6)
>60
>60

or together

alprazolam,
with

1.7
(1.2-2.5)
2.6
(1.5-4.6)

(1 .6-9.0)
>60
>60

lmidazenr

2). Imidazenil

Thiopent
iv.

(1 .8-4.6)
1 .8
(1 .3-2.5)

100

pmol/kg,o.s.

activity

>60

,in/kg

ED,,,,-0.75

given

0.32

>60

Ethad

None

(0.21-2.6)

motor

iv. 10 mm before the test. Values in parentheses

IMIDAZENIL

.1

from

0.46

Drugs

when

4.1

(8.3-37)

fail to
righting

4.2

indicate 95% CL.

3.8 0.32

(1.7-3.1)
2.9
(1.0-5.9)

Imidazenil

The BDZs

19

ED,,,,-

I-

2.2 0.27

TABLE 3
Loss of righting reflex induced by various positive allosteric
modulators
of GABAA receptors in rats treated with doses of
ethanol and thiopental which per se fall to produce
loss of righting
reflex

3.

chemical

4.3
(3.6-5.2)
2.4

Bretazenil
action

BIC-induced
seizures. Each bar is the mean S.E.M.
.05 when imidazenil-treated
rats are compared with
rats.

<

bretazenil-treated

2.4 0.25

(2.3-6.9)
10

imidazenil

iv.

2.6 0.32

(1.4-2.8)

taken

effe

U)
C

severity

activity

Drugs were Injected 10 mm before the test. Each ED,, value of locomotor activity
and ataxia is the mean S.E.M. calculated from dose-response curves (Tallailda
and Murray, 1987). For the anticonflict effeCtS, each value is the mean Obtalned
from dose-response curves calculated with the method of Finney et a!. (1971).
Confidence
limits in parentheses.

>
0

1025

of GABAA Receptors

Modulators

<

.05 or #{149}P
of imidazen.

duced
loss
flumazenil

<

with rats treated

of righting
recognition

reflex,
sites;

occupy
about
in contrast,
the

bretazenil,

when

given

dazenil

and

of

[3H]flumazenil

the

.01 when compared

recognition

(see

in doses
sites

50%
two

that
located

Materlals

and

with dIazepam

of the
PAMs,

occupy
on

[3H]
imi-

100%
GABAA

in

1026

GiustI et al.

Vol. 266

TABLE 5
of BZD recognition
sites on GABAA
and anticonflict effect by various BZDS
Occupancy

and extent of motor coordination

receptors

D
Test

A
Receptor

ED

50

mof/kg

Anticonflict
Thiopental
potentiation
Ethanol potentiation
Locomotor activity
Ethanol potentiation

2.0

53

4.0

63

1 .7
1 .9

47
51

2.6
1 .8

54
46

>60
>50

-1 00
-1 00

2.6

63

2.2

50

>60

--100

1 .9

51

1 .8

46

>50

Locomotoractivity

2.6
2.4

63
60

2.2
3.8

50
62

>60
>50

-100
-100
-100

fail

to alter

ethanol-

motor

activity,

elicit

or thiopental-induced

ataxia

,rnof/kg

2.4

50% of specifically

loss of righting

reflex.

Massotti

et aL,

presence

of

receptors

or

receptors

aL (1993)

can
be classified
to criteria
proposed

to differentiate

population
drugs
are

of
used

to relieve

psychoses

the anxiety

therapeutic
by

sedation,

action

as

of various

use

a large

ataxia,

associated

with
Rickels

and

of diazepam

and

alprazolam,

spectrum
amnesia

and

side

psychotic

disorders,

and

potent

and recombinant
to the chemical

native

belongs
boxamides

erties

and

1987).

however,
effects

such

(FAM)

(figs.

considered

the

anxiolytic

drugs

imidazenil

anticonflict

to those
6-11).

and

PAM

and

of diazepam

However,

class

similar

to

the

devoid

of

potentiation

side
of

flumazenil,

ethanol

such
and

of

classes,

as

whereas

that

slightly
belongs

a4,ha-2
is always
(fig.

is largely

potency

cerebellar

so far

tested
list).

for

the

zenil
for
diazepam,

and
sites

the
for

the

from their binding

In the spinal cord,
[3Hjflumazenil
but smaller
than

Hill coefficient
BZDs
in the

characteristics.
placing

flumazenil

In

potency

binding

the

and

sites in the brain

(see results
however,
the affinity
of imida-

binding
sites
is similar
that
of bretazenil
or

is below unity (table

spinal
cord appear
case

of alpidem

heterogeneity
(Santi

et

ligands

and

for
at.,

1988;

to that
alprazolam

GABA
supporting
the
PAM
class.
GABAA
receptor

inferior

(30-50%)

reduces

the

GABA-elicited

1). Thus,
to have
zolpidem,

the
Langer

low

spinal

cord
et

al.,

dis-

[3H]
1990;

ligands

with

by

in which

positive
C1

intrinsic
Conto displace
[3H]

and

was

shifted

that
imidazenil
tested
on various
imidazenil
efficacy

oftniazolam

(a FAM)
et at.,

1991;

(Ducic
Ducic

et

with diazepam,
imiefficacy
of diazepam

modulatory

currents

no

GABA.

membranes

the conclusion
Indeed,
when
subtypes,
the
to that

appears

to act

as an

allo-

antagonist.
Finally,
imidazenil
is a potent
of BICand PTZ-induced
seizures,
but its anticonaction
has an efficacy
lower than that of diazepam
or

proper
of PAM.
In several
pharmacological
ative and muscle
relaxant
haves as a truly allostenic
antagonistic

fractional
fails

binding
peculiar

sub-

modulator

intrinsic
of

that the low

cord is due

receptors,

of imidazenil

vulsant
alprazolam.
When
given
together
again
acts as an allosteric
modulator
reducing
diazepams
anticonvulsant

receptor

electrophys-

gamma-i

unaffected

or cortical

antagonist

neurotransmitter

the

including

of GABAA

with

known

GABA-

containing

to conclude
to the spinal

receptors,

effects

or all the

on

subunits
in combination
less active
in receptors

4). However,

region.
sites

Consistent

cells

of the

GABAA

efficacy

receptors

GABAA

or allostenic

the

from
by
to

steric

its PAM profile,

imidazenil
reduces
the CNS
of SAM
(i.e., diazepam,
fig. 8; table 4).
Imidazenil
shows high (subnanomolar)
affinity
for the brain
allostenic
binding
sites located
on the extracellular
domain
of
the GABAA
receptors
in which
the BZDs
also bind,
but 1O
M
imidazenil
fails to displace
4Cl diazepam
or PK 11195 from
the mitochondrial
diazepam
binding
inhibitor
receptor
located

indication

recombinant

low

et a!., 1993) or diazepam

(a SAM)
(Puia
al., 1993).
Moreover,
when administered
on

barbiturates.

of PAM
flumazenil)

to diazepam,

trary

dazenil

ataxia,

of

sufficient
in the binding

of GABAA

(i.e.,

activity

recombinant

the

an

BZD binding
sites
are located,
are influenced
by the presence
of GABA
in the assay
medium
(GABA
shift)
(Braestrup
et at.,
1982).
The binding
of SAM
and FAM
is enhanced
by GABA,

flumazenil

is

and
but

present
in this CNS
The allostenic
modulatory

is greatly

which

on

depressant

in glial

is the slope factor (see fig.

considered

equally

elicited

of imidazenil

units

alprazolam

with

gamma-i
subunits
data do not appear

to an abundance

prop-

and

effects

--100
-100
-100

in vivo,

spectrum

acts

currents

is

Imidazenil
car-

anticonvulsant

(SAM)

C1

as

in several

pure antagonist
(Bonetti
et at., 1982)
belonging
to the SAM, FAM and PAM

is virtually

sedation

the

of

gated

in addition

GABAA receptor
subtypes.
family
of imidazobenzodiazepine

possesses

comparable

drug

>50

on gamma-2

a broad

subtypes,

including
iological
potency

other

degree oftolerance
and dependence
liability
(Garratt
et at., 1988;
Oswald,
1991).
The diazepam
and alprazolam
potentiation
of
the CNS
depressant
effects
of ethanol
and
barbiturates,
imposes
a further
caveat
in the clinical
use of
these agents
(Haefely
et at., 1990).
In this report
we have described
the pharmacological
profile
a new

apha-3

alpha-i,
alpha-3,
apha-5
with gamma-2
subunits,

on

to a higher

of imidazenil,

-100
-100

containing
GABAA
subunits
on gamma-i
containing
GABAA
et at., 1989; Puia
et aL, 1991). Imidazenil,

BZDs

Schweizer,

of unwanted

98

>60
>50

>60
>50

has been
subunits

receptor

depression

et aL, 1988;

1991)

SAM
and
by Ducic
et

structurally
different
GABAA
receptors.
Both
in the treatment
of anxiety
and panic
disorders

(Ballenger

plagued

the

2.9

bound [3Hjflumazenil

alpha-5

(Pnitchett
tested
on

when

alprazolam
according

96

or to poten-

Discussion
Diazepam
and
FAM,
respectively,

occupancy

#{176}

smof/kg

the dose displacing

occupancy

receptors

or ethanol potentlation
InidaZen

mo1/kg

Ataxia

The

ED
#{176}

Receptor
occupancy,
100/[(lC,,,,/EDCU)C
+ 1], where
ED,,, denotes
2) and ED,,, refers to the oftective dose from tables 2 and 3.

and

thiopental

Bretazeni
Receptor
occupancy

ED,,,

occupatcy

tiate

impairment,

to have

BZD

tests which are indicative

of sedeffects
in patients,
imidazenil
bemodulator
of GABAA receptor
with

action

recognition

an intrinsic

with
diazepam,
imidazenil
with antagonistic
activity
efficacy
to the level of that

because

site
action

at

occupancy

(table

5), but

doses

that

cause

close

to

100%,

blocks

the

sedative

and muscle
relaxant
effects
of large doses of SAM and FAM
(table 4).
In contrast,
although
alprazolam
and diazepam
induce
sedation, ataxia and potentiate
the effects of ethanol
at a fractional
occupancy
(approximately
50% of the brain
BZD recognition

a
it

Partial Modulators

1993

sites)

which

is also

(table
5),
potentiate
flict

one

imidazenil
the effect

actions

and

binding
sites.
and monkeys
failed

the

to

required

fails to
of ethanol
occupy

also
here)

sedative

anticonflict

induce
ataxia,
in doses that

virtually

Imida.zenil,
(not shown

produce

for their

100%

effects

sedation
or to
produce
anticon-

of

the

brain

BZD

when injected
i.v. to cats, dogs
at doses as high as 35 ,mol/kg,
effects,

ataxia

or

to

potentiate

ethanol-

or barbiturate-induced
loss of righting
reflex.
In our studies,
imidazenil
has a pharmacological
profile
similar to that ofbretazenil
(table
5), which
also possesses
a profile
in

pharmacological

and

tients

seizures

with

half-life.

Thus,

side

effects.

or sedation

of the
imidazenil

receptor
potent

binding
sites
antagonizes

occupancy
and

has

has

a better

it is virtually

devoid

imidazenil

because

anxiolytic

ataxia

100%

lower

it is more

bretazenil

predict

not produce

occupy
approximately
flumazenil.
However,
though

that

tests

does

Moreover,

the

data

so

action

in pa-

at doses

which

labeled
by [3HJ
BIC
and
PTZ

than

bretazenil,

longer

separation

index

of unwanted
far

a!-

biological

lasting

than

locomotor

available

(P.

Giusti,

unpublished
observations)
indicate
that imidazenil
has
low level of tolerance
and dependence
liability.
In considering
the mechanisms
by which high-potency
of GABAA
receptors
achieve
it must
be kept in mind that
tors

determined

is

by

the

pharmacological

the efficacy
maximal

range

efficacy

PAM

selectivity,

of the

modula-

of GABA

on

SAM,

facilitating
receptors,

and
in

GABA
action
in the majority
or in selective
GABAA
tend
to minimize,
or at times
to obliterate,
the

physiologically

graded

receptor

quanta

of GABA
released
PAM
such as imidazenil,
because
of their intrinsic
GABA

action

graded

responses

(figs.

4 and

given

ing

therapeutic

and

not

ideally

suited

rodents

the

nonexistent

they

5) tend

to GABA

a consequence,

responsiveness

to

a range

of

from nerve terminals.

By contrast,
even at maximally
efficacious
doses,
low efficacy
in positively
modulating
to preserve

released

the virtual
unwanted

from

overlapping
side effects,

for therapeutic

the

physiologically

nerve

terminals.

application;

unwanted

side

appear

to have

As

of the doses
causSAM
and FAM
are

effects

however,
of

PAM

because

are

a brighter

possible
therapeutic
of the unwanted

use is concerned.
side effects
cannot

future
as far as their
Probably
in rodents
some
even be elicited
with PAM

because

ability

as antagonists

of their

modulation

when

the

which
can be projected
efficacy
of the neurally

to function
efficacy

surpasses

a certain

to be not greater
released
GABA.

of allostenic
limiting

than

value

doubling

the

H.,

FERRARESE,

C.,

VIcINI,

S.

neurons
in dissociated
cell cultures
localization
with specific
modulator

AND VACCARINO,

of neonatal
peptides.

F.:

Subsets

of GABAergic

rat cerebral
cortex
Dcv. Brain
Res. 39:

show

co-

193-204,

1988.
J. A. AND ITO, Y.: Semi-preparative
purification
of an endogenous
ligand
for brain serotonin-2-receptors
by coil planet
centrifuge
counter-current
chromatography.
J. Chromatogr.
538:
177-185,
1991.
BALLENGER,
J. C., BURROWS,
G. D., DUPONT,
R. L., LESSER, I. M., Novas,
R.,
PECKNOLD,
J. C., RIFKIN,
A. AND SwINsoN,
R. P.: Alprazolani
in panic
disorders
and agoraphobia:
Results
from a multicenter
trial. I. Efficacy in short
term treatment.
Arch. Gen. Psychiatry
45: 413-422,
1988.
APUD,

BARKER,

J. L.,

MCBURNEY,

R. N.

AND

MATHERS,

D. A.: Convulsant-induced

depression
of amino
acid responses
in cultured
mouse
spinal
neurones
under
voltage
clamp. Br. J. PharmacoL
80: 619-629,
1983.
BoNErri, E. P., PlEiti, L., CUMIN, R., SCHAFNER, R., PIERs, M., GAMzU, E. R.,
MULLER,
R. K. M. AND HAEFELY, W.: Benzodiaxepine
antagonist
Re 15-1788.
Neurological
and behavioral effects. Psychopharmacology
78: 8-18, 1982.
BOWERY,
N. G., HILL, D. R. AND HUDSON,
A. L.: [HjBaclofen:
An improved
ligand for GABAB 5t8.
Neuropharmacology
24: 207-210,
1985.
BRAESTRUP,
C., SCHMIECHEN,
R., NEFF, G., NIELSEN,
M. AND PETERSEN,
E.

Science

modulation

of [SJ-t-butylbicyclophorothionate

binding
to cerebellar
granule
cells. J. Neurosci. 5: 2432-2438,
1985.
Giutrn,
J. C., Gnwr, J. P., FEELY, M. AND HAIGH, J. R. M.: Can benzodiazepines be classified
by characterizing
their anticonvulsant
tolerance-inducing
potential?
Eur. J. Pharmacol.
145: 75-80, 1988.
GIORGI,
0., ORLANDI,
M., LECcA, D. AND CORDA, M. G.: MK-801 prevents
chemical
kindling
induced
by pentylenetetrazol
in rats. Eur. J. Pharmacol.
193: 363-365,
1991.
GIUSTI, P., GUmrrni,
G., COSTA,
E. AND GUIDOTFI,
A.: The preferential
antagonism
of pentylenetetrazole
proconflict
responses
differentiates
a class of
anxiolytic
benzodiazepines
with potantial
antipanic
action. J. Pharmacol.
Exp.
Ther. 257: 1062-1068,
1991.
HAEFELY, W. E., MARTIN,
J. R. AND ScHOCH,
P.: Novel anxiolytics
that act as
partial
agonists
at benzodiazepine
receptors.
Trends
Pharmacol.
Sci. 1 1: 452456, 1990.
JENcK,

F., M0REAU, J. L., BoNrrri,

Ro 19-8022,

a nonbenzodiazepine

Neuropharmacological

Ther. 262:
KOE, B. K.,

E. P.,

MARTIN,

J. R.

AND

HAEFELY,

W. E.:

partial
agonist
at benzodiazepine
receptors:
of a potential
anxiolytic.
J. Pharmacol.
Exp.

profile

1121-1127,

1992.

C. A.

BURKHART,

AND

L. A.: (+)-[HJ3-(3-Hydroxyphenyl)-

LEBEL,

to sigma receptors
in mouse brain in vivo. Eur.
J. Pharmacol.
161: 263-266, 1989.
LANCER, S. Z., ARBILLA,
S., TAN, S., LLoyD,
K. G., GEORGE,
P., ALLEN, J. AND
WICK, A.: Selectivity
for omega receptor
subtypes
as a strategy
for the development of anxiolytic drugs. Pharmacopsychiatry
23: (Suppl). 103-107,
1990.
MASSOTFI,
M., SCHLICHTING,
J. L., ANTONACCI,
M. D., GIUSTI, P., MEMO,
M.,
COSTA,
E. AND GUIDOTFI,
A.: -y-Aminobutyric
acidA receptor heterogeneity
in
rat central
nervous
system:
Studies
with clonazepam
and other benzodiazepine
liganda.
J. Pharmacol.
Exp. Ther. 256: 1154-1160,
1991.
MCPHERSON,
G. I.: Ligand (release 2.0), Elsevier Biosoft, Cambridge,
1987.
NIELSEN,
E. 0., Danjan,
J., CHA, J. H., YOUNG,
A. B. AND H0N0RE,
T.:
Autoradiographic
characterization
and localization
of quisqualate
binding
sites

in rat brain

References
ALHO,

receptors.

From pharmacology
to molecular
biology.
Fed. Am. Soc. Ezp. Biol. J. 5:2916-2923,1991.
BYLUND, D. B. AND YAMAMURA,
H. I.: Methods for receptor binding. In Methods
in Neurotransmitter
Receptor
Analysis,
ed. by H. I. Yamamura,
S. J. Enna and
M. J. Kuhar, pp. 1-35, Raven Presa, New York, 1990.
CHANG,
R. S. L AND Loini, V. J.: Biochemical
and pharmacological
character#{149}
ization
of an extremely
potent
and selective
nonpeptide
cholecystokinin
antagonist. Proc. NatI. Acad. Sci. U.S.A. 83: 4923-4926,
1986.
COSTA,
E.: The allosteric
modulation
of GABAA receptors:
Seventeen
years of
research.
Neuropsychopharmacology
4: 225-235,
1991.
DANY5Z,
W. A., FADDA, E., WROBLEWSKI,
J. T. AND COSTA, E.: [H]D-serine
labels strychnine-insensitive
glycine recognition
sites of rat central
nervous
system.
Life Sci. 46: 155-164,
1990.
DEvANE,
W., Dysutz,
F. A. Ill, JOHNSON,
M. R., MziviN,
L. S. AND HOWLEIF,
A. C.: Determination
and characterization
of a cannabinoid
receptor in rat
brain. MoL Pharmacol.
34: 605-613,
1988.
DUCIC,
I., PUIA, G., VICINI, S. AND COSTA, E.: TTiaZO1aIn
is more efficacious
than diazepam
in a broad spectrum
of recombinant
GABAA receptors.
Eur. J.
PharmacoL
244: 29-35, 1993.
FACKLAM,
M., SCHOCH,
P., B0NErn,
E. P., JENCK, F., MARTIN,
J. R., M0REAU,
J. L AND HAEFELY,
W. E.: Relationship
between
benzodiazepine
receptor
occupancy
and functional
effects
in vivo in four ligands
of differing
intrinsic
efficacy.
J. PharmacoL
Exp. Ther. 261: 1113-1121,
1992.
FINNEY, D. J.: Probit Analysis,
3rd ed., Cambridge
University
Press, Cambridge,
1971.
FRITSCHY,
J. M., BENKE, D., MERTENS,
S., OERTEL,
W. H., BACH!, T. AND
MOEHLER,
H.: Five subtypes
of type A -y-aminobutyric
acid receptors
identified
in neurons
by double
and triple
immunofluorescence
staining
with subunitspecific
antibodies.
Proc. Natl. Acad. Sci. U.S.A. 89:6726-6730,1992.
GALLO,
V., WISE, B. C, VAcciuNo,
F. AND GUIDOTII,
A.: -y-Aminobutyric
acid

N-(1-propyl)-piperidine

virtually

1027

Receptors

N.: Interaction
of convulsive
liganda
with benzodiazepine
(Wash.
DC) 216: 1241-1243,
1982.
BURT, D. R. AND
KAMATCHI,
G.: GABAA
receptor
subtypes:

and benzodiazepine-induced

any

GABAA receptor
subtype.
FAM, such as alprazolam,
such
as diazepam,
because
of their
high efficacy

given

in

their

a very

of GABA

Comparison
pionic acid

using the antagonists

with
binding

R. W.

OLSEN,

binding

AND

[3H]6-cyano-7-nitroquinoxaline-2,3-dione:

(R,S)-[3HIa-amino-3-hydroxy-5
sites. J. Neurochem.
54: 686-695,
T0BIN,

A. J.: Molecular

Am. Soc. Exp. Biol. J. 4: 1469-1480,

OSWALD, I.: Safety of triazolain.
Lancet
PRITCHE1F,

D. B.,

benzodiazepine
245: 1389-1392,

D. B.

PRITCHETF,

point
Proc.

H.

LUDDEN5,

receptors
1989.
AND

AND

biology

338:
SEEBURG,

516-517, 1991.
P. H.: Type I and type II GABAA

in transfected

H.,

PRITCHETF,

PuIA, G.,

Science

(Wash.

DC)

acid type A receptor

for the benzodiazepine
site.

B. D., YMER,
P. H.: Importance

SHWERS,

SEEBURG,

for benzodiazepine

VICINI,
S., SEEBURG,
-y-aminobutyric
acidA-receptor
modulators
of y-aminobutyric
691-696,
1991.
RICKELS,
K. AND SCHWEIZER,

cells.

P. H.: -y-Aminobutyric

SEEBURG,

increases
the affinity
of compounds
NatL Acad. Sci. U.S.A. 88: 1421-1425,
1991.
mutation

receptor
subunit
585, 1988.

Fed.

1990.

produced

D. B., SONTHEIMER,
H., SCHOFIELD,
P. R. AND

methyl-4-isoxazolepro1990.
of GABAA receptors.

pharmacology.

Nature

S., KEVFENMANN,
of a novel GABAA
(Lond.)
338: 582-

P. H. AND COSTA, E.: Influence

of recombinant
subunit
composition
on the action of allosteric

acid-gated
E. E.:

Current

Cl channels.
pharmacotherapy

Mol. Pharmacol.

39:

of anxiety

and

1028

Giusti et al.

Vol. 266

panic.

anxioselective

Y. Meltzer,

In Psychopharmacology,
The Third
Generation
of Progress,
ed. by H.
pp. 1193-1203,
Raven Press, New York, 1987.
RoMEo,
E., AUrA, J., K0zIK0WSKI,
A. P., MA, D., PAPADOPOULOS,
V., PUi,
G., COSTA, E. AND GUIDOTTI,
A.: 2-Aryl-3-indoleacetamides
(FGIN-1): A new
class ofpotent
and specific Uganda for the mitochondrial
DBI receptor
(MDR).
J. Pharmacol.
Exp. Ther. 262: 971-978, 1992.
SANGER,
D. J. AND Zlvxovlc,
B: Differential
development
of tolerance
to the
depressant
effects
of benzodiazepine
and non-benzodiazepine
agonists
at the
omega
(BZ) modulatory
sites of GABAA receptors.
Neuropharmacology
31:
693-700,
1992.
SANTI,
M. R., Cox, D. H. AND GUmorn,
A.: Heterogeneity
of -y- aminobutyric/
benzodiazepine/beta
carboline
receptor
complex
in rat spinal
cord. J. Neuro
chem.
50: 1080-1086,
1988.

Exp. Ther. 253:

STEPHENS,
D. N., SCHNEIDER,
H. H., KEHR,
J., TURSKI,
L., SCHMIECHEN,
R., TURNER,
E. N., HoNoRS,
T. AND BONDO HANSEN,

W.,

ANDREWS,
J. D., JENSEN,

J.: Abecarnil,

J. S., RErric,
L. H.,

K.-

PETERSEN,

a metabolically

stable,

TALLARIDA,

beta-carboline

334-343,

R. J. AND

acting
1990.

MURRAY

at benzodiazepine

R. B.: Manual

receptors.

of Pharmacological

J. Pharmacol.

Calculations

with

Computer
Programs,
Springer-Verlag,
New York, 1987.
S. R., BEER, B. AND CLODY, D. E.: A simple reliable conflict procedure
for testing
anti-anxiety
agents.
Psychopharmacology
21: 1-7, 1971.
ZIvKovIc,
B., MOREL, E., JOLY, D., PERRAULT,
G. H., SANGER,
D. J. AND LLOYD,
K. G.: Pharmacological
and behavioral
profile of alpidem
as an anxiolytic.
Pharmacopsychiatry
23: (SuppL) 108-113,
1990.
VoGEL,

Pharmacopsychiatry

23:

(SuppL)

108-113,

-_____________________________________
Send reprint
requests
to: Dr. Erminio
the Neurosciences,
Georgetown
University
Rd., NW, Washington,
DC 20007.

1990.

Costa,
School

Fidia-Georgetown
of Medicine,

Institute
3900

Reservoir

for