Research Departments, H. Lundbeck A/S (C.S., J.A., E.M., J.P.),
Copenhagen-Valby, Denmark and
University of Bradford (B.C., M.E.K.,
R.J.N.), Bradford West Yorkshire, United Kingdom
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Introduction |
The
benzodiazepines remain the predominant pharmacotherapy for anxiety
disorders. However, much effort is still directed at the development of
new non-benzodiazepine anxiolytics (e.g., review by
Perregaard et al., 1993
). The reason for this is mainly the side effects related to treatment with benzodiazepines. There is a
potential risk that benzodiazepine treatment leads to physical dependence, and withdrawal symptoms are described in relation to
discontinuation of prolonged treatment (review by Woods et al., 1992). Sedation, especially during the first period of
treatment, is another drawback of benzodiazepine treatment.
Lu 28-179 (fig. 1) is a novel 
ligand
with subnanomolar affinity and specificity for the 2
binding site (Perregaard et al., 1995). Two subtypes of binding sites, the 1 and the 2 site, have
been characterized, but the functions of these sites still remain to be
clarified (Quirion et al., 1992). The binding sites have
mainly been hypothesized to play a role in schizophrenia. A number of
neuroleptics (e.g., haloperidol and remoxipride) have affinity for binding sites, and ligands are described as
interacting with midbrain dopamine neurons (Walker et al.,
1990; Monnet, 1993; Zhang et al., 1993). A number of
-selective compounds have been suggested as antipsychotics on the
basis of preclinical data; examples include DuP 734 and XJ 448 (Gilligan et al., 1992), NPC 16377 (Clissold et
al., 1993; Karbon et al., 1993) and NE-100 (Okuyama
et al., 1993; Chaki et al., 1994). These
compounds either are nonselective or show preference for the
1 binding site. The 2 binding site has
been discussed in relation to dystonic effects in rats (Walker et
al., 1993).
Limited evidence suggests involvement of binding sites in anxiety.
The two ligands DTG and (+)-pentazocine show an anxiogenic-like profile in rats tested in a modified version of the Vogel conflict test
(Lai et al., 1989). DTG is non-selective with respect to 1 and 2 binding sites, whereas
(+)-pentazocine has preference for the 1 binding site
(Quirion et al., 1992).
In the present study, we assess the anxiolytic potential of a
2 ligand (Lu 28-179) after single and repeated treatment
in various models of anxiety in rodents. Preliminary data on some of
the anxiolytic-like effects of Lu 28-179 were presented at the 24th
Annual Meeting of the Society for Neuroscience (Sánchez et
al., 1994). In addition, the in vitro binding profile
and ex vivo binding data for Lu 28-179 are presented.
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Materials and Methods |
General animal housing conditions.
All rodents were housed
at a temperature of 21 ± 2°C and had free access to water and
standard rodent laboratory chow.
Receptor binding studies and amine reuptake inhibition in
vitro.
2 binding site: Inhibition
of 3H-DTG binding to 2 binding sites in
homogenates from rat brain minus cerebellum was determined as described
by Meier et al. (1997). 1 binding site: Inhibition of binding of 3H(+)-pentazocine to
1 receptors in homogenates from rat brain minus
cerebellum was determined as described by Meier et al.
(1997). DA D1 receptors: Inhibition of
3H-SCH 23390 binding to DA D1 receptors in rat
striatal membranes was determined as described by Hyttel (1982).
DA D2 receptors: Inhibition of
3H-spiperone binding to DA D2 receptors in rat
striatal membranes was determined as described by Hyttel (1987).
Serotonin1A (5-HT) receptor: Inhibition of
3H-8-OH-DPAT binding to 5-HT1A receptors in
membranes from rat brain minus cerebellum was determined as described
by Hyttel et al. (1988). 5-HT2A
receptors: Inhibition of 3H-ketanserin binding to
5-HT2A receptors in membranes from rat cortex was
determined as described by Hyttel et al. (1988).
5-HT2C receptors: Inhibition of
3H-mesulergine (0.5 nM) binding to a cloned rat
5-HT2C receptor expressed in 3T3 cells was determined
in vitro as described by Bøgesø et al. (1995).
Alpha-1 adrenoceptors: Inhibition of
3H-prazosin binding to alpha-1 adrenoceptors in
membranes from rat whole brain was determined as described by Hyttel
and Larsen (1985). Alpha-2 adrenoceptors:
Inhibition of 3H-idazoxan binding to alpha-2
adrenoceptors in membranes from rat brain cortex was determined as
described by Megens et al. (1986). Beta
adrenoceptors: Inhibition of 3H-dihydroalprenolol
binding to beta adrenoceptors in membranes from rat cortex
was determined as described by Hyttel et al. (1984). Histamine H1 receptors: Inhibition of
3H-mepyramine binding to histamine H1 receptors
in membranes from rat brain minus cerebellum was determined as
described by Hall and Ögren (1984). Muscarine cholinergic
receptors: Inhibition of 3H-QNB binding to muscarinic
cholinergic receptors in membranes from rat whole brain was determined
as described by Meier et al. (1997). Inhibition of
3H-DA uptake in rat striatal synaptosomes was
determined as described by Hyttel (1982). Inhibition of
3H-noradrenaline (NA) uptake in rat cortical
synaptosomes was determined as described by Hyttel (1982).
Inhibition of 3H-5-HT uptake in synaptosomes
from rat brain minus cerebellum was determined as described by Hyttel
(1982).
Estimation of 2 binding site activity in the CNS
using ex vivo binding.
Ex vivo binding
studies were performed essentially as described by Freedman et
al. (1989). Briefly, mice (NMRI, Bomholtgård, Denmark) were given
test compound either s.c. or p.o., and were sacrificed after 3 h
(Lu 28-179) or after 0.5 h (DTG or haloperidol). The forebrain was
quickly removed and homogenized in 100 vol of assay buffer (50 mM
Tris-buffer, pH 7.4). Binding activity (3H-DTG) was
estimated on the forebrain homogenate, and 2 binding site activity of the compound was quantified by the relative inhibition of binding compared with saline-treated control animals. For absorption and elimination experiments, mice were given 3.3 µmol/kg Lu 28-179 s.c. At different time intervals after drug administration, mice were
decapitated, and ex vivo binding (3H-DTG) was
determined in brain homogenates and expressed as 100 minus percent
binding compared with saline-treated controls. Absorption and
elimination data were fitted to the equation F(t) = F0 exp(
ka t) + exp(ket), where F0 is
the total binding activity, ex vivo, extrapolated to time 0, ka and ke are the
absorption respective elimination constants and t is the
time in days.
Black and white test box, rats.
We used male Wistar WU rats
(Charles River, Hannover, Germany) housed in groups of four in macrolon
type III cages and kept on a reversed light/dark cycle (dark period
7.00-19.00 h). The two-compartment test model is formerly described by
Sánchez et al. (1995). A white, open-topped
compartment (80 cm × 65 cm × 33 cm) with the floor divided
into nine squares was connected to a closed black box (25 cm × 21 cm) by an opening (10 cm × 10 cm) located in the center of one of
the short sides of the white compartment. The white compartment was
illuminated by bright light (2000 Lux). The test system was fully
automated by 2 rows of 11 infrared light sources and photocells in the
transverse direction and 1 row of 18 in the longitudinal direction
(lower row). The lower row of photocells (5.5 cm above the cage floor)
detected horizontal locomotor activity, whereas the upper row of
photocells (13 cm above the cage floor) detected rearing activity.
The rats were taken from the dark holding room in a dark container to a
dimly illuminated (red light) room. Treatment with test substance was
performed after a 1 to 2-h period of adaptation. During the test,
individual animals were placed in the center of the white compartment
and observed for 7 min for number of rearings and number of line
crossings between squares in the two compartments, number of entries
into the black compartment and time spent in the white compartment.
Each treatment group consisted of 8 to 24 rats. The dose-response
relationship for single doses of Lu 28-179 was assessed, along with the
duration of action of a single dose (1 µg/kg) of Lu 28-179.
Black and white test box, mice.
We used male BKW mice
(Bradford strain, University of Bradford, UK) weighing 30 to 36 g,
housed in groups of 10 in polypropylene cages and kept on a reversed
light/dark cycle (dark period 7.00-19.00 h).
The test box is described in detail by Costall et al.
(1989). Briefly, the box (45 cm × 27 cm × 27 cm) was
open-topped, and the base was lined into 9-cm squares. Two-fifths of
the box was painted black, illuminated under a dim red light and
partitioned from the remainder of the box, which was painted white and
brightly illuminated. The compartments were connected by an opening 7.5 cm × 7.5 cm located at floor level in the center of the
partition.
The mice were taken from the dark holding room in a dark container to a
dimly illuminated (red light) room. Treatment with test substance was
performed after a 1 to 2-h period of adaptation. During the test,
individual animals were placed in the center of the white compartment,
and their behavior was recorded by remote video recording for 5 min.
The recordings were subsequently evaluate, by a person unaware of the
drug treatments, for time spent in each compartment, number of
exploratory rearings in each compartment, number of crossings between
squares in each compartment, and latency of the initial movement from
the white to the black compartment. Each treatment group consisted of 5 to 10 mice. We assessed the dose-response relationship for single doses
of Lu 28-179, as well as the effects of daily treatment with 0.0018 µmol/kg (1 µg/kg i.p.) for 3, 7, 10 or 14 days. Furthermore,
separate groups of animals treated with Lu 28-179 daily for 14 days
were assessed 8 h, 12 h and 1, 2, 4 and 10 days after
withdrawal of treatment.
Social interaction test.
We used male Hooded-Lister rats
(Bradford strain, University of Bradford, UK) weighing 225 to 275 g, housed in groups of five in polypropylene cages and kept on a 12-h
light/dark cycle with lights on at 7.00 h.
The apparatus was an open-topped perspex box (51 cm × 51 cm × 20 cm) with 17 × 17 cm areas marked on the floor. The box was illuminated with bright white light. Tests were conducted in an illuminated room using a methodology based on the model of File (1980).
Two naive rats, from separate housing cages, were both treated with
test drug and placed into the brightly illuminated test box. Their
behavior was recorded by remote video recording over a 10-min period.
Social interaction between animals (sniffing of partner, crawling under
or climbing over partner, genital investigation of partner and
following partner) was determined by timing in seconds, and exploratory
locomotion was measured as the number of crossings of the lines marked
on the floor of the test box. The video recordings were evaluated by a
person unaware of the drug treatments. A total of 6 to 12 pairs of rats
were used per dose.
We assessed the dose-response relationship for single doses of Lu
28-179, as well as the effects of daily treatment with 0.0018 µmol/kg
(1 µg/kg) for 1, 2, 3 and 4 weeks, respectively. Furthermore, separate groups of animals treated with Lu 28-179 for 4 weeks were
assessed 24 h, 48 h and 7, 10 and 14 days after withdrawal of
treatment.
Inhibition of shock-induced suppression of drinking.
We used
male Wistar rats (Mol:Wist, Møllegård, Denmark) that weighed 150 to
175 g at the beginning of the study. The rats were housed in
groups of four in macrolon type III cages and were kept on a 12-h
light/dark cycle with lights on at 7.00.
The test chambers were stainless steel boxes (13 cm × 21 cm × 14 cm) with wire mesh floor and walls. A water bottle with a metal
tube was mounted on one side of the chamber. The chambers were
contained in a sound-attenuating cabinet. Shocks were provided by a
two-pole shocker connected to a metal drinking tube and the floor of
the box. A shock was delivered every 20 licks, or, at constant licking,
a shock was delivered every 7 sec (equivalent to approximately 20 licks). The numbers of shocks and licks were recorded by means of a
computer program.
Rats were deprived of water for 24 h before the first training
session. Then the drinking behavior was assessed in a nonpunished 6-min
session. Only rats that licked during this session progressed to a
further 24-h deprivation of water. The rats were tested again 24 h
later, this time receiving a shock for every 20 licks. The trial
session time was 6 min, starting automatically when the rat completed
20 licks and received the first shock. If a rat had not licked after
the first 3 min, the recording started automatically, thus resulting in
a maximum duration of 9 min for a test session. The shock intensity was
0.6 mA, and the duration of the shock was 0.1 sec. Immediately after
the second session, drugs were administered and the punished session
was repeated 1 h or 2 h later. Each treatment group consisted
of 8 to 24 animals.
Isolation-induced aggression.
We used male NMRI mice (BOM,
SPF, Møllegård, Denmark) with starting weight 18 to 20 g. The
isolated mice were single-housed in Macrolon type II cages, and the
socially housed intruder mice were kept in groups of 10 in opaque
plastic cages (35 cm × 30 cm × 12 cm). Lights were on 7.00 to 19.00 h.
The test was conducted as described by Sánchez et al.
(1993). Briefly, the mice were kept isolated for about 21 days. After the isolation period the mice were trained to attack, initially by
introducing a single housed intruder into the test cage, and subsequently by means of a nonaggressive intruder mouse. An attack was
defined as biting or attempting to bite the intruder mouse. The
training and the testing sessions took place in the home cage of the
isolated mouse. Only mice with attack latencies of less than 10 sec
were included in the studies. In the test sessions, the mice were
tested immediately before drug treatment and 30 min or 2 h later.
The attack latency was measured with a maximum observation time of 180 sec. Each group consisted of 8 to 16 aggressive mice and 16 to 32 nonaggressive mice (for testing before and after drug).
Inhibition of footshock-induced ultrasonic vocalization.
We
used male Wistar WU rats (Charles River, Hannover, Germany) that
weighed 150 to 175 g at the beginning of the study. The rats were
housed in groups of four in macrolon type III cages and lights were on
7.00 to 19.00 h.
The test cages and procedure are described in detail by Sánchez
(1993). Briefly, the test cages (22 cm × 22 cm × 22 cm)
were made of grey perspex and equipped with a metal grid floor.
Footshocks were delivered from a two-pole shocker. A microphone
sensitive to ultrasounds in the range of 20 to 30 kHz was placed in the center of the lid of the test cages. The ultrasounds were sent from the
microphone to a preamplifier and converted from AC signals to DC
signals in a signal rectifier. The accumulated time when the voltage of
the rectified signal was larger than the voltage of a previously
determined threshold level was recorded.
Test procedure: Briefly, 24 h before the first test
session, the animals were primed. The rat was placed in the test cage and received immediately thereafter four inescapable 1.0-mA footshocks, each lasting 10 sec with an intershock interval of 5 sec. The animals
were left in the test cages for a total of 6 min after the last shock.
On the test day, rats received the same shock regimen, and recording of
ultrasonic vocalization started 1 min after the last shock and lasted
for 5 min. The total time spent vocalizing was recorded. Each treatment
group consisted of 8 to 16 animals.
Motor side effects.
We used male NMRI mice (BOM, SPF,
Møllegård, Denmark) that weighed 18 to 25 g (locomotor activity)
or 25 to 35 g (horizontal wire test) and were kept in groups of 10 to 15 in opaque plastic cages (35 cm × 30 cm × 12 cm).
Lights were on 7.00 to 19.00 h.
Spontaneous locomotor activity was measured in automated activity
cages. Each cage consisted of an open transparent perspex arena (20 cm × 32 cm × 20 cm) placed on a black screen with 40 photocells. A light source was placed above the cage, and the number of
light-beam interruptions was recorded. Groups of three mice were
treated with test substance or saline and, 1 h later, placed
groupwise in the activity cages. The number of light-beam interruptions
was recorded for 15 min. Each dose level was assessed in 2 to 4 activity cages, and the locomotor activity was expressed as percent
activity relative to the activity of animals from parallel saline-treated control groups.
Performance on the horizontal wire test was assessed in the following
way: The mouse was lifted by the tail and allowed to grasp with its
forepaws a horizontally strung wire (35 cm long and 0.55 mm in
diameter) 30 cm above the table level; it was then released. Animals
that were unable to grasp the wire with the hindpaws within 15 sec were
scored as responders (all-or-none criterion). A total of 3 to 6 animals
were used per treatment group.
Statistics.
In the in vitro studies,
IC50 values were estimated on the basis of two full
concentration-response curves, each containing five concentrations of
drug covering 3 to 5 log units (each data point in triplicate).
Estimation of IC50 values was performed by means of the
receptor program Ligand and/or Multicalc from Wallac. If the log ratio
(logR) between the two determinations was greater than corresponding to
3 × S.D. (99% confidence interval), then extra determinations
were performed and outliers discarded. The S.D. values were calculated
from a series of n determinations of logR between double
determinations. Antilog (S.D.) values applied for the individual
binding assays are shown in table 1.
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TABLE 1
In vitro receptor binding. IC50 values were
estimated on the basis of two full concentration-response curves, each
containing five concentrations of drug covering 3 to 5 log units (each
data point in triplicate).
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In the in vivo studies, one-way analysis of variance was
used, followed by post-hoc comparisons of means (Dunnett's
test) when the outcome of the analysis of variance was significant
(P < .05). Inhibitory potencies on footshock-induced ultrasonic
vocalization, isolation-induced aggression, locomotor activity and
performance on the horizontal wire test were expressed as
ED50 values, calculated by means of log-probit analysis.
Drugs.
Lu 28-179 hemifumarate or oxalate, mw 571 or 545, respectively (H. Lundbeck A/S, Copenhagen, Denmark), 10 mg, was
dissolved in a mixture of 1 ml propyleneglycol and 0.2 ml 0.1 M
methanesulphonic acid by heating. The solution was diluted by slowly
adding distilled water (40-50 parts). Diazepam, mw 285 (Roche, Basel,
Switzerland) was dissolved in a minimum amount of polyethylene glycol
and diluted into saline or provided as ampules (Apozepam, Apothekernes
Laboratorium A/S, Oslo, Norway) and diluted with saline. Lorazepam, mw
320 (Ferrosan, Copenhagen, Denmark) was suspended in 0.5%
methylcellulose saline dissolution. Haloprendol (Tanson, Belgium) was
dissolved in minimum amounts of tartaric acid and diluted with saline.
DTG (Tocris Cookson, UK) was dissolved in 96% alcohol and propylene glycol and diluted with water.
The injection volumes were 10 ml/kg in mice, and 5 ml/kg (black and
white box test, footshock-induced ultrasonic vocalization and conflict
test) or 1 ml/kg (social interaction) in rats. Treatment times, doses
and routes of administration are given with the results of the
individual studies.
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Results |
In vitro receptor binding and amine reuptake inhibitory
potency.
Lu 28-179 had very high affinity for 2
sites labeled by 3H-DTG (table 1). In contrast, the
affinities for the various receptor sites, including the site labeled
by 3H-(+)-pentazocine, were much lower (table 1). The
reference compounds DTG, haloperidol and (+)-3-PPP had considerably
lower (90 to several thousand-fold) affinities for the 2
binding site.
Lu 28-179 had negligible effects on the uptake of labeled amines into
synaptosomes compared with their affinity for sites (table 1).
Ex vivo receptor binding.
Lu 28-179 easily
penetrated the blood-brain barrier and reached its maximum value within
1 to 2 h after administration (fig. 2). It was slowly eliminated from the
brain. The elimination constant was estimated to
ke = 0.838 ± 0.0423 day
1,
from which T1/2 = 0.827 days, or approximately 20 h,
was calculated. Maximum inhibition of binding was seen 2 to 3 h
after administration, but 60 to 70% inhibition was seen at 15 to 60 min after administration.
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Fig. 2.
Absorption and elimination of Lu 28-179 (3.3 µmol/kg) from murine brain after s.c., p.o. and i.v. administration
as estimated by 3H-DTG ex vivo binding. Values
represent the mean ± S.E.M. of five determinations.
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Lu 28-179 was effective after s.c. and p.o. administration, haloperidol
displaced 3H-DTG at high doses and DTG was ineffective
(table 2).
Black and white test box, rats and mice.
The effects of Lu
28-179 and diazepam are shown in figures 3 and 4 for the rat and for
the mouse, respectively.
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Fig. 3.
The effect of Lu 28-179 (s.c. 2 h before test)
and diazepam (0.035 µmol/kg, s.c. 30 min before test; hatched bars)
on rat rearing behavior, line crossings, time spent in the white
compartment and number of entries into the black part of a
two-compartment black and white test box. C indicates the response of
vehicle-treated control animals. Values represent the mean ± S.E.M. The numbers of animals per group were 24 controls; 8, 16, 16, 16, 8 and 8 Lu 28-179-treated rats; 16 diazepam-treated rats.
Significant increases or decreases in responding compared with control
values are indicated by * (P < .05; one-way analysis of variance
followed by Dunnett's t test).
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Fig. 4.
The effect of Lu 28-179 (i.p. 40 min before test;
double-hatched bars) and diazepam 4.6 µmol/kg = 1.3 mg/kg (i.p.
40 min before test; hatched bars) on mouse rearing behavior, line
crossings, the percentage of time spent in the white compartment and
latency of first movement from the white to the black part of a
two-compartment black and white test box. C (open bars) indicates the
response of vehicle-treated control animals. Values represent the
mean ± S.E.M. The numbers of animals were 10 controls,
n = 5 in each of the Lu 28-179-treated groups, and 10 diazepam-treated. Significant increases or decreases in responding
compared with control values are indicated by * (P < .05; one-way
analysis of variance followed by Dunnett's t test).
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In the rat, treatment with Lu 28-179 significant increased exploratory
rearing in the white compartment without affecting the rearing activity
in the black compartment (fig. 3).
Similarly, the number of entries into the black compartment and the
time spent in the white compartment increased significantly. The
potency of Lu 28-179 was high, with a MED of 0.00018 µmol/kg (0.0001 mg/kg s.c., 2 h before test), and Lu 28-179 was active over a wide
dose range, i.e., 0.00018 to 1.8 µmol/kg (0.0001-1.0
mg/kg). Diazepam 0.035 µmol/kg (0.01 mg/kg s.c., 30 min before test)
induced a significant increase in number of rearings, line crossings
and time spent in the white compartment.
In the mouse, treatment with Lu 28-179 (i.p., 40 min before test)
caused a significant increase in rearings and line crossings in the
white compartment and a corresponding significant reduction in the
black compartment (fig. 4). Similarly,
time spent in the black compartment decreased significantly. The
potency of Lu 28-179 was extremely high, with a MED of 0.00018 nmol/kg
(0.10 ng/kg), and Lu 28-179 was active over a very large dose range,
i.e., 0.00018 nmol/kg to 1800 nmol/kg (0.1 ng/kg-1.0
mg/kg). Diazepam 4.6 µmol/kg (1.3 mg/kg i.p., 40 min before test)
induced a significant increase in number of rearings and line crossings
and time spent in the white compartment (fig. 4).
Lu 28-179 (0.0018 µmol/kg i.p.) administered daily for 1, 3, 7, 10 or
14 days to separate groups of mice, significantly increased number of
rearings and line crossings and time spent in the white compartment
(fig. 5, left half of each graph). A
similar anxiolytic-like effect was obtained after 10 days of treatment
with diazepam 4.6 µmol/kg/day (1.3 mg/kg/day i.p., test 40 min after
the last dose). Groups of mice treated with Lu 28-179 (0.0018 µmol/kg = 1 µg/kg/day i.p.) for 14 days maintained an
increased level of rearings and line crossings and time spent in the
white compartment, 1 to 2 days after withdrawal from treatment (fig. 5,
right half of each graph). At 4 and 10 days after withdrawal, the
behavior was indistinguishable from that of control animals. A group
treated daily with diazepam for 14 days and assessed 24 h after
the last dose showed a significant decrease in number of rearings and
line crossings and in time spent in the white compartment.
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Fig. 5.
Left half of graphs) The effects of treatment with Lu
28-179 (0.0018 µmol/kg = 1 µg/kg/day i.p.; double-hatched
bars) for 1, 3, 7, 10 or 14 days or of treatment with diazepam 4.6 µmol/kg (1.3 mg/kg/day i.p.) for 10 days (hatched bars) on mouse
exploratory behavior in a two-compartment black and white test box. C
(open bars) indicates the response of control animals treated with
vehicle for 14 days. The mice were tested 40 min after the last
injection. Right half of graphs) The effects of Lu 28-179 0.0018 µmol/kg/day (1 µg/kg/day i.p.) (double-hatched bars) or of diazepam
18 µmol/kg (5 mg/kg/day) for 14 days (hatched bars) on mouse
exploratory behavior in a two-compartment black and white test box
8 h, 12 h or 1, 2, 4 or 10 days and 24 h after
withdrawal from treatment with Lu 28-179 and diazepam, respectively.
Each value is the mean ± S.E.M. of five determinations.
Significant increases or decreases in responding compared with control
values are indicated by * (P < .05; one-way analysis of variance
followed by Dunnett's t test).
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Social interaction in the rat.
Lu 28-179 increased the time
that pairs of rats spent in active social interaction over a wide dose
range without modifying locomotor activity (fig.
6). Lu 28-179 was extremely potent;
MED = 0.00018 nmol/kg (0.1 ng/kg i.p., 40 min before test). The
number of line crossings was not affected by Lu 28-179 at the highest dose tested, 1.8 µmol/kg (1.0 mg/kg). Diazepam 4.6 µmol/kg (1.3 mg/kg i.p., 40 min before test) induced a maximum increase in social
interaction of the same magnitude as that induced by treatment with Lu
28-179.
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Fig. 6.
The effect of Lu 28-179 (i.p. 40 min before test;
double-hatched bars) and diazepam 4.6 µmol/kg = 1.3 mg/kg (i.p.
40 min before test; hatched bars) to increase rat social interaction
under high-light unfamiliar conditions. C (open bars) indicates the
response of vehicle-treated control animals. Pairs of rats received
acute drug or vehicle administrations. Each value is the mean ± S.E.M. The numbers of animals were 12 pairs of control rats,
n = 6 pairs in each of the Lu 28-179-treated groups,
and 12 diazepam-treated pairs. Significant increases or decreases in
responding compared with control values are indicated by * (P < .05; one-way analysis of variance followed by Dunnett's t
test).
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Daily treatment for 7, 14, 21 or 28 days with 0.0018 µmol/kg (1 µg/kg) significantly increased the time spent in active social interaction compared with the behavior of vehicle-treated animals (fig.
7, left half). Diazepam treatment (35 µmol/kg = 10 mg/kg) for 7 days increased the time that pairs of
rats spent in active social interaction. However, locomotor activity
was significantly decreased.
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Fig. 7.
Left) The effect of treatment with Lu 28-179 (0.0018 µmol/kg/day = 1 µg/kg/day i.p.) for 1, 2, 3 or 4 weeks
(double-hatched bars) or of treatment with diazepam (35 µmol/kg/day = 10 mg/kg/day i.p.) for 7 days (hatched bar) on the
time that pairs of rats spend in social interaction under high-light
unfamiliar conditions. C (open bar) indicates the response of control
animals treated with vehicle for 4 weeks. Pairs of rats were tested 40 min after the last injection. Right) The effect of Lu 28-179 (0.0018 µmol/kg/day = 1 µg/kg/day i.p. × 1) on rat social interaction
24 h or 2, 7, 10 or 14 days after withdrawal of a 4-week treatment
with Lu 28-179 (double-hatched bars). The effect of diazepam (35 µmol/kg/day = 10 mg/kg/day i.p.) on rat social interaction
24 h after withdrawal of a 1-week treatment with diazepam is also
shown (hatched bar). Each value is the mean ± S.E.M. of six
determinations. Significant increases or decreases in responding
compared to control values are indicated by * (P < .05; one-way
analysis of variance followed by Dunnett's t test).
+Significantly different from day 7 (t test).
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Rats treated for 4 weeks with Lu 28-179 0.0018 µmol/kg/day (1 µg/kg/day) maintained a significantly increased response 24 h
after withdrawal from treatment (fig. 7, right half). The response recorded at 2, 7, 10 or 14 days after withdrawal was comparable to
vehicle responses. A group of rats treated with diazepam 35 µmol/kg
(10 mg/kg) for 7 days showed a significant decrease in time spent in
social interaction, without change in number of line crossings, 24 h after withdrawal (fig. 7).
Inhibition of shock-induced suppression of drinking.
Lu 28-179 significantly increased the number of shocks received during a 6-min
test period (fig. 8). The effective doses
were much higher than those necessary to achieve anxiolytic-like
effects with Lu 28-179 in the aforementioned models (MED = 18 µmol/kg = 10 mg/kg s.c., 2 h before test), but the doses
were comparable to the effective doses of the benzodiazepine lorazepam
(fig. 8). Furthermore, the maximum effects obtained with Lu 28-179 and
lorazepam were of similar magnitude.
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Fig. 8.
The effect of Lu 28-179 and lorazepam on
shock-induced suppression of drinking behavior in rats. Rats were water
deprived for 48 h. Subsequently, drinking behavior was assessed
while rats received a shock (0.6 mA for 0.1 sec) for every 20 licks on
the metal drinking tube. Immediately after this session, Lu 28-179 (s.c.) or lorazepam (p.o.) was administered, and the punished session
was repeated 2 h or 1 h later, respectively. Each value is
the mean ± S.E.M. The numbers of animals per group were 24 controls; 8, 8, 24, 16 and 8 Lu 28-179-treated rats; 8, 16, 16, 8 and 8 lorazepam-treated rats. Significant increases in responding compared
with control values are indicated by * (P < .05; one-way analysis
of variance followed by Dunnett's t test).
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Inhibition of footshock-induced ultrasonic vocalization in the rat
and inhibition of isolation-induced aggression in the mouse.
Lu
28-179 did not inhibit footshock-induced ultrasonic vocalization in the
rat, and Lu 28-179 did not inhibit isolation-induced aggression in male
mice (0.01-20 mg/kg s.c., 2 h before test; table
3). The effects of diazepam are included
in the table for comparison.
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TABLE 3
Effects of Lu 28-179 and diazepam on footshock-induced ultrasonic
vocalization in the rat and isolation-induced aggressive behavior of
male mice. For details, see "Materials and Methods"
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Motor effects.
In mice, Lu 28-179 neither inhibited
spontaneous locomotor activity nor affected motor performance on a
horizontal wire test, even at very high doses (table
4). Diazepam significantly decreased spontaneous locomotor activity and significantly impaired performance on the horizontal wire test (table 4).
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TABLE 4
Effects of Lu 28-179 and diazepam on spontaneous locomotor activity and
performance on a horizontal wire in mice. For details, see "Materials
and Methods"
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Discussion |
The in vitro binding studies demonstrated that Lu
28-179 is a highly selective and very potent 2 ligand
compared with other ligands that have affinities for the
2 binding site. Furthermore, the ex vivo
binding studies suggested that Lu 28-179 penetrates the blood-brain
barrier easily, that it is slowly eliminated with a half-life of about
20 h and that the compound is effective after s.c. as well as p.o.
administration. The lack of effect of DTG in the ex vivo
binding experiments may be due to poor CNS penetration of DTG. However,
it should be emphasized that the tissue is diluted in 100 vol of
buffer. Thus the ex vivo binding potency may underestimate the actual binding affinity as a result of dissociation of the test
compound from its binding sites during incubation. For this reason,
comparisons of potency between different compounds should be made with
caution.
The present data demonstrate an anxiolytic-like profile of Lu 28-179 in
a number of rodent models of anxiety. Lu 28-179 had an extremely high
potency in the rat social interaction test and in the mouse black and
white box test, and it also had a very potent effect in the rat black
and white box test. The MED ranged from 0.01 to 100 ng/kg in these
tests. It should be stressed that these high potencies are not
reflected in the ex vivo binding studies. Methods for
measuring such low drug concentration in tissue are presently not
available. Furthermore, the functional correlate to stimulation of the
sigma binding site is unknown. The anxiolytic-like effect
had also a very long duration of action, as shown by the
anxiolytic-like effects in the mouse black and white test box and the
rat social interaction test persisting for 1 to 2 days upon withdrawal
after 2 and 4 weeks of treatment, respectively. Furthermore, a single
injection (1 µg/kg s.c.) of Lu 28-179 produces significantly
anxiolytic-like effects in the rat black and white test box 24 h
after administration (C. Sánchez, unpublished observations). The
high potency might suggest that the anxiolytic-like effects are
mediated by binding sites, because affinities for a variety of
other receptors are at least 1000 times weaker. Lu 28-179 has recently
been described as a ligand with selectivity for the
2 binding site (Perregaard et al., 1995). The
in vitro affinities for the 1 binding site
(3H-(+)-pentazocine) and for the 2 binding
site (3H-DTG) are IC50 = 17 nM and
IC50 = 0.19 nM, respectively, which corresponds to a ratio
of about 90. This might suggest a role for the 2 binding
site in anxiety. However, we cannot exclude the possibility that the
1 binding site is involved in the anxiolytic-like effects. Thus the high 1/2 selectivity
ratio is due to an extremely high affinity for the 2
binding site in the subnanomolar range, whereas the affinity for the
1 binding site is in the nanomolar range. The
1 binding site has formerly been associated with an anxiogenic-like effect; the 1 ligand (+)-pentazocine
induced an anxiogenic-like effect in a conflict model in rats (Lai
et al., 1989). However, in the rat black and white test box
(+)-pentazocine is found to induce an anxiolytic-like effect
[effective doses from 0.035 to 3.5 µmol/kg (0.01-1 mg/kg)] without
affecting locomotor activity (C. Sánchez, unpublished
observations). Because the functional characterization of ligands
with respect to intrinsic activity is hampered by the lack of
appropriate models, additional information about the subtypes
involved in the action of Lu 28-179 remains to be provided.
Lu 28-179 was about 100 times more potent than diazepam in the rat
black and white test box and several thousand-fold more potent than
diazepam in the mouse black and white test box and the rat social
interaction test. Diazepam was effective at doses from 0.035 µmol/kg
(0.010 mg/kg) in the former and from 0.46 µmol/kg (0.13 mg/kg) in the
latter (Sánchez et al., 1995). Lu 28-179 does not have
affinity for benzodiazepine receptors (E. Meier unpublished
observation). Also, 5-HT1A receptor agonists
(e.g., buspirone) show potent effects in this model (C. Sánchez, unpublished observations). However, the effect of Lu
28-179 can be explained by effects on neither 5-HT1A nor
5-HT3 receptors, because Lu 28-179 is devoid of affinity
for these receptors. CCK receptors are also involved in mediating
anxiety (review by Harro et al., 1993), and CCK antagonists
facilitate the exploratory behavior in the mouse black and white test
box (Costall et al., 1991; Hendrie et al., 1993).
However, Lu 28-179 does not have affinity for either CCKA
or CCKB receptors (E. Meier, unpublished observation).
The neurobiological mechanisms involved in mediation of the
anxiolytic-like activity of Lu 28-179 are not known. Both
1 and 2 binding sites are widely
distributed in the brain, particularly in the limbic system
(e.g., hippocampus and amygdala) and in brain stem motor
areas (Gundlach et al., 1986). The 1 and
2 binding sites differ in distribution; the former is at
its highest level in the brain stem, the latter in hippocampal
membranes (McCann et al., 1994). This might suggest that the
2 binding sites are involved in modulation of emotional
responses. However, a recent study suggests that 1 sites
are involved in mediation of conditioned fear stress (Kamei et
al., 1996).
Lu 28-179 was also active against shock-induced suppression of
drinking, which in the present form is a modification of the classical
Vogel conflict test of anxiety (Petersen and Lassen, 1981). The
effective doses of Lu 28-179 were much higher than those necessary to
achieve anxiolytic-like effects in the black and white test box and the
social interaction test, but the doses were comparable to the effective
doses of the benzodiazepine lorazepam (fig.
9). Furthermore, the maximum effects
obtained with Lu 28-179 and lorazepam were of similar magnitude. This
is quite remarkable, because the responses of non-benzodiazepine
anxiolytics in conflict models are generally variable and smaller than
those of benzodiazepines (Perregaard et al., 1993). The
model-dependent and very large differences among the MEDs of Lu 28-179 are not readily explained, although some differences in sensitivity are
observed with diazepam, too. For example, reports from different
laboratories on the MEDs in the mouse black and white test box range
from 0.01 mg/kg (Onaivi and Martin, 1989) to 1.0 mg/kg (Young and
Johnson, 1991). The intensity or type of stressor in anxiogenic stimuli
may interfere with the MEDs needed. A recent study of the mouse black
and white test box showed that tail suspension for 5 min immediately
before test enhanced the anxiolytic-like response diazepam
significantly (Sánchez, in press).
On the other hand, Lu 28-179 did not inhibit footshock-induced
ultrasonic vocalization in adult rats, another paradigm suggested as a
model of anxiety (Cuomo et al., 1988; Sánchez, 1993;
De Vry et al., 1993). Like the conflict model, this model
applied a relatively stressful secondary aversive stimulus. But no
effect was observed with Lu 28-179, even at very high doses.
Anxiolytics such as the 5-HT1A receptor agonist buspirone
are much more effective inhibitors of footshock-induced ultrasonic
vocalization in the rat than diazepam (Sánchez, 1993), whereas
5-HT1A receptor agonists have no or rather weak effects in
the present version of the conflict test (C. Sánchez, unpublished
observation). This model dependence of anxiolytic activity is a rather
common finding for non-benzodiazepine anxiolytics (Lister, 1990; File,
1992; Perregaard et al., 1993). The type of stressor in the
anxiogenic stimuli may interfere with the type of response in the
different test models. Different neuronal pathways have been shown to
be involved in mediating the behavioral responses to different
anxiogenic stimuli. For example, injection of a 5-HT1A
receptor agonist in the basolateral part of the amygdala enhanced the
anxiety response in the social interaction test but not in the elevated
plus maze, whereas midazolam induced an anxiolytic response in the
social interaction test but not in the elevated plus maze (Gonzalez
et al., 1996). Another study demonstrated an anxiolytic
response in the elevated plus maze after midazolam injection into
basolateral but not central amygdala, whereas the opposite result was
observed in the shock-probe burying test (Pesold and Treit, 1995).
Furthermore, a recent study of different types of fear in the elevated
T-maze suggested that stimulation of the ascending serotonergic pathway
facilitated learned fear whereas it inhibited unconditioned fear
(Graeff et al., 1996). Perhaps Lu 28-179 is effective only
against a specific subtype of anxiety. However, this is pure
speculation, because no clear relation between the different clinical
diagnoses of anxiety and the different types of animal models has been
established so far.
The anxiolytic-like responses of Lu 28-179 were observed from doses far
below nmol/kg up to 1.8 µmol/kg, and no sedation was induced by any
of these doses. In contrast, diazepam induced sedation and motor
impairment. In the mouse two-compartment black and white test box, the
anxiolytic-like profile was indistinguishable for mice treated daily
for up to 2 weeks with Lu 28-179 and mice treated with a single dose.
Furthermore, unlike the results with diazepam-treated mice, no
anxiogenic-like effects were observed upon withdrawal after 2 weeks of
treatment with Lu 28-179. In the rat social interaction test, a
significant anxiolytic-like effect was seen after up to 4 weeks of
daily treatment with Lu 28-179. The effect after 4 weeks of treatment
was significantly lower than after 3 weeks but was similar to the
effect after 1 week. The possibility that tolerance starts to develop
after 4 weeks cannot be excluded, but this is unlikely because no signs
of withdrawal-induced anxiogenesis were detectable after 4 weeks of
treatment with Lu 28-179. The lack of sedative effects and withdrawal
anxiogenesis after treatment with Lu 28-179 may indicate a more
favorable side effect profile than that of the benzodiazepines.
In conclusion, Lu 28-179 exerts potent and very long-lasting
anxiolytic-like effects in animal studies using mice and rats. Future
clinical studies will show whether this new class of compounds is
effective in relieving anxiety without inducing the unwanted effects of
the benzodiazepines.
We thank Karin Larsen, Lone Hanne Petersen, Dorit Skov (H. Lundbeck A/S), Deborah Murphy and Ben Grayson (Postgraduate Studies in
Pharmacology, University of Bradford) for excellent technical help, and
Hanne Albertsen for typing the manuscript.
Lu 28-179, 1-(4-fluorophenyl)-3-[4-[4-(4-fluorophenyl)-1-piperidinyl]-1]butyl]-1H-indole;
MED, minimal effective dose;
DTG, 1,3-di-o-tolylguanidine;
DA, dopamine;
5-HT, serotonin, 8-OH-DPAT,
8-hydroxy-2-(di-n-propylamino)tetraline;
QNB, quinyclidinyl benzilate;
NA, noradrenaline;
mw, molecular weight;
3-PPP, N-n-propyl-3-(3-hydroxyphenyl)-piperidine;
CCK, cholecystokinin.
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2-Ligand Lu 28-179 Has Potent
Anxiolytic-Like Effects in Rodents
Abstract
Introduction
Abstract
pharmacological profile of a specific serotonin-uptake inhibitor with antidepressant activity.
Prog. Neuro. Psychopharmacol.
6: 277-295, 1982.
1/
-Aminoalkyl)-1H-indoles.
J. Med. Chem.
38: 1998-2008, 1995[Medline].
