- Email: [email protected]
5-HT2 receptor antagonist
fkg.
Pergamon
Nruro-Psycho~~f~armacol
&. r3lOl. Pssycfual. CopyrIght fnn~ed
0278-5846(95)00237-5
1995.
Vol
c‘ 1995
19. p,‘. Elsrvrr
in CreaC Bntain
12OlL 12 16 Sc~rncr
All nghts
0278
5X46/95
529.00
PHARMACOLOGICAL PROFILE OF (-)HT-SOB, A NOVEL 5-HTIA RECEPTOR AGONIST/5-HT2 RECEPTOR ANTAGONIST HARUHISA
UCHIDA, KENTARO INAGAWA,
CHIHO TAMEDA AND TATSUO MIYAIJCHI
Fuji Gotemba Research Labs., Chugai Pharmaceutical
Co., Ltd.. Gotemba-shi,
Shizuoka, Japan.
(Final form, May 1995)
Abstract Pharmacological Profile Uchida, Haruhisa, Kentaro Inagawa, Chiho Tameda and Tatsuo Miyauchi: of (-)HT-90B, A Novel 5-HTIA Receptor Agonist / 5HTz Receptor Antagonist. Prog. NeuroPsychopharmacol. & Biol. Psychiat. 1995, 19(7): 1201-1216. 1. HT-90B ( (-)-N-( [2-(8.methyl-l, 4-benzodioxane-2-ylmethyl)amino]ethyl)tricyclo[3,3,1,1(3,7)] decane- 1-carboxamide) had high affinities for the 5-HT,,, (Ki = 0.18 nM) and 5-HTz (Ki = 9.2 nM) receptors. 2. HT-90B inhibited forskolin activated adenylate cyclase in rat hippocampal membranes as a 5-HT,, full agonist (I& = 2 nM), and the potency of the drug was higher than that of 8-hydroxy-2-(di-npropylamino) tetralin (8-OH-DPAT), a standard 5-HT,A agonist. 3. In the serotonin syndrome test, HT-90B behaved as a weak partial 5-HT,, agonist in reserpinized rats. 4. 5-HTz receptor-mediated potentiation of rabbit platelet aggregation by serotonin (5.HT) was reduced by HT-90B (IC,, = 1.73 PM). 5. Head twitch response induced by 1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane HT? agonist, was inhibited by HT-90B in mice. receptor agonist as well as 6. It is concluded that HT-BOB has potent 5HT,, antagonist properties in vitro and in vivo. Kev words: buspirone, HT-90B, 5-HT,, receptors, 5-HT? receptors, 8-OH-DPAT,
(DOl). a 5 S-HT, receptor
ritanserin
Abbreviations: 1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane (DOI), dimethyl sulfoxide (DMSO), 8-hydroxy-2-(di-n-propylamino) tetralin (8.OH-DPAT). (-)-N-( [2-(8-methyl-1.4.benzodioxane-2ylmethyl)amino]ethyl) tricyclo[3,3,1,1(3,7)]decane1-carboxamide (HT-90B), serotonin (5-HT)
5-HT distributes
widely in the mammalian
of several neuropsychological elaboration
and expression
processes, of affective
CNS as a neurotransmitter.
including
feeding,
aggression,
and roles in the modulation sexual behavior,
states (Bevan et al., 1989). Currently,
and the
5-HT receptors
are
classified into 7 families, 5-HT, through 5-HT,, based on amino acid sequence and intracellular signal transduction
mechanism.
A S-HT, receptor
family has six members,
S-HT,,~,, (Julius, 1991: Hamblin and Metcalf.
1201
In<
wservtd
1991:
1202
H. Uchida et al.
McAllister
et al., 1992; Adham et al., 1993). In comparison
5-HT,, receptor has been particularly well characterized HT,A receptor Fozard,1983; of animals
ligand,
to the other 5-HT, receptor subtypes, the
because of the availability of the selective 5-
%hydroxy-2-(di-n-propylamino)
tetralin
(8OH-DPAT)
Hamon et a1.,1984). The 5-HTIA receptor has a heterogeneous and humans,
being found in high density
cortex, amygdala and raphe nuclei shown to be coupled
adenylate
forskolin-stimulated
adenylate cyclase by the agonists
behaviors
by the stimulation
induced
behavioral syndrome”,
and frontal
in the brain have been
and to be involved
(De Vivo and Maayani,
of 5-HT receptors
in the inhibition
the activation of 5-HT,,
of
1986). Characteristic
in rats have been termed
and these behaviors have been used as indexes of physiological
HT receptor. In particular,
the anxiolytic
entorhinal
in the hippocampus,
cyclase
and
in the brains
(Palacios et al., 1987). 5-HTIA receptors
to the enzyme
(Middlemiss
distribution
the “5-HT
functions of 5-
flat body posture and forepaw treading have been shown to be induced by receptors (Tricklebank
effects of buspirone
et al., 1984; Smith and Peroutka,
and related arylpiperazine
1986). In addition,
derivatives
are thought to be mediated
of high affinity antagonists,
5-HT2 receptors have been
by 5-HT,, receptors (Taylor et al., 1984; Traber et al., 1984). On the other hand, due to the development
studied for much longer period of time than 5-HT, receptors. concentrations
5-HT, receptors
distribute
at high
in the Iv layer of the frontal cortex and the caudate nucleus (Hoyer et al., 1986; Pazos
et al., 1985). The neuronal excitation which follows 5-HT, receptor activation results from the closure of an inwardly rectifying that 5-HTz receptors
K’conductance
which induces a slow depolarization.
It has been suggested
could be linked to protein kinase C through inositol phospholipid
(North and Uchimura
1989). Stimulation
shake or wet-dog shake responses
hydrolysis
of 5-HTz receptors induces head twitches in mice and head
in rats (Lucki et al., 1984; Yap and Taylor, 1983). 5-HT2 receptors
are present also in the platelet,
and mediate 5-HT-induced
inositol phospholipid
and increase in intracellular calcium (Affolter et al., 1984; De Clerck
hydrolysis
et al., 1984).
Also,
antidepressants,
because chronic treatment
5-HT2 receptors
have been
shape change and aggregation
implicated
with antidepressants
in the mechanism
through
of action
produces down-regulation
of
of 5-HTz
receptors (Peroutka and Snyder, 1980).
0 NH CH3
Fig. 1 Chemical structure of HT-90B, (-)-N-l [2 (8-methyl-l,4-benzodioxane-2-ylmethyl)amino] ethyl)tricyclo[3,3,1,1(3,7)]decane-l-carboxamide. This report describes the pharmacological
properties of a novel benzodioxane
(Fig. l), which exhibits a high affinity for both 5-HT,,
and 5-HT2 receptors.
compound, The properties
HT-90B of HT-
90B have been studied in vitro by means of radioligand binding assay, forskolin stimulated adenylate cyclase assay and 5-HT induced platelet aggregation.
In vivo activities
of HT-90B have also been
Pharmacological examined
by means of the 5HT
syndrome
profile of HT-SOB
test and the head-twitch
agonist,l-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane 90B has been compared
to those of reference
1203 response
(DOI) (Glenon,
compounds,
8-OH-DPAT
induced by a S-HTz
1987). The activity of HTand buspirone
in the case of
5-HTIA and ritanserin in the case of 5-HTz
Methods Animals Male Sprague Dawley rats (Charles River), 200-300 g, male Japanese White rabbit (CSK), 2.0-3-O kg, and male ddY mice (SLC), 20-30 g, were maintained
on a 12-hr light/dark cycle and housed 5
(rats), 1 (rabbits) and 10 (mice) per cage with food and water ad libitum. The animals were brought into the laboratory at least 1 week before the experiments. Drugs The following drugs were used: HT-90B and ritanserin were synthesized 8-OH-DPAT
was obtained
from Amersham,
England Nuclear; 8-OH-DPAT,
and other radioligand
DOI and methysergide
in our laboratories.;
were purchased
[‘HI-
from New
were from Research Biochemical
Inc.; ATP,
CAMP and GTP from Yamasa Shoyu K.K.; DMSO and Clear-sol I from Nacalai Tesque Inc.; Other chemicals were purchased from Sigma Chemical Co. In all the in vitro studies, HT-90B, buspirone. DMSO, and subsequent aggregation
8-OH-DPAT,
5-HT and ritanserin were dissolved in
dilutions were made in either the same solvent (in binding assay and platelet
tests) or distilled water (adenylate cyclase assays). In the in vivo experiments,
and DO1 were dissolved
in saline (1 ml/kg), while other drugs were suspended
reserpine
in 1 % gum arabic (2
ml/kg). Binding Assav The affinity of the different
compounds
for 5-HT,,
receptors
was assessed
by use of the [‘HI-g-
OH-DPAT binding assay described by Hall et al. (1985) and Peroutka (1986). Rats were sacrificed by decapitation
and their brains were rapidly removed
cortex were dissected
out and homogenized
buffer (pH7.7 at 25 C) using a Polytron centrifuged
and put on ice. The hippocampus
in 40 volumes disrupter
(vol./wt)
(Kinematica
of ice-cold
CH-6010).
and cerebral
50mM Tris-HCI
The homogenate
was
40,000 X g for 20 min. The supernatant was discarded and the pellet was washed twice by
resuspension
in 40 volumes of Tris-HCI and centrifugation,
homogenized
stored at -80 43 until needed. On the day of study, the homogenate and incubated at 37 “c for 20 min to remove endogenous then collected by centrifugation
in 40 volumes Tris-HCI and
was thawed at room temperature
5-HT (Nelson et a1.,1978). Membranes
and washed twice before final resuspension
Tris-HCI. pH7.7. The protein concentration
was measured
were
in 65 volumes of 50 mM
by the method of Bradford
(1976) with
bovine serum albumin as the standard. Membranes containing
were incubated
at 25 “C for 30 min in a final volume
0.5 nM [‘HI-&OH-DPAT,
10 ul of the compound
of lml Tris-HCI.
to be tested in DMSO,
pH7.7,
and 0.1%
1204
H. Urhida et ul
ascorbic acid. The membranes
were then rapidly vacuum-filtered
through Whatman GF/B filters and
washed three times with 3 ml of ice-cold Tris-HCl buffer. The filters were placed in polyethylene vials with 5 ml of Clear-sol I, a scintillation to the measurement Nonspecific
of radioactivity
binding was determined
All determinations
cocktail, and stored at 4 “C overnight to equilibrate prior
by liquid scintillation by incubating
spectrometry
at an efficiency
similar samples with unlabelled
of 40-47 %.
5HT (IO FM).
were made in triplicate.
The 5HTz receptor binding assay was carried out on frozen crude membrane preparations as SHT,,
receptors.
Membranes
HCI, pH7.7, containing Nonspecific
as well
were incubated at 30 “c for 30 min in a final volume of 1 ml Tris-
0.5 nM [‘HI-ketanserin
binding was determined
and 10 ~1 of the compound
by incubating
to be tested in DMSO.
similar samples with unlabelled
methysergide
(1
i.tM). Other binding assays were carried out as indicated used in the references
quoted.
The ICo
binding) values were determined
in Table I. based essentially
(i. e., the concentration
on the methods
that inhibits 50 o/o of the specific
by non-linear regression analysis using an EBDA-LIGAND
program
on a Macintosh computer. Table 1 Receptor Binding Methodology Receotor 5-HTIA ~-HTIc
Tissues cortex+hippocampus cortex
5-HT2 5-HT? S-HTJ
cortex+hrppocampus cortex
D2
CII cf.2 P BZD
strlatum strlatum whole bram(-cerebellum) cortex cortex cortex
Species lieand rat [‘HI&OH-DPAT rat [‘H]mesulergine
rat rat
[jH]ketanserin t’HlBRL43694
rat [‘H]GRlI3808 rat [JH]spiperone rat [‘Hlprazosin rat [7H]rauwolscine rat [“Hldihydroalprenolol ratI’HIRol5-1788
Rct’erence Peroutka (1986) Pazos ( 19X.5) Leysen et al. (1982) Nelson and Thomas (1989) Grossman et al. ( 1993) Sanger and Nelson (1989) Greengrass and Brcmner (I 979) Broadhurst and Wyllie (I 986) Bylund and Snyder (I 976) Brown and Martm (1984)
Adenvlate Cvclase Assavs The assay conditions (1985).
were modified
Rats were sacrificed
homogenized
from those published
by decapitation
in a 9 volume (v/w) ice-cold solution containing
EDTA, 5 mM dithiothreitol
The hippocampi
were
300 mM sucrose, 1 mM EGTA, 5 mM
was diluted &fold in homogenization
500 X g at 0 “C for 5 min. The pellet from this centrifugation
membranes
(De Vivo and Maayani,
removed.
and 20 mM Tris-HC1 (pH7.4 at 23 C) in a tissue homogenizer
strokes by hand). The homogenate centrifuged
previously
and the hippocampi
solution and centrifuged
was discarded and the supernatant
(20 at was
at 39,000 X g for 10 min at 0 “C. The supernatant from the second spin was discarded and were resuspended
by vortexing
Aliquots (100 ~1) of the membrane suspension
in 40 volumes of ice-cold
homogenization
solution.
were added to 400 ~1 of cyclase assay medium to start
the assay. The cyclase assay medium was incubated for 5 min at 30 “C before the start of the assay.
Pharmacological
The final components mM magnesium
1205
of the assay medium were : 10 PM forskolin,
100 mM NaCI, 10 PM GTP. 2
acetate, 0.2 mM ATP, 1 mM CAMP, 80 mM Tris-HCl (pH7.4), 60 mM sucrose. 0.2
1 to 2 ptci of [a-‘?P]ATP (specific activity. 20-40
mM EGTA. 1 mM EDTA, 1 mM dithiothreitol, Ci/mmol),
profile of HT-SOB
2 pg of membrane
protein, 4 mM theophylline.
creatine phosphate and various concentrations
0.1 mg/ml creatine phosphokinase.
5 mM
of agonists. The medium was incubated at 30 “C for IO
min and the reaction was stopped by heating the mixture to 95 “c for 5 min. After cooling the sample to 4 % , 100 pl of 45 mM ATP and 1.3 mM cAMP containing (30.000 dpm) was added to the medium. The conversion described
by Salomon
(1979). Proteins
an internal standard of [ ‘HIcAMP
of [?:P]ATP to [‘?P]cAMP was measured ax
were determmed
( 1976) with
by the method of Bradford
bovine serum albumin as the standard. Forskolin was added from a 50 mM stock solution in ethanol and diluted such that the ethanol concentration exchange
in the assay mixture was less than 0.02 %. C‘atmn
resin (AG 5OW-X4, 200-400 mesh) and aluminium
Laboratories
(Richmond.
oxide were obtained
from Blo-Rad
CA).
Platelet Aggregation The rabbit was anesthetized obtained citrate
from the common to 9 parts blood)
centrifugation
with sodium thiopental (50 mg/kg. i.v.), and arterial blood (50 ml) was carotid artery with a syringe containing
as anticoagulant.
and platelet-rich
at 1.300rpm for 10 min. Platelet aggregation
method of Born ( 1962) using an aggregometer
3.8 % sodium citrate (I part
plasma
(PRP)
was prepared
by
was studied in PRP with the turbidimetric
(Aggretec TE-500. Erma). A cuvette containing 200 ~1
of PRP, 5 ~1 of 192 mM CaCI, in saline, and 5 ~1 of the drug to be tested in DMSO was placed in the aggregometer
and allowed to incubate for 3 min. Platelet aggregation
5-HT (final concentration
4.2 PM) and 20 1.11of collagen
cuvette. and recorded for 5 min. The concentrations sub-threshold
was initiated by adding IO ~1 of
(final concentration
4 - 10 pg/ml,
to the
of 5-HT and collagen were selected to produce a
response by each inducer alone and 40 - 60 % aggregation
by both inducers together.
IC50values were calculated by Probit analysis. 5-HT Behavioral Syndrome Following
the method described
by Tricklebank
mg/k g, KC.) 18 hr before drug administration.
et al. (1984), rats were treated with reserpine
For measurement
of the behavioral
syndrome,
animals were placed in individual clear Plexiglass cages 5 min before drug administration. posture and forepaw treading were scored during an observation ranked intensity
scale (0 = absent,
1978). Observations administration. DOI-Induced
were
made
1 = equivocal, every
Scores for each compotent
12 min.
Flat body
period of 45 set per rat on a 4-point
2 = definite,
3 min for
(I the
3 = extreme) beginning
(Deakin and Green.
5 min after
were summed over the 5 observation
the drug
periods.
Head Twitch
Mice were injected with DOI (5 mg/kg, i.p.) 30 and 60 min before the administration and ritanserin.
respectively.
The number of head twitch responses
administration
of DOI was counted.
of HT-90B
during the first 10 min after the
H. Uchida
1206
In DOI-induced
head-twitches.
ritanserin
cat al.
was used to confirm
activation in the behavior. Therefore, ritanserin was administered 90B is a candidate (unpublished
of anxiolytic/antidepressant
of behaviorally
of 5-HT, receptor
which is orally active 111various behavioral
data; Inagawa et al., 199.5). Therefore,
action after p.0. administration
the involvement
i.p. as usual. On the other hand, HTtests
we aimed to test its in vivo 5-HT? antagonistic
effective doses (higher than 10 mg/kg).
Data Analvsis Statistical
differences
variance followed
between
experimental
groups
by Dunnet’s multiple comparison
were evaluated
either
by an analysis
of
test or Student’s t-test. P values less than 0.05
were regarded as significant.
Results L Binding Assay The apparent affinity (Ki) of HT-90B and the reference for the 5-HT,,
receptors
was determined.
labeled by [3H]-8-OH-DPAT
compounds,
8-OH-DPAT
and buspirone,
in rat cortical plus hippocampal
membranes
As shown in Table 2, HT-90B was the most potent agent tested with a Ki value of
0.18 nM. %OH-DPAT
was slightly less potent than HT-90B with a Ki value of 0.49 nM. Buspirone
was the weakest agent analyzed with a Ki value of 7.7 nM. HT-90B exhibited a high affinity for the 5-HT, recognition membranes
labelled with [‘Hlketanserin,
site in the rat cortical plus hippocampal
with a Ki value of 9.2 nM. Buspirone
potent than HT-90B (Ki = 490 nM). 8-OH-DPAT
displayed
a
was 50-fold less
very low affinity
for the 5-HTz
receptors (Ki > IO PM). In the rat cortical membranes, [7H]-mesulergine receptors
HT-90B was less active at the 5-HT,e binding sites labelled with
(Ki = 260 nM), and displayed
labelled with [‘HI-BRL43694
Both &OH-DPAT
and buspirone
a very low affinity
and for the 5-HT, receptors
displayed
a very low affinity
(Ki > 3300) for the 5-HT, labelled with [‘HI-GR113808.
for the 5-HTlc
and the 5-HT,
receptors. With respect
to the other neurotransmitter
receptors,
dopamine D2 receptors (Ki = 49 nM) and a, adrenoceptors
HT-90B
had moderate
affinities
for the
(Ki = 54 nM). At the a, adrenoceptor,
HT-
90B displayed a low affinty (Ki = 170 nM). HT-90B was less active m binding to the P-adrenoceptors (Ki = 170 nM). At the benzodiazepine Buspirone exhibited
receptors,
HT-90B showed negligible
some affinity for the dopamine
affinity (Ki > 5.9 FM).
Dz receptor, similar to that of HT-90B (Ki = 63
nM), but showed very low affinities for the a,,a?,p and BZD receptors. 8-OH-DPAT bmding to the D2. a,, ~1~and p adrenoceptors To compare
the receptor
various receptors examined,
selectivity
(Table 2).
of HT-90B, buspirone
were divided by those for 5-HT,,, receptors
HT-90B had a high selectivity
was inactive in
and &OH-DPAT,
the Ki values for
(Table 2). Among the 5-HT receptors
for 5_HT,,, and 5HTz receptors
(Ki ratio was I:5 1). It had
Pharmacological
profile
of HT-9013
more than a lOO@fold less potent affinity for the other 3 subtypes.
1207
For the other neurotransmtttet
receptors, HT-90B exhibited 300-fold (DI and a,) and more than 900-fold (a,, p and BZD) less potent affinities than tt did for 5-HT,, receptors. With respect to buspirone,
receptor selectivity
for SHT, , and S-HT, ( 1:63) was similar to that of
HT-90B. However, buspirone had a much higher selectivity HTI receptors.
H-OH-DPAT exhibited exclusive selectivity
for the D, receptors (1:8) than for the 5for the S-HT,,, receptors.
Table 2 Receptor Binding Profile of HT-90B. Buspirone and %OH-DPAT
Receptor
HT-90B
Ki (nM) Buspirone
( / 5-HTIA)
( / S-HTI A)
5-HTI 5-HTIc
0.18
7.1
(1)
(1)
260
2000
(1400)
5-HTJ
9.2 (51) na. b
5-HTJ
n.a. b
5HT>
D? ai a2
C/5-HTIA)
0.49 IIJ
7x00 c
I IhIM),
1260)
11.a.b.c
490 (61)
n.a. b
na. b.c
n.d. a
n.d. a
49
63
(270)
(8)
2200 c (4SO0,
2700 c
1700
54 (3(K)) 170
3400
(940)
iJ40)
2100
P
SOH-DPAT
(5500)
1220)
300 c (hl0,
n.3. b.c
na. b
(12000)
n.d. a n.a. b n.a. b BZD a: n.d. = not determined, b: n.a. = no or weak affinity observed at the highest concentration. c: data from Van Wijingaaden et al (1990). Number in parentheses represent the ratto of Ki value for a given receptor to that for 5-HT,,\ receptor.
Adenvlate Cvclase Assay As shown in Fig.‘, 5HT inhibited foskolin-stimulated
cyclase activity. and a maximal inhibition of
25-30 % was achieved routinely at IO FM. X-OH-DPAT, a 5-HT,,, receptor full agonist. inhibited the cyclase activity to the same degree as did 5-HT. HT-90B also acted as a full agonist. since maximal inhibition by HT-90B was similar to those by 5.HT and R-OH-DPAT.
However, the potency of HT-
90B was much higher than that of the two other full agonists (Fig. 2. Table 3). Figure 2 shows that the inhibition antagonist.
curve produced
by HT-90B was shifted to the right by IOpM of pindolol.
Pindolol by itself had no effect on the forskolin-stimulated
contrast wtth the effects of full agonists, was only 608 of that produced by 5HT.
maximal inhibition
a S-HT,,
adenylate cyclase activity.
by huspirone,
In
a 5-HT, , partial agonist.
1208
H. Uchda <'I nl.
0
--O+ *
S-HT (n=2) HT-90B (n=4) 8-OH-DPAT (n=2)
-t_
Buspirone (n=2)
-9
-8
-7 log
-6
+
HT-90B (n=4) HT-90B + pindolol (IO@I) (n=2)
a-
0
-5
-9
-8
M
log
Fig. 2 Inhibition of forskolin-stimulated Each point represent the mean f S.E.M.
-7
-6
M
adenylate cyclase activity in rat hippocampal * : P < 0.05, ** : P < 0.01 (vs HT-90B).
membranes.
Table 3 I& Values of Inhibition of Forskolin-Stimulated Compound HT-90B
IG
Adenylate Cyclase by 5-HT,, Receptor Agonists.
(nM) 3.4
HT-90B+ Pindolol (10 FM) 210 5-HT 38 8-OH-DPAT 11 Busnirone 650 I& values were expressed as 50 % of maximum inhibition by 5-HT, a 5-HT,,t receptor full agonist.
Platelet Aggregation Although 5-HT (4.2 pM) alone did not induce the aggregation amplified the aggregation HT-90B Inhibition
attenuated
induced by a subthreshold
the amplifying
by ritanserin
produced by HT-90B.
(I&,
effect
of rabbit platelets, it synergistically
concentration
of collagen (4 - 10 pM) (Fig. 3).
of 5-HT in a dose-dependent
= 25 nM), a 5-HT2 antagonist,
manner (IC,, = 1.73 PM).
was 200-fold
more potent than that
Buspirone exhibited a less potent inhibition than did HT-90B and ritanserin.
5-HT Behavioral Svndrome Subcutaneous syndrome
administration
response
forepaw treading.
(Tricklebank
of 8-OH-DPAT
to reserpine-treated
rats elicited a typical 5-HT motor
et al., 1984; Mir et al., 1988) consisting
When compared with the effect of 8-OH-DPAT,
of flat body posture and
buspirone and HT-90B produced
Pharmacological only mild to moderate behavioral
profile of HT-SOB
effects in this model, buspirone
1209
being slightly more effective
than
HT-90B in this regard (Fig. 4).
Ritanserine (n=3) HT-90B (n=4) Buspirone (n=2)
I””
-10
-9
-8
-1
-6
-5
-4
log M Fig.3 Inhibition of platelet aggregation induced by collagen (4-10 pg/ml) and 5-HT (4.2 t.tM). Platelet-rich plasma from rabbits was pre-incubated for 3 min with different concentration of drugs. Results were expressed as % of maximum aggregation induced by collagen and 5-HT. Each point represent the mean + S.E.M. * : P < 0.05, ** : P < 0.01 (vs HT-90B).
Flat body posture (n=8- I I )
Forepaw treading (n=8- I I 1
R-OH-DPAT
8-OH-DPAT
dose (mgkg, SC.)
dose (mgkg,
s.c.)
Fig.4 5-HT syndromes in reserpine-treated rats. Flat body posture and forepaw during observation period of 45 set per rat on a 4-point ranked intensity scale 1978). Observations were made every 3 min for 12 min. beginning at 5 administration. Scores for each component were summed over the 5 observation represent the mean f S.E.M. ** : P < 0.01 (vs HT-90B).
treading were scored (Deakin and Green, min after the drug periods. Each point
1210
H. Uchida et al
+
HT-90B (n=l2-1.3) Ritanserine (O.Smgkg,
A
o-
0
IO
20
30
40
i.p.) (n=8)
50
dose (mgkg, p.0.) Fig. 5 Number of head twitch response during the first 10 min after the administration of DOI. Mice were injected with DO1 (5 mg/kg, i.p.) 0.5 and 1 hr after the administration of HT-90B and ritanserin, respectively. Each point represent the mean + S.E.M. * : P < 0.05, ** : P c 0.01 (vs 0 mg/kg).
DOI-Induced
Head-Twitches
The effect of HT-90B on head-twitches HT-90B inhibited the DOI-induced administration.
induced by DOI, a 5-HT, agonist, is presented
head-twitches
in a dose-dependent
Ritanserin (0.5mg/kg, i.p.) completely
in Fig. 5.
manner in the mouse after oral
abolished the DOI-induced
head-twitches.
Discussion The present studies have demonstrated
that HT-90B, a novel benzodioxane
both 5-HT,, receptor agonistic and 5-HT? receptor antagonistic
derivative,
possesses
activities in vitro and in vivo.
Binding Assav In radioligand binding assay, HT-90B exhibited an approximately OH-DPAT
for the 5-HT,* receptors
3-fold greater affinity than did 8-
labelled with [3H]-&OH-DPAT.
for 5-HT, receptors labeled with [?H]-ketanserin. HT,c, 5-HT3 and 5HT4, were virtually for 5-HT,,
negligible.
receptors,
but the selectivity
receptors.
It has been reported that buspirone,
HT-90B also had high affinity
Its affinity for the other 5-HT receptor subtypes, 5-
receptors
It displayed
some affinities
was approximately
a non-benzodiazepine
for the D, and CI,
300-fold greater than these anxiolytic with 5-HT,,
activity (Eison and Temple 1986; Traber and Glaser, 1987; Carli et al, 1989), has side-effects to be caused by anti-dopamine
agonist thought
actions of the drug such as sedation, akathisia and perioral dyskinesia
(Goa and Ward, 1986). HT-90B and buspirone exhibited similar affinities for D, receptors. However, the selectivity
ratio of HT-90B for 5-HT,,
over D, receptors was 270, which was far greater than that
of buspirone.
These results indicate that HT-90B has a higher affinity for 5-HTIA receptors than does
8-OH-DPAT
and is more selective than is buspirone.
Pharmacological
profile of HT-SOB
1211
Adenvlate Cvclase Assay In adenylate cyclase assay, employed
as a functional test of 5HT, 4 receptor activity in vitro, HT-
90B showed potent 5-HT,, receptor agonist properties.
For example, HT-BOB inhibited the forskolin-
activated adenylate cyclase to the same extent as did 5-HT and S-OH-DPAT. was 3-fold higher than that of 8-OH-DPAT,
which parallels the difference
receptors. The effect of HT-90B was suppressed reported
that pindolol
has 5-HTIA antagonist
DPAT on forskolin-stimulated results
suggest
hippocampal
adenylate
that HT-90B
The potency of HT-90B in the affinity for 5-HT,,\
by pindolol, which was inactive by itself. It has been activity and reverses
cyclase activity
acts as a full agonist
the inhibitory
(Oksenberg
action of &OH-
and Peroutka,
1988). These
and is more potent than 8-OH-DPAT
in rat
neurons in vitro.
5-HT Behavioral Svndrome The activity to induce flat body posture and forepaw treading was used to explore the central 5HTIA agonistlc action of HT-90B in vivo. HT-90B induced these behaviors with a lower efficacy than did 8-OH-DPAT bioavailability
and buspirone.
If less potent in vivo activity of HT-90B was only due to a lower
of the compound,
then maximum effect of HT-90B at high dose would be even bigger
than those of the reference maximum
compounds
because of HT-90B’s higher in vitro potency. However,
effect of HT-90B was much weaker than those of X-OH-DPAT
and buspirone
the
in the
present study. In contrast, HT-90B exhibited more potent and efficient anxiolytic- and antidepressantlike behavioral
action than did 8-OH-DPAT
1995). Furthermore, accumulation
and buspirone
in rat brain in vivo (unpublished
90B in eliciting
in rats (unpublished
HT-90B was more potent than 8-OH-DPAT
serotonin
syndrome
and buspirone
data; Inagawa et al., in inhibiting
5-HTP
data), These results suggest that lower potency of HT-
could not be explained
by a lower bioavailability
of the
compound. Thus, it appears that HT-90B acts as a partial agonist in these behavioral tests. This result may look incompatible explained
with the data obtained by presumed
mechanisms
differences
in 5-HT,,,
cyclase assay. However,
receptors
coupling
this can be
to different
effector
and existing in different locations in the CNS. It has been shown that buspirone
efficacious
than MDL73005EF,
hyperpolarizations inhibition
subtle
in the in vitro adenylate
another
in hippocampal
of forskolin-activated
5-HT,,
pyramidal adenylate
partial
agonist,
in 5-HT, ,-mediated
cells, while the reverse
cyclase
is more
membrane
is true in 5-HT,,-mediated
even in the same tissue (Van den Hooff and
Galvan. 199 I: Cornfield et al., 1989). Both actions have been shown to be mediated by a G protein (Andrade et al.,l986; Clarke et al., 1987), and yet they appear to be independent levels of CAMP do not affect membrane 1988). Furthermore,
potential changes produced by S-HT,, agonists (Innis et al.,
it has also been reported that the intrinsic activity of 5-HT,,
buspirone (De Vivo and Maayani, 1986; VanderMaelen and Galvan, producing
of one another since
1991; Sprouse,
hyperpolarization
et al., 1991). MDL73005EF
agonists, such as (Van den Hooff
1991) and others (Millan et a1..1993; Greuel and Glaser. does in fact change when measured
present studies, rat hippocampal
membranes
in different
1992). in
brain regions.
In the
were used for the adenylate cyclase assay, while the flat
1212
H. Uchida et al.
body posture and forepaw treading are postulated to be triggered by the stimulation of 5HT receptors in the brainstem Therefore,
and / or spinal cord (Jacobs
it seems
hippocampus
probable
that HT-90B
and Klemfuss,
1975; Lucki and Frazer,
acts as a full agonist
on 5-HT,,
receptors
coupled to adenylate cyclase, while it has a partial agonistic action on 5HT,,
1982). in the
receptors
in the brain stem and / or spinal cord, probably coupled to other mechanisms. 5_HT, Antagonistic Inhibition
Action in Vitro and in Vivo
of 5HT-potentiated
activity of the compound
platelet aggregation
by HT-BOB revealed the 5-HT, antagonistic
in vitro. Although HT-90B was lOO-fold less potent than ritanserin, it was
more potent than buspirone.
The 5-HTZ antagoistic
vivo after oral administration,
since this compound
action of HT-90B in the CNS was detected in inhibited the DOI-induced
head-twitch
response,
which is mediated by an activation of central 5-HT2 receptors (Goodwin and Green, 1985). The effect of HT-90B was much weaker than that of ritanserin. antagonizing
DOI-induced
HT2 antagonistic
Lower potency
of HT-90B in
head twitches may be a simple reflection of 100.fold less potent in vitro 5-
action of HT-90B when compared with ritanserin.
Conclusion The present antagonistic buspirone
study
has demonstrated
that HT-90B
profile in vitro and good bioavailability and related
arylpiperazines
display
has a potent
in vivo.
anxiolytic
5HT,,
5-HT,,
agonistic
/ 5-HT,
agonist such as 8-OH-DPAT,
and antidepressive
models (Engel et a1.,1984; Riblet et a1.,1984; Taylor et al.,l984;Traber
activities
in animal
et a1.,1984), and in humans
(Robinson et al, 1989; Beneke et al., 1988; Cott et al., 1988). On the other hand, ritanserin,
a 5-HTz
antagonist, has also been reported to produce anxiolytic effect in certain models of anxiety (Critchley & Handley,
1987) and also anxiolytic
which possesses
both .5-HT,, agonistic
effect in humans (Ceulemans and 5-HTz antagonistic
et al., 1985). Thus, HT-90B,
activities,
may be a promising
candidate as a therapeutic agent for these disorders.
References ADHAM, N., KAO, H. T., SCHECHTER, L. E., BARD, J., OLSEN, M., URQUHART, D., DURKIN, M., HARTIG, P. R., WEINSHANK, R. L. AND BRANCHECK, T. A. (1993) Cloning of another human serotonin (5.HTlF): a fifth 5-HTl receptor subtype coupled to the inhibition of adenylate cyclase. Proc. Natn. Acad. Sci. U.S.A. 90: 408-412. AFFOLTER, H., ERNE, P., BURGISSER, E. AND PLETSCHER, A. (1984) Ca2+ as messenger of 5HT2-receptor stimulation in human blood platelets, Naunyn. Schmiedebergs Arch. Pharmacol. 325: 337-342 ANDRADE, R., MALENKA, R. C. AND NICOLL, R. A. (1986) A G protein couples serotonin and GABAB receptors to the same channels in hippocampus. Science 234: 126 1- 1265. BENEKE, M., KUMMEL, B., ROAD, I. S. AND SPECHTMEYER, neurosis with ipsapirone. Psychopharmacol. @(suppI): S353.
H. (1988) Treatment of anxiety
BEVAN, P., LORENS, S. AND ARCHER, T. (1989) Behavioural pharmacology of 5-HT: possible progress in the laterality of concepts. In: Behavioural Pharmacology of 5-HT., P. Bevan, A. R.
Pharmacological Cools and T. Archer (Eds.), Jersey.
pp.459-474,
BORN, G. V. R. (1962) Aggregation Nature 194: 143-155.
profile of HI-SOB
Lawrence
1219
Erlbaum Associates,
of blood platelets by adenosine
Inc., Publishers.
diphosphate
New
and its reversal.
BRADFORD, M. M. (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dyebinding. Anal. Biochem. 12: 248254. BROADHURST, A. M. AND WYLLIE, M. G. (1986) A reassessment of the binding [3H]rauwolscine to membranes from the rat cortex. Neuropharmacology 25: 287-295. BROWN, C. L. AND MARTIN, bound rat brain benzodiazepine
of
I. L. (1984) Kinetics of [3H]Ro 15-1788 binding to membranereceptors. J. Neurochem. 42: 9 18-923.
BYLUND, D. B. AND SNYDER, S. H. (1976) Beta adrenergic receptor preparations from mammalian brain. Mol. Pharmacol. 12: 568-S80. CARLI, M., PRONTERE, C AND SAMANIN, R. (1989) hydroxytryptaminergic neurones are involved in the anxiolytic Pharmacol. 96: 829-836.
binding
in membrane
Evidence that central 5activity of buspirone. Br. J.
CEULEMANS, D. L., HOPPENBROUWERS, M. L., GELDERS, Y. G. AND REYNTJENS, A. J. (1985) The Influence of Ritanserin, a Serotonin Antagonist, in Anxiety Disorders: A Double-blind Plasebo-controlled Study versus Lorazepam. Pharmacopsychiat. 18: 303-305. CLARKE. W. P., DEVIVO, M., BECK, S. G., MAAYANI, S. AND GOLDFARB, J. (1987) Serotonin decreases population spike amplitude in hippocampal cells through a pertussis toxin substrate. Brain Res. 410: 357-361. CORNFIELD, L. J., NELSON, D. L., TAYLOR, E. W. AND MARTIN, A. R. ( 1989) MDL 73005EF: partial agonist at the 5-HTIA receptor negatively limed to adenylate cyclase. Eur. J. Pharmacol. 173: 189-192. COTT. J. M., KURTZ, N. M., ROBINSON, 5-HTIA ligand with both antidepressant 164-167.
D. S., LANCASTER, S. P. AND COPP, J. E. (1988) A and anxiolytic properties. Psychopharmacol. Bull. 2:
CRITCHLEY. M. A. E. AND HANDLEY, S. L. (1987) Effect in the X-maze anxiety model of agents acting at 5-HTl and 5-HT2 receptors. Psychopharmacology 93: 502-506. DEAKIN. J. F. AND Green, A. R. (1978) The effects of putative 5-hydroxytryptamine antagonists on the behaviour produced by administration of tranylcypromine and L-tryptophan or tranylcypromine and L-DOPA to rats. Br. J. Pharmacol. 64: 20 I-209 DE CLERCK, F., XHONNEUX, B., LEYSEN. J. AND JANSSEN, P.A. (1984) Evidence for functional 5-HT2 receptor sites on human blood platelets. Biochem. Pharmacol. 33: 2807-28 I 1. DE VIVO, M. AND MAAYANI, S. (1985) Inhibition of forskolin-stimulated activity by 5-HT receptor agonist. Eur. J. Pharmacol. 119: 231-234.
adenylate
cyclase
DE VIVO, M. AND MAAYANI, S. (1986) Characterization of the 5-hydroxytryptaminelA receptor-mediated inhibition of forskolin-stimulated adenylate cyclase activity in guinea pig and rat hippocampal membranes. J. Pharmacol. Exp. Ther. 238: 248-253. EISON, A. S. AND TEMPLE, D. L. (1986) Buspirone:Review of its pharmacology perspectives on its mechanism of action. Am. J. Med. @(SuppI 3B): I-9.
and current
ENGEL, J. A.. HJORTH, S., SVENSSON, K., CARLSSON, A. AND LILJEQUIST, S. (1984) Anticonflict effect of the putative serotonin receptor agonist 8-hydroxy-2-(di-npropylamino)trtralin (R-OH-DPAT). Eur. J. Pharmacol. 10$: 365-368. GLENON, R. A. (1987) Central serotonin receptors as targets for drug research. l-12. GOA, K. L. AND WARD. A. (1986) Buspirone.
Drugs _12: 113-129
J. Med. C’hem. 30:
1214
tf. Urhicla
cjf al
GOODWIN, G. M. AND GREEN. A. R. (1985) A behavioural and biochemical srudy in mice and rats of putative selectivje agonists and antagonists for S-HTI and S-HT2 receptors. Br. J. Pharmacol. 84: 743-753. GREENGRASS, a-adrenergic
P. AND BREMNER, receptors.
R. (1979) Binding characteristics
of 3H-prazosin
to rat brain
Eur. J. Phamacol. 55: 323-326.
GRELJEL, J. M. AND GLASER, T. (1992) The putative S-HTlA receptor antagonist NAN-190 and BMY 7378 are partial agonists in the rat dorsal raphe nucleus in vitro. Eur. J. Pharmacol. 211: 211-219. GROSSMAN, C. J., KILPATRICK, G. J. AND BUNCE, K. T. (1993) Development of a radioligand binding assay for S-HT4 receptors in guinea-pig and rat brain. Br. J. Pharmacol. 109: 618-624. HALL, M. D., EL MESTIKAWY, S., EMERIT, M. B., PICHAT, L., HAMON, M. AND GOZLAN, H. (1985) [3H]8-Hydroxy-2-(di-n-propylamino)tetralin binding to pre- and postsynaptic Shydroxytryptamine sites in various regions of the rat brain. J. Neurochem. 44: 1685-1696. HAMBLIN. M. W. AND METCALF, M. A. (1991) Primary structure and functional characterization of a human S-HT 1D-type serotonin receptor. Mol. Pharmac. 40: 143- 148. HAMON, M., BOURGOIN, S., GOZLAN, H., HALL, M. D., GOETZ, C., ARTAUD, F. AND HORN, AS. (1984) Biochemical evidence for the S-HT agonist properties of PAT (8-hydroxy-2(di-n-propylamino)tetraIin) in the rat brain. Eur. J. Phamacol. 100: 263-276. HOYER, D., PAZOS, A., PROBST, A. AND PALACIOS, J. M. (1986) Serotonin receptors in the human brain. II. Characterization and autoradiographic localization of S-HTIC and S-HT2 recognition sites, Brain Res. 376: 97-107. INNIS, R. B., NESTLER, E. J. AND AGHAJANIAN, G. K. (1988) Evidence for G protein mediation of serotonin- and GABAB-induced hyperpolarization of rat dorsal raphe neurons. Brain Res. Q: 27-36. JACOBS, B. L. AND KLEMFUSS, H. (1975) Brain stem and spinal serotonergic behavioral syndrome. Brain Res. 100: 450-457. JULIUS, D. (1991) Molecular biology of serotonin receptors.
cord mediation
Annu. Rev. Neurosci.
of a
14: 335-360.
LEYSEN, J. E., NIEMEGREERS, C. J. E., VAN NUETEN, J. M. AND LADURON, P. M. (1982) [3H]Ketanserin (R 41 468), a Selective 3H-Ligand for Serotonin2 Receptor Binding Sites. Mol. Phramacol. 21: 30 l-3 14. LUCKI, I. AND FRAZER, A. (1982) Prevention of the serotonin syndrome in rats by repeated administration of monoamine oxidase inhibitors but not tricyclic antidepressants. Psychopharmacology n: 205-2 1 I. LUCKI, I., NOBLER, M.S. AND FRAZER, A. (1984) Differential actions of serotonin antagonists on two behavioral models of serotonin receptor activation in the rat. J. Pharmacol. Exp. Ther. 228: 133-139. MC ALLISTER, G., CHARLESWORTH, A., SNODIN, C., BEER, M. S., NOBLE, A. J., MIDDLEMISS, D. N., IVERSEN L. L. AND WHITING, P. (1992) Moleculer cloning of a serotonin receptor from humon brain (S-HTlE): a fifth S-HTl-like subtype. Proc. Natn. Acad. Sci. U.S.A. 89: 5.517-5521. MIDDLEMISS, discriminates
D. N. AND FOZARD, J. R. (1983) %Hydroxy-2-(di-n-propylamino) tetralin between subtypes of the S-HT1 recognition site. Eur. J. Pharmacol. 9QlSO-153.
MILLAN, M. J., RIVET, J. -M.. CANTON, H., LEJEUNE, F., GOBERT, A., WIDDOWSON, P., BERVOETS, K., BROCCO, M. AND PEGLION, J. -L. (1993) SlSS3S:a highly selective benzodioxopiperadine S-HTlA receptor ligand which acts as an agonist and an antagonist at presynaptic and postsynaptic sites respectively. Eur. J. Pharmacol. 230: 99- 102. MIR, A. K., HIBERT, M., TRICKLEBANK, M. D., MIDDLEMISS, D. N., KIDD, E. J. AND FOZARD, J. R. (1988) MDL 72832: a potent, selective and stereospecific ligand with mixed
Pharmacological agonist and antagonist properties Pharmacol. 149: 107-120.
profile of
at both central
HT-SOB
and peripheral
1215
S-HTIA
receptors.
Eur. J.
NELSON, D. R. AND THOMAS, D. R. (1989) [3H]-BRL 43694 (Granisetron), a specific ligand for 5-HT3 binding sites in rat brain cortical membranes. Biochem. Pharmac. 18: 1693-1695. NELSON, D. L., HERBET, A., BOURGOIN, S., GLOWINSKI, J. AND HAMON, M. (1978) Characteristics of central 5-HT receptors and their adaptive changes following intracerebral 5. 7dihydroxytryptamine administration in the rat. Mol. Pharmacol. 14: 983-995. NORTH, R. A. AND UCHIMURA, N. (1989) 5-Hydroxytryptamine acts at S-HT2 receptors decrease potassium conductance in rat nucleus accumbens neurones. J. Physiol. 417: I- 12.
to
OKSENBERG, D. AND PEROUTKA, S. J. (1988) Antagonism of S-hydroxytryptaminelA (5. HT 1A) receptor-mediated modulation of adenylate cyclase activity by pindolol and propranolol isomers. Biochem. Pharmac. 37:3429-343.3. PALACIOS, J. M., PAZOS, A. AND HOYER, D. (1987) Characterisation and mapping of S-HTIA sites in the brain of animals and man. In: Brain .5-HTlA Receptors, C. T. Dourish. S. Ahleniui; and P. H. Huston (Eds.), pp 67-8 1, Ellis Horwood Ltd.. Chichester. England. PAZOS, A., HOYER, D. AND PALACIOS, J. M. (1985) Mesulergine. a selective serotonin-2 ligand in the rat cortex, does not label these receptors in porcine and human cortex: evidence for species differences in brain serotonin-2 receptors. Eur. J. Pharmacol. 106: 53 l-538. PEROUTKA, S. J. (1986) Pharmacological differentiation and characterization and 5-HTlC binding sites in rat frontal cortex. J.Neurochem. 37: 529-540.
of 5-HT I A. 5-HT I B.
PEROUTKA, S. J. AND SNYDER, S. H. (1980) Long-term antidepressant spiroperidol-labeled serotonin receptor binding. Science 210: 88-90.
treatment
decreases
RIBLET, L. A., EISON, A. S., EISON, M. S., TAYLOR, D. P., TEMPLE, D. L. AND VANDERMAELEN, C. P. (1984) Neuropharmacology of buspirone. Psychopathology 12: 6978. ROBINSON, D. S., ALMS, D. R., SHROTRIYA, R. C., MESSINA, M. AND WICKRAMARATNE, M. (1989) Serotonergic anxiolytics and treatment of depression. Psychopathol. 22(suppl): 27-36. SALOMON.
Y. (1979) Adenylate cyclase assay. Adv.Cyclic
Nucleotide Res. lo: 35-55
SANGER,G.J. AND NELSON,D.R. (1989) Selective and functional 5-hydroxytryptamine3 antagonism by BRL 43694 (granisetron). Eur. J. Phamacol, 159: 113-124.
receptor
SMITH, L. M. AND PEROUTKA, S. J. (1986) Differential effects S-hydroxytryptaminela drugs on the .5-HT behavioral syndrome. Phamacol. Biochem. Behav. 3: 15 13. I5 19.
selective
SPROUSE, J.S. (1991) Inhibition of dorsal raphe cell firin, a by MDL 7300SEF. a novel S-HT I A receptor ligand. Eur. J. Pharmacol. 201: 163-169. TAYLOR, D. P., ALLEN, L. E., BECKER, J. A., CRANE. M., HYSLOP, D. K. AND RIBLET, L. A. (1984) Changing concepts of the biochemical action of the anxioselective drug,buspirone. Drug Dev. Res. 4: 95-108. TRABER. J. M.. DAVIES, M. A., DOMPERT. W. U., GLASER, T.. SCHUURMAN, T. AND SEIDEL, P. R. (1984) Brain serotonin receptors as a target for the putative anxiolytic TVX Q 782 1. Brain Res. Bull. 12: 74 I-744. TRABER. J. AND GLASER, T. (1987) 5-HTI A receptor-related
anxiolytics.
TIPS 8: 432-437.
TRICKLEBANK, M. D., FORLER, C. AND FOZARD. J. R. (1984) The involvement of subtypes of the 5-HTl receptor and of catecholaminergic systems in the behavioural response to S-hydroxy2(di-n-propyl-amino)tetralin (R-OH-DPAT) by the putative S-HTlA receptor. Eur. J. Pharmacol. 106: 27 l-282. VAN DENHOOFF. P. AND GALVAN. M. (1991) Electrophysiology of the S-HTIA MDL73005EF in the rat hippocampal slice. Eur. I. Pharmacol. 196: 291-298.
llgand
1216
H. Uchida et al
VANDERMAELEN, C. P., WILDERMAN, R. C. AND GEHLBACH, G. (1991) Electrophysiological studies of the effect of buspirone on serotonergic neurons. In: buspirone. Mechanisms and Clinical Aspects, G. Tunnicliff, A. S. Eison and D. P. Taylor (Eds.), pp 215230, Academic Press, Inc., California. VAN WIJNGAARDEN, I., TULP, M. TH. M. AND SOUDIJN, W. (1990) selectivity in 5HT receptor research. Eur.J.Pharmacol. 188: 301-3 12. YAP, C. Y. AND TAYLOR, D. A. (1983) behaviour induced by 5hydroxytryptophan
of
Involvement of 5-HT2 receptors in the wet-dog shake in the rat. Neuropharmacology 2: 801-804.
Inquiries and reprint requests should be addressed to: Haruhisa Uchida Department of Pharmacology Fuji Gotemba Research Labs. Chugai Pharmaceutical Co., Ltd Gotemba-shi, Shizuoka 412, Japan
The concept
Pergamon
Nruro-Psycho~~f~armacol
&. r3lOl. Pssycfual. CopyrIght fnn~ed
0278-5846(95)00237-5
1995.
Vol
c‘ 1995
19. p,‘. Elsrvrr
in CreaC Bntain
12OlL 12 16 Sc~rncr
All nghts
0278
5X46/95
529.00
PHARMACOLOGICAL PROFILE OF (-)HT-SOB, A NOVEL 5-HTIA RECEPTOR AGONIST/5-HT2 RECEPTOR ANTAGONIST HARUHISA
UCHIDA, KENTARO INAGAWA,
CHIHO TAMEDA AND TATSUO MIYAIJCHI
Fuji Gotemba Research Labs., Chugai Pharmaceutical
Co., Ltd.. Gotemba-shi,
Shizuoka, Japan.
(Final form, May 1995)
Abstract Pharmacological Profile Uchida, Haruhisa, Kentaro Inagawa, Chiho Tameda and Tatsuo Miyauchi: of (-)HT-90B, A Novel 5-HTIA Receptor Agonist / 5HTz Receptor Antagonist. Prog. NeuroPsychopharmacol. & Biol. Psychiat. 1995, 19(7): 1201-1216. 1. HT-90B ( (-)-N-( [2-(8.methyl-l, 4-benzodioxane-2-ylmethyl)amino]ethyl)tricyclo[3,3,1,1(3,7)] decane- 1-carboxamide) had high affinities for the 5-HT,,, (Ki = 0.18 nM) and 5-HTz (Ki = 9.2 nM) receptors. 2. HT-90B inhibited forskolin activated adenylate cyclase in rat hippocampal membranes as a 5-HT,, full agonist (I& = 2 nM), and the potency of the drug was higher than that of 8-hydroxy-2-(di-npropylamino) tetralin (8-OH-DPAT), a standard 5-HT,A agonist. 3. In the serotonin syndrome test, HT-90B behaved as a weak partial 5-HT,, agonist in reserpinized rats. 4. 5-HTz receptor-mediated potentiation of rabbit platelet aggregation by serotonin (5.HT) was reduced by HT-90B (IC,, = 1.73 PM). 5. Head twitch response induced by 1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane HT? agonist, was inhibited by HT-90B in mice. receptor agonist as well as 6. It is concluded that HT-BOB has potent 5HT,, antagonist properties in vitro and in vivo. Kev words: buspirone, HT-90B, 5-HT,, receptors, 5-HT? receptors, 8-OH-DPAT,
(DOl). a 5 S-HT, receptor
ritanserin
Abbreviations: 1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane (DOI), dimethyl sulfoxide (DMSO), 8-hydroxy-2-(di-n-propylamino) tetralin (8.OH-DPAT). (-)-N-( [2-(8-methyl-1.4.benzodioxane-2ylmethyl)amino]ethyl) tricyclo[3,3,1,1(3,7)]decane1-carboxamide (HT-90B), serotonin (5-HT)
5-HT distributes
widely in the mammalian
of several neuropsychological elaboration
and expression
processes, of affective
CNS as a neurotransmitter.
including
feeding,
aggression,
and roles in the modulation sexual behavior,
states (Bevan et al., 1989). Currently,
and the
5-HT receptors
are
classified into 7 families, 5-HT, through 5-HT,, based on amino acid sequence and intracellular signal transduction
mechanism.
A S-HT, receptor
family has six members,
S-HT,,~,, (Julius, 1991: Hamblin and Metcalf.
1201
In<
wservtd
1991:
1202
H. Uchida et al.
McAllister
et al., 1992; Adham et al., 1993). In comparison
5-HT,, receptor has been particularly well characterized HT,A receptor Fozard,1983; of animals
ligand,
to the other 5-HT, receptor subtypes, the
because of the availability of the selective 5-
%hydroxy-2-(di-n-propylamino)
tetralin
(8OH-DPAT)
Hamon et a1.,1984). The 5-HTIA receptor has a heterogeneous and humans,
being found in high density
cortex, amygdala and raphe nuclei shown to be coupled
adenylate
forskolin-stimulated
adenylate cyclase by the agonists
behaviors
by the stimulation
induced
behavioral syndrome”,
and frontal
in the brain have been
and to be involved
(De Vivo and Maayani,
of 5-HT receptors
in the inhibition
the activation of 5-HT,,
of
1986). Characteristic
in rats have been termed
and these behaviors have been used as indexes of physiological
HT receptor. In particular,
the anxiolytic
entorhinal
in the hippocampus,
cyclase
and
in the brains
(Palacios et al., 1987). 5-HTIA receptors
to the enzyme
(Middlemiss
distribution
the “5-HT
functions of 5-
flat body posture and forepaw treading have been shown to be induced by receptors (Tricklebank
effects of buspirone
et al., 1984; Smith and Peroutka,
and related arylpiperazine
1986). In addition,
derivatives
are thought to be mediated
of high affinity antagonists,
5-HT2 receptors have been
by 5-HT,, receptors (Taylor et al., 1984; Traber et al., 1984). On the other hand, due to the development
studied for much longer period of time than 5-HT, receptors. concentrations
5-HT, receptors
distribute
at high
in the Iv layer of the frontal cortex and the caudate nucleus (Hoyer et al., 1986; Pazos
et al., 1985). The neuronal excitation which follows 5-HT, receptor activation results from the closure of an inwardly rectifying that 5-HTz receptors
K’conductance
which induces a slow depolarization.
It has been suggested
could be linked to protein kinase C through inositol phospholipid
(North and Uchimura
1989). Stimulation
shake or wet-dog shake responses
hydrolysis
of 5-HTz receptors induces head twitches in mice and head
in rats (Lucki et al., 1984; Yap and Taylor, 1983). 5-HT2 receptors
are present also in the platelet,
and mediate 5-HT-induced
inositol phospholipid
and increase in intracellular calcium (Affolter et al., 1984; De Clerck
hydrolysis
et al., 1984).
Also,
antidepressants,
because chronic treatment
5-HT2 receptors
have been
shape change and aggregation
implicated
with antidepressants
in the mechanism
through
of action
produces down-regulation
of
of 5-HTz
receptors (Peroutka and Snyder, 1980).
0 NH CH3
Fig. 1 Chemical structure of HT-90B, (-)-N-l [2 (8-methyl-l,4-benzodioxane-2-ylmethyl)amino] ethyl)tricyclo[3,3,1,1(3,7)]decane-l-carboxamide. This report describes the pharmacological
properties of a novel benzodioxane
(Fig. l), which exhibits a high affinity for both 5-HT,,
and 5-HT2 receptors.
compound, The properties
HT-90B of HT-
90B have been studied in vitro by means of radioligand binding assay, forskolin stimulated adenylate cyclase assay and 5-HT induced platelet aggregation.
In vivo activities
of HT-90B have also been
Pharmacological examined
by means of the 5HT
syndrome
profile of HT-SOB
test and the head-twitch
agonist,l-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane 90B has been compared
to those of reference
1203 response
(DOI) (Glenon,
compounds,
8-OH-DPAT
induced by a S-HTz
1987). The activity of HTand buspirone
in the case of
5-HTIA and ritanserin in the case of 5-HTz
Methods Animals Male Sprague Dawley rats (Charles River), 200-300 g, male Japanese White rabbit (CSK), 2.0-3-O kg, and male ddY mice (SLC), 20-30 g, were maintained
on a 12-hr light/dark cycle and housed 5
(rats), 1 (rabbits) and 10 (mice) per cage with food and water ad libitum. The animals were brought into the laboratory at least 1 week before the experiments. Drugs The following drugs were used: HT-90B and ritanserin were synthesized 8-OH-DPAT
was obtained
from Amersham,
England Nuclear; 8-OH-DPAT,
and other radioligand
DOI and methysergide
in our laboratories.;
were purchased
[‘HI-
from New
were from Research Biochemical
Inc.; ATP,
CAMP and GTP from Yamasa Shoyu K.K.; DMSO and Clear-sol I from Nacalai Tesque Inc.; Other chemicals were purchased from Sigma Chemical Co. In all the in vitro studies, HT-90B, buspirone. DMSO, and subsequent aggregation
8-OH-DPAT,
5-HT and ritanserin were dissolved in
dilutions were made in either the same solvent (in binding assay and platelet
tests) or distilled water (adenylate cyclase assays). In the in vivo experiments,
and DO1 were dissolved
in saline (1 ml/kg), while other drugs were suspended
reserpine
in 1 % gum arabic (2
ml/kg). Binding Assav The affinity of the different
compounds
for 5-HT,,
receptors
was assessed
by use of the [‘HI-g-
OH-DPAT binding assay described by Hall et al. (1985) and Peroutka (1986). Rats were sacrificed by decapitation
and their brains were rapidly removed
cortex were dissected
out and homogenized
buffer (pH7.7 at 25 C) using a Polytron centrifuged
and put on ice. The hippocampus
in 40 volumes disrupter
(vol./wt)
(Kinematica
of ice-cold
CH-6010).
and cerebral
50mM Tris-HCI
The homogenate
was
40,000 X g for 20 min. The supernatant was discarded and the pellet was washed twice by
resuspension
in 40 volumes of Tris-HCI and centrifugation,
homogenized
stored at -80 43 until needed. On the day of study, the homogenate and incubated at 37 “c for 20 min to remove endogenous then collected by centrifugation
in 40 volumes Tris-HCI and
was thawed at room temperature
5-HT (Nelson et a1.,1978). Membranes
and washed twice before final resuspension
Tris-HCI. pH7.7. The protein concentration
was measured
were
in 65 volumes of 50 mM
by the method of Bradford
(1976) with
bovine serum albumin as the standard. Membranes containing
were incubated
at 25 “C for 30 min in a final volume
0.5 nM [‘HI-&OH-DPAT,
10 ul of the compound
of lml Tris-HCI.
to be tested in DMSO,
pH7.7,
and 0.1%
1204
H. Urhida et ul
ascorbic acid. The membranes
were then rapidly vacuum-filtered
through Whatman GF/B filters and
washed three times with 3 ml of ice-cold Tris-HCl buffer. The filters were placed in polyethylene vials with 5 ml of Clear-sol I, a scintillation to the measurement Nonspecific
of radioactivity
binding was determined
All determinations
cocktail, and stored at 4 “C overnight to equilibrate prior
by liquid scintillation by incubating
spectrometry
at an efficiency
similar samples with unlabelled
of 40-47 %.
5HT (IO FM).
were made in triplicate.
The 5HTz receptor binding assay was carried out on frozen crude membrane preparations as SHT,,
receptors.
Membranes
HCI, pH7.7, containing Nonspecific
as well
were incubated at 30 “c for 30 min in a final volume of 1 ml Tris-
0.5 nM [‘HI-ketanserin
binding was determined
and 10 ~1 of the compound
by incubating
to be tested in DMSO.
similar samples with unlabelled
methysergide
(1
i.tM). Other binding assays were carried out as indicated used in the references
quoted.
The ICo
binding) values were determined
in Table I. based essentially
(i. e., the concentration
on the methods
that inhibits 50 o/o of the specific
by non-linear regression analysis using an EBDA-LIGAND
program
on a Macintosh computer. Table 1 Receptor Binding Methodology Receotor 5-HTIA ~-HTIc
Tissues cortex+hippocampus cortex
5-HT2 5-HT? S-HTJ
cortex+hrppocampus cortex
D2
CII cf.2 P BZD
strlatum strlatum whole bram(-cerebellum) cortex cortex cortex
Species lieand rat [‘HI&OH-DPAT rat [‘H]mesulergine
rat rat
[jH]ketanserin t’HlBRL43694
rat [‘H]GRlI3808 rat [JH]spiperone rat [‘Hlprazosin rat [7H]rauwolscine rat [“Hldihydroalprenolol ratI’HIRol5-1788
Rct’erence Peroutka (1986) Pazos ( 19X.5) Leysen et al. (1982) Nelson and Thomas (1989) Grossman et al. ( 1993) Sanger and Nelson (1989) Greengrass and Brcmner (I 979) Broadhurst and Wyllie (I 986) Bylund and Snyder (I 976) Brown and Martm (1984)
Adenvlate Cvclase Assavs The assay conditions (1985).
were modified
Rats were sacrificed
homogenized
from those published
by decapitation
in a 9 volume (v/w) ice-cold solution containing
EDTA, 5 mM dithiothreitol
The hippocampi
were
300 mM sucrose, 1 mM EGTA, 5 mM
was diluted &fold in homogenization
500 X g at 0 “C for 5 min. The pellet from this centrifugation
membranes
(De Vivo and Maayani,
removed.
and 20 mM Tris-HC1 (pH7.4 at 23 C) in a tissue homogenizer
strokes by hand). The homogenate centrifuged
previously
and the hippocampi
solution and centrifuged
was discarded and the supernatant
(20 at was
at 39,000 X g for 10 min at 0 “C. The supernatant from the second spin was discarded and were resuspended
by vortexing
Aliquots (100 ~1) of the membrane suspension
in 40 volumes of ice-cold
homogenization
solution.
were added to 400 ~1 of cyclase assay medium to start
the assay. The cyclase assay medium was incubated for 5 min at 30 “C before the start of the assay.
Pharmacological
The final components mM magnesium
1205
of the assay medium were : 10 PM forskolin,
100 mM NaCI, 10 PM GTP. 2
acetate, 0.2 mM ATP, 1 mM CAMP, 80 mM Tris-HCl (pH7.4), 60 mM sucrose. 0.2
1 to 2 ptci of [a-‘?P]ATP (specific activity. 20-40
mM EGTA. 1 mM EDTA, 1 mM dithiothreitol, Ci/mmol),
profile of HT-SOB
2 pg of membrane
protein, 4 mM theophylline.
creatine phosphate and various concentrations
0.1 mg/ml creatine phosphokinase.
5 mM
of agonists. The medium was incubated at 30 “C for IO
min and the reaction was stopped by heating the mixture to 95 “c for 5 min. After cooling the sample to 4 % , 100 pl of 45 mM ATP and 1.3 mM cAMP containing (30.000 dpm) was added to the medium. The conversion described
by Salomon
(1979). Proteins
an internal standard of [ ‘HIcAMP
of [?:P]ATP to [‘?P]cAMP was measured ax
were determmed
( 1976) with
by the method of Bradford
bovine serum albumin as the standard. Forskolin was added from a 50 mM stock solution in ethanol and diluted such that the ethanol concentration exchange
in the assay mixture was less than 0.02 %. C‘atmn
resin (AG 5OW-X4, 200-400 mesh) and aluminium
Laboratories
(Richmond.
oxide were obtained
from Blo-Rad
CA).
Platelet Aggregation The rabbit was anesthetized obtained citrate
from the common to 9 parts blood)
centrifugation
with sodium thiopental (50 mg/kg. i.v.), and arterial blood (50 ml) was carotid artery with a syringe containing
as anticoagulant.
and platelet-rich
at 1.300rpm for 10 min. Platelet aggregation
method of Born ( 1962) using an aggregometer
3.8 % sodium citrate (I part
plasma
(PRP)
was prepared
by
was studied in PRP with the turbidimetric
(Aggretec TE-500. Erma). A cuvette containing 200 ~1
of PRP, 5 ~1 of 192 mM CaCI, in saline, and 5 ~1 of the drug to be tested in DMSO was placed in the aggregometer
and allowed to incubate for 3 min. Platelet aggregation
5-HT (final concentration
4.2 PM) and 20 1.11of collagen
cuvette. and recorded for 5 min. The concentrations sub-threshold
was initiated by adding IO ~1 of
(final concentration
4 - 10 pg/ml,
to the
of 5-HT and collagen were selected to produce a
response by each inducer alone and 40 - 60 % aggregation
by both inducers together.
IC50values were calculated by Probit analysis. 5-HT Behavioral Syndrome Following
the method described
by Tricklebank
mg/k g, KC.) 18 hr before drug administration.
et al. (1984), rats were treated with reserpine
For measurement
of the behavioral
syndrome,
animals were placed in individual clear Plexiglass cages 5 min before drug administration. posture and forepaw treading were scored during an observation ranked intensity
scale (0 = absent,
1978). Observations administration. DOI-Induced
were
made
1 = equivocal, every
Scores for each compotent
12 min.
Flat body
period of 45 set per rat on a 4-point
2 = definite,
3 min for
(I the
3 = extreme) beginning
(Deakin and Green.
5 min after
were summed over the 5 observation
the drug
periods.
Head Twitch
Mice were injected with DOI (5 mg/kg, i.p.) 30 and 60 min before the administration and ritanserin.
respectively.
The number of head twitch responses
administration
of DOI was counted.
of HT-90B
during the first 10 min after the
H. Uchida
1206
In DOI-induced
head-twitches.
ritanserin
cat al.
was used to confirm
activation in the behavior. Therefore, ritanserin was administered 90B is a candidate (unpublished
of anxiolytic/antidepressant
of behaviorally
of 5-HT, receptor
which is orally active 111various behavioral
data; Inagawa et al., 199.5). Therefore,
action after p.0. administration
the involvement
i.p. as usual. On the other hand, HTtests
we aimed to test its in vivo 5-HT? antagonistic
effective doses (higher than 10 mg/kg).
Data Analvsis Statistical
differences
variance followed
between
experimental
groups
by Dunnet’s multiple comparison
were evaluated
either
by an analysis
of
test or Student’s t-test. P values less than 0.05
were regarded as significant.
Results L Binding Assay The apparent affinity (Ki) of HT-90B and the reference for the 5-HT,,
receptors
was determined.
labeled by [3H]-8-OH-DPAT
compounds,
8-OH-DPAT
and buspirone,
in rat cortical plus hippocampal
membranes
As shown in Table 2, HT-90B was the most potent agent tested with a Ki value of
0.18 nM. %OH-DPAT
was slightly less potent than HT-90B with a Ki value of 0.49 nM. Buspirone
was the weakest agent analyzed with a Ki value of 7.7 nM. HT-90B exhibited a high affinity for the 5-HT, recognition membranes
labelled with [‘Hlketanserin,
site in the rat cortical plus hippocampal
with a Ki value of 9.2 nM. Buspirone
potent than HT-90B (Ki = 490 nM). 8-OH-DPAT
displayed
a
was 50-fold less
very low affinity
for the 5-HTz
receptors (Ki > IO PM). In the rat cortical membranes, [7H]-mesulergine receptors
HT-90B was less active at the 5-HT,e binding sites labelled with
(Ki = 260 nM), and displayed
labelled with [‘HI-BRL43694
Both &OH-DPAT
and buspirone
a very low affinity
and for the 5-HT, receptors
displayed
a very low affinity
(Ki > 3300) for the 5-HT, labelled with [‘HI-GR113808.
for the 5-HTlc
and the 5-HT,
receptors. With respect
to the other neurotransmitter
receptors,
dopamine D2 receptors (Ki = 49 nM) and a, adrenoceptors
HT-90B
had moderate
affinities
for the
(Ki = 54 nM). At the a, adrenoceptor,
HT-
90B displayed a low affinty (Ki = 170 nM). HT-90B was less active m binding to the P-adrenoceptors (Ki = 170 nM). At the benzodiazepine Buspirone exhibited
receptors,
HT-90B showed negligible
some affinity for the dopamine
affinity (Ki > 5.9 FM).
Dz receptor, similar to that of HT-90B (Ki = 63
nM), but showed very low affinities for the a,,a?,p and BZD receptors. 8-OH-DPAT bmding to the D2. a,, ~1~and p adrenoceptors To compare
the receptor
various receptors examined,
selectivity
(Table 2).
of HT-90B, buspirone
were divided by those for 5-HT,,, receptors
HT-90B had a high selectivity
was inactive in
and &OH-DPAT,
the Ki values for
(Table 2). Among the 5-HT receptors
for 5_HT,,, and 5HTz receptors
(Ki ratio was I:5 1). It had
Pharmacological
profile
of HT-9013
more than a lOO@fold less potent affinity for the other 3 subtypes.
1207
For the other neurotransmtttet
receptors, HT-90B exhibited 300-fold (DI and a,) and more than 900-fold (a,, p and BZD) less potent affinities than tt did for 5-HT,, receptors. With respect to buspirone,
receptor selectivity
for SHT, , and S-HT, ( 1:63) was similar to that of
HT-90B. However, buspirone had a much higher selectivity HTI receptors.
H-OH-DPAT exhibited exclusive selectivity
for the D, receptors (1:8) than for the 5for the S-HT,,, receptors.
Table 2 Receptor Binding Profile of HT-90B. Buspirone and %OH-DPAT
Receptor
HT-90B
Ki (nM) Buspirone
( / 5-HTIA)
( / S-HTI A)
5-HTI 5-HTIc
0.18
7.1
(1)
(1)
260
2000
(1400)
5-HTJ
9.2 (51) na. b
5-HTJ
n.a. b
5HT>
D? ai a2
C/5-HTIA)
0.49 IIJ
7x00 c
I IhIM),
1260)
11.a.b.c
490 (61)
n.a. b
na. b.c
n.d. a
n.d. a
49
63
(270)
(8)
2200 c (4SO0,
2700 c
1700
54 (3(K)) 170
3400
(940)
iJ40)
2100
P
SOH-DPAT
(5500)
1220)
300 c (hl0,
n.3. b.c
na. b
(12000)
n.d. a n.a. b n.a. b BZD a: n.d. = not determined, b: n.a. = no or weak affinity observed at the highest concentration. c: data from Van Wijingaaden et al (1990). Number in parentheses represent the ratto of Ki value for a given receptor to that for 5-HT,,\ receptor.
Adenvlate Cvclase Assay As shown in Fig.‘, 5HT inhibited foskolin-stimulated
cyclase activity. and a maximal inhibition of
25-30 % was achieved routinely at IO FM. X-OH-DPAT, a 5-HT,,, receptor full agonist. inhibited the cyclase activity to the same degree as did 5-HT. HT-90B also acted as a full agonist. since maximal inhibition by HT-90B was similar to those by 5.HT and R-OH-DPAT.
However, the potency of HT-
90B was much higher than that of the two other full agonists (Fig. 2. Table 3). Figure 2 shows that the inhibition antagonist.
curve produced
by HT-90B was shifted to the right by IOpM of pindolol.
Pindolol by itself had no effect on the forskolin-stimulated
contrast wtth the effects of full agonists, was only 608 of that produced by 5HT.
maximal inhibition
a S-HT,,
adenylate cyclase activity.
by huspirone,
In
a 5-HT, , partial agonist.
1208
H. Uchda <'I nl.
0
--O+ *
S-HT (n=2) HT-90B (n=4) 8-OH-DPAT (n=2)
-t_
Buspirone (n=2)
-9
-8
-7 log
-6
+
HT-90B (n=4) HT-90B + pindolol (IO@I) (n=2)
a-
0
-5
-9
-8
M
log
Fig. 2 Inhibition of forskolin-stimulated Each point represent the mean f S.E.M.
-7
-6
M
adenylate cyclase activity in rat hippocampal * : P < 0.05, ** : P < 0.01 (vs HT-90B).
membranes.
Table 3 I& Values of Inhibition of Forskolin-Stimulated Compound HT-90B
IG
Adenylate Cyclase by 5-HT,, Receptor Agonists.
(nM) 3.4
HT-90B+ Pindolol (10 FM) 210 5-HT 38 8-OH-DPAT 11 Busnirone 650 I& values were expressed as 50 % of maximum inhibition by 5-HT, a 5-HT,,t receptor full agonist.
Platelet Aggregation Although 5-HT (4.2 pM) alone did not induce the aggregation amplified the aggregation HT-90B Inhibition
attenuated
induced by a subthreshold
the amplifying
by ritanserin
produced by HT-90B.
(I&,
effect
of rabbit platelets, it synergistically
concentration
of collagen (4 - 10 pM) (Fig. 3).
of 5-HT in a dose-dependent
= 25 nM), a 5-HT2 antagonist,
manner (IC,, = 1.73 PM).
was 200-fold
more potent than that
Buspirone exhibited a less potent inhibition than did HT-90B and ritanserin.
5-HT Behavioral Svndrome Subcutaneous syndrome
administration
response
forepaw treading.
(Tricklebank
of 8-OH-DPAT
to reserpine-treated
rats elicited a typical 5-HT motor
et al., 1984; Mir et al., 1988) consisting
When compared with the effect of 8-OH-DPAT,
of flat body posture and
buspirone and HT-90B produced
Pharmacological only mild to moderate behavioral
profile of HT-SOB
effects in this model, buspirone
1209
being slightly more effective
than
HT-90B in this regard (Fig. 4).
Ritanserine (n=3) HT-90B (n=4) Buspirone (n=2)
I””
-10
-9
-8
-1
-6
-5
-4
log M Fig.3 Inhibition of platelet aggregation induced by collagen (4-10 pg/ml) and 5-HT (4.2 t.tM). Platelet-rich plasma from rabbits was pre-incubated for 3 min with different concentration of drugs. Results were expressed as % of maximum aggregation induced by collagen and 5-HT. Each point represent the mean + S.E.M. * : P < 0.05, ** : P < 0.01 (vs HT-90B).
Flat body posture (n=8- I I )
Forepaw treading (n=8- I I 1
R-OH-DPAT
8-OH-DPAT
dose (mgkg, SC.)
dose (mgkg,
s.c.)
Fig.4 5-HT syndromes in reserpine-treated rats. Flat body posture and forepaw during observation period of 45 set per rat on a 4-point ranked intensity scale 1978). Observations were made every 3 min for 12 min. beginning at 5 administration. Scores for each component were summed over the 5 observation represent the mean f S.E.M. ** : P < 0.01 (vs HT-90B).
treading were scored (Deakin and Green, min after the drug periods. Each point
1210
H. Uchida et al
+
HT-90B (n=l2-1.3) Ritanserine (O.Smgkg,
A
o-
0
IO
20
30
40
i.p.) (n=8)
50
dose (mgkg, p.0.) Fig. 5 Number of head twitch response during the first 10 min after the administration of DOI. Mice were injected with DO1 (5 mg/kg, i.p.) 0.5 and 1 hr after the administration of HT-90B and ritanserin, respectively. Each point represent the mean + S.E.M. * : P < 0.05, ** : P c 0.01 (vs 0 mg/kg).
DOI-Induced
Head-Twitches
The effect of HT-90B on head-twitches HT-90B inhibited the DOI-induced administration.
induced by DOI, a 5-HT, agonist, is presented
head-twitches
in a dose-dependent
Ritanserin (0.5mg/kg, i.p.) completely
in Fig. 5.
manner in the mouse after oral
abolished the DOI-induced
head-twitches.
Discussion The present studies have demonstrated
that HT-90B, a novel benzodioxane
both 5-HT,, receptor agonistic and 5-HT? receptor antagonistic
derivative,
possesses
activities in vitro and in vivo.
Binding Assav In radioligand binding assay, HT-90B exhibited an approximately OH-DPAT
for the 5-HT,* receptors
3-fold greater affinity than did 8-
labelled with [3H]-&OH-DPAT.
for 5-HT, receptors labeled with [?H]-ketanserin. HT,c, 5-HT3 and 5HT4, were virtually for 5-HT,,
negligible.
receptors,
but the selectivity
receptors.
It has been reported that buspirone,
HT-90B also had high affinity
Its affinity for the other 5-HT receptor subtypes, 5-
receptors
It displayed
some affinities
was approximately
a non-benzodiazepine
for the D, and CI,
300-fold greater than these anxiolytic with 5-HT,,
activity (Eison and Temple 1986; Traber and Glaser, 1987; Carli et al, 1989), has side-effects to be caused by anti-dopamine
agonist thought
actions of the drug such as sedation, akathisia and perioral dyskinesia
(Goa and Ward, 1986). HT-90B and buspirone exhibited similar affinities for D, receptors. However, the selectivity
ratio of HT-90B for 5-HT,,
over D, receptors was 270, which was far greater than that
of buspirone.
These results indicate that HT-90B has a higher affinity for 5-HTIA receptors than does
8-OH-DPAT
and is more selective than is buspirone.
Pharmacological
profile of HT-SOB
1211
Adenvlate Cvclase Assay In adenylate cyclase assay, employed
as a functional test of 5HT, 4 receptor activity in vitro, HT-
90B showed potent 5-HT,, receptor agonist properties.
For example, HT-BOB inhibited the forskolin-
activated adenylate cyclase to the same extent as did 5-HT and S-OH-DPAT. was 3-fold higher than that of 8-OH-DPAT,
which parallels the difference
receptors. The effect of HT-90B was suppressed reported
that pindolol
has 5-HTIA antagonist
DPAT on forskolin-stimulated results
suggest
hippocampal
adenylate
that HT-90B
The potency of HT-90B in the affinity for 5-HT,,\
by pindolol, which was inactive by itself. It has been activity and reverses
cyclase activity
acts as a full agonist
the inhibitory
(Oksenberg
action of &OH-
and Peroutka,
1988). These
and is more potent than 8-OH-DPAT
in rat
neurons in vitro.
5-HT Behavioral Svndrome The activity to induce flat body posture and forepaw treading was used to explore the central 5HTIA agonistlc action of HT-90B in vivo. HT-90B induced these behaviors with a lower efficacy than did 8-OH-DPAT bioavailability
and buspirone.
If less potent in vivo activity of HT-90B was only due to a lower
of the compound,
then maximum effect of HT-90B at high dose would be even bigger
than those of the reference maximum
compounds
because of HT-90B’s higher in vitro potency. However,
effect of HT-90B was much weaker than those of X-OH-DPAT
and buspirone
the
in the
present study. In contrast, HT-90B exhibited more potent and efficient anxiolytic- and antidepressantlike behavioral
action than did 8-OH-DPAT
1995). Furthermore, accumulation
and buspirone
in rat brain in vivo (unpublished
90B in eliciting
in rats (unpublished
HT-90B was more potent than 8-OH-DPAT
serotonin
syndrome
and buspirone
data; Inagawa et al., in inhibiting
5-HTP
data), These results suggest that lower potency of HT-
could not be explained
by a lower bioavailability
of the
compound. Thus, it appears that HT-90B acts as a partial agonist in these behavioral tests. This result may look incompatible explained
with the data obtained by presumed
mechanisms
differences
in 5-HT,,,
cyclase assay. However,
receptors
coupling
this can be
to different
effector
and existing in different locations in the CNS. It has been shown that buspirone
efficacious
than MDL73005EF,
hyperpolarizations inhibition
subtle
in the in vitro adenylate
another
in hippocampal
of forskolin-activated
5-HT,,
pyramidal adenylate
partial
agonist,
in 5-HT, ,-mediated
cells, while the reverse
cyclase
is more
membrane
is true in 5-HT,,-mediated
even in the same tissue (Van den Hooff and
Galvan. 199 I: Cornfield et al., 1989). Both actions have been shown to be mediated by a G protein (Andrade et al.,l986; Clarke et al., 1987), and yet they appear to be independent levels of CAMP do not affect membrane 1988). Furthermore,
potential changes produced by S-HT,, agonists (Innis et al.,
it has also been reported that the intrinsic activity of 5-HT,,
buspirone (De Vivo and Maayani, 1986; VanderMaelen and Galvan, producing
of one another since
1991; Sprouse,
hyperpolarization
et al., 1991). MDL73005EF
agonists, such as (Van den Hooff
1991) and others (Millan et a1..1993; Greuel and Glaser. does in fact change when measured
present studies, rat hippocampal
membranes
in different
1992). in
brain regions.
In the
were used for the adenylate cyclase assay, while the flat
1212
H. Uchida et al.
body posture and forepaw treading are postulated to be triggered by the stimulation of 5HT receptors in the brainstem Therefore,
and / or spinal cord (Jacobs
it seems
hippocampus
probable
that HT-90B
and Klemfuss,
1975; Lucki and Frazer,
acts as a full agonist
on 5-HT,,
receptors
coupled to adenylate cyclase, while it has a partial agonistic action on 5HT,,
1982). in the
receptors
in the brain stem and / or spinal cord, probably coupled to other mechanisms. 5_HT, Antagonistic Inhibition
Action in Vitro and in Vivo
of 5HT-potentiated
activity of the compound
platelet aggregation
by HT-BOB revealed the 5-HT, antagonistic
in vitro. Although HT-90B was lOO-fold less potent than ritanserin, it was
more potent than buspirone.
The 5-HTZ antagoistic
vivo after oral administration,
since this compound
action of HT-90B in the CNS was detected in inhibited the DOI-induced
head-twitch
response,
which is mediated by an activation of central 5-HT2 receptors (Goodwin and Green, 1985). The effect of HT-90B was much weaker than that of ritanserin. antagonizing
DOI-induced
HT2 antagonistic
Lower potency
of HT-90B in
head twitches may be a simple reflection of 100.fold less potent in vitro 5-
action of HT-90B when compared with ritanserin.
Conclusion The present antagonistic buspirone
study
has demonstrated
that HT-90B
profile in vitro and good bioavailability and related
arylpiperazines
display
has a potent
in vivo.
anxiolytic
5HT,,
5-HT,,
agonistic
/ 5-HT,
agonist such as 8-OH-DPAT,
and antidepressive
models (Engel et a1.,1984; Riblet et a1.,1984; Taylor et al.,l984;Traber
activities
in animal
et a1.,1984), and in humans
(Robinson et al, 1989; Beneke et al., 1988; Cott et al., 1988). On the other hand, ritanserin,
a 5-HTz
antagonist, has also been reported to produce anxiolytic effect in certain models of anxiety (Critchley & Handley,
1987) and also anxiolytic
which possesses
both .5-HT,, agonistic
effect in humans (Ceulemans and 5-HTz antagonistic
et al., 1985). Thus, HT-90B,
activities,
may be a promising
candidate as a therapeutic agent for these disorders.
References ADHAM, N., KAO, H. T., SCHECHTER, L. E., BARD, J., OLSEN, M., URQUHART, D., DURKIN, M., HARTIG, P. R., WEINSHANK, R. L. AND BRANCHECK, T. A. (1993) Cloning of another human serotonin (5.HTlF): a fifth 5-HTl receptor subtype coupled to the inhibition of adenylate cyclase. Proc. Natn. Acad. Sci. U.S.A. 90: 408-412. AFFOLTER, H., ERNE, P., BURGISSER, E. AND PLETSCHER, A. (1984) Ca2+ as messenger of 5HT2-receptor stimulation in human blood platelets, Naunyn. Schmiedebergs Arch. Pharmacol. 325: 337-342 ANDRADE, R., MALENKA, R. C. AND NICOLL, R. A. (1986) A G protein couples serotonin and GABAB receptors to the same channels in hippocampus. Science 234: 126 1- 1265. BENEKE, M., KUMMEL, B., ROAD, I. S. AND SPECHTMEYER, neurosis with ipsapirone. Psychopharmacol. @(suppI): S353.
H. (1988) Treatment of anxiety
BEVAN, P., LORENS, S. AND ARCHER, T. (1989) Behavioural pharmacology of 5-HT: possible progress in the laterality of concepts. In: Behavioural Pharmacology of 5-HT., P. Bevan, A. R.
Pharmacological Cools and T. Archer (Eds.), Jersey.
pp.459-474,
BORN, G. V. R. (1962) Aggregation Nature 194: 143-155.
profile of HI-SOB
Lawrence
1219
Erlbaum Associates,
of blood platelets by adenosine
Inc., Publishers.
diphosphate
New
and its reversal.
BRADFORD, M. M. (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dyebinding. Anal. Biochem. 12: 248254. BROADHURST, A. M. AND WYLLIE, M. G. (1986) A reassessment of the binding [3H]rauwolscine to membranes from the rat cortex. Neuropharmacology 25: 287-295. BROWN, C. L. AND MARTIN, bound rat brain benzodiazepine
of
I. L. (1984) Kinetics of [3H]Ro 15-1788 binding to membranereceptors. J. Neurochem. 42: 9 18-923.
BYLUND, D. B. AND SNYDER, S. H. (1976) Beta adrenergic receptor preparations from mammalian brain. Mol. Pharmacol. 12: 568-S80. CARLI, M., PRONTERE, C AND SAMANIN, R. (1989) hydroxytryptaminergic neurones are involved in the anxiolytic Pharmacol. 96: 829-836.
binding
in membrane
Evidence that central 5activity of buspirone. Br. J.
CEULEMANS, D. L., HOPPENBROUWERS, M. L., GELDERS, Y. G. AND REYNTJENS, A. J. (1985) The Influence of Ritanserin, a Serotonin Antagonist, in Anxiety Disorders: A Double-blind Plasebo-controlled Study versus Lorazepam. Pharmacopsychiat. 18: 303-305. CLARKE. W. P., DEVIVO, M., BECK, S. G., MAAYANI, S. AND GOLDFARB, J. (1987) Serotonin decreases population spike amplitude in hippocampal cells through a pertussis toxin substrate. Brain Res. 410: 357-361. CORNFIELD, L. J., NELSON, D. L., TAYLOR, E. W. AND MARTIN, A. R. ( 1989) MDL 73005EF: partial agonist at the 5-HTIA receptor negatively limed to adenylate cyclase. Eur. J. Pharmacol. 173: 189-192. COTT. J. M., KURTZ, N. M., ROBINSON, 5-HTIA ligand with both antidepressant 164-167.
D. S., LANCASTER, S. P. AND COPP, J. E. (1988) A and anxiolytic properties. Psychopharmacol. Bull. 2:
CRITCHLEY. M. A. E. AND HANDLEY, S. L. (1987) Effect in the X-maze anxiety model of agents acting at 5-HTl and 5-HT2 receptors. Psychopharmacology 93: 502-506. DEAKIN. J. F. AND Green, A. R. (1978) The effects of putative 5-hydroxytryptamine antagonists on the behaviour produced by administration of tranylcypromine and L-tryptophan or tranylcypromine and L-DOPA to rats. Br. J. Pharmacol. 64: 20 I-209 DE CLERCK, F., XHONNEUX, B., LEYSEN. J. AND JANSSEN, P.A. (1984) Evidence for functional 5-HT2 receptor sites on human blood platelets. Biochem. Pharmacol. 33: 2807-28 I 1. DE VIVO, M. AND MAAYANI, S. (1985) Inhibition of forskolin-stimulated activity by 5-HT receptor agonist. Eur. J. Pharmacol. 119: 231-234.
adenylate
cyclase
DE VIVO, M. AND MAAYANI, S. (1986) Characterization of the 5-hydroxytryptaminelA receptor-mediated inhibition of forskolin-stimulated adenylate cyclase activity in guinea pig and rat hippocampal membranes. J. Pharmacol. Exp. Ther. 238: 248-253. EISON, A. S. AND TEMPLE, D. L. (1986) Buspirone:Review of its pharmacology perspectives on its mechanism of action. Am. J. Med. @(SuppI 3B): I-9.
and current
ENGEL, J. A.. HJORTH, S., SVENSSON, K., CARLSSON, A. AND LILJEQUIST, S. (1984) Anticonflict effect of the putative serotonin receptor agonist 8-hydroxy-2-(di-npropylamino)trtralin (R-OH-DPAT). Eur. J. Pharmacol. 10$: 365-368. GLENON, R. A. (1987) Central serotonin receptors as targets for drug research. l-12. GOA, K. L. AND WARD. A. (1986) Buspirone.
Drugs _12: 113-129
J. Med. C’hem. 30:
1214
tf. Urhicla
cjf al
GOODWIN, G. M. AND GREEN. A. R. (1985) A behavioural and biochemical srudy in mice and rats of putative selectivje agonists and antagonists for S-HTI and S-HT2 receptors. Br. J. Pharmacol. 84: 743-753. GREENGRASS, a-adrenergic
P. AND BREMNER, receptors.
R. (1979) Binding characteristics
of 3H-prazosin
to rat brain
Eur. J. Phamacol. 55: 323-326.
GRELJEL, J. M. AND GLASER, T. (1992) The putative S-HTlA receptor antagonist NAN-190 and BMY 7378 are partial agonists in the rat dorsal raphe nucleus in vitro. Eur. J. Pharmacol. 211: 211-219. GROSSMAN, C. J., KILPATRICK, G. J. AND BUNCE, K. T. (1993) Development of a radioligand binding assay for S-HT4 receptors in guinea-pig and rat brain. Br. J. Pharmacol. 109: 618-624. HALL, M. D., EL MESTIKAWY, S., EMERIT, M. B., PICHAT, L., HAMON, M. AND GOZLAN, H. (1985) [3H]8-Hydroxy-2-(di-n-propylamino)tetralin binding to pre- and postsynaptic Shydroxytryptamine sites in various regions of the rat brain. J. Neurochem. 44: 1685-1696. HAMBLIN. M. W. AND METCALF, M. A. (1991) Primary structure and functional characterization of a human S-HT 1D-type serotonin receptor. Mol. Pharmac. 40: 143- 148. HAMON, M., BOURGOIN, S., GOZLAN, H., HALL, M. D., GOETZ, C., ARTAUD, F. AND HORN, AS. (1984) Biochemical evidence for the S-HT agonist properties of PAT (8-hydroxy-2(di-n-propylamino)tetraIin) in the rat brain. Eur. J. Phamacol. 100: 263-276. HOYER, D., PAZOS, A., PROBST, A. AND PALACIOS, J. M. (1986) Serotonin receptors in the human brain. II. Characterization and autoradiographic localization of S-HTIC and S-HT2 recognition sites, Brain Res. 376: 97-107. INNIS, R. B., NESTLER, E. J. AND AGHAJANIAN, G. K. (1988) Evidence for G protein mediation of serotonin- and GABAB-induced hyperpolarization of rat dorsal raphe neurons. Brain Res. Q: 27-36. JACOBS, B. L. AND KLEMFUSS, H. (1975) Brain stem and spinal serotonergic behavioral syndrome. Brain Res. 100: 450-457. JULIUS, D. (1991) Molecular biology of serotonin receptors.
cord mediation
Annu. Rev. Neurosci.
of a
14: 335-360.
LEYSEN, J. E., NIEMEGREERS, C. J. E., VAN NUETEN, J. M. AND LADURON, P. M. (1982) [3H]Ketanserin (R 41 468), a Selective 3H-Ligand for Serotonin2 Receptor Binding Sites. Mol. Phramacol. 21: 30 l-3 14. LUCKI, I. AND FRAZER, A. (1982) Prevention of the serotonin syndrome in rats by repeated administration of monoamine oxidase inhibitors but not tricyclic antidepressants. Psychopharmacology n: 205-2 1 I. LUCKI, I., NOBLER, M.S. AND FRAZER, A. (1984) Differential actions of serotonin antagonists on two behavioral models of serotonin receptor activation in the rat. J. Pharmacol. Exp. Ther. 228: 133-139. MC ALLISTER, G., CHARLESWORTH, A., SNODIN, C., BEER, M. S., NOBLE, A. J., MIDDLEMISS, D. N., IVERSEN L. L. AND WHITING, P. (1992) Moleculer cloning of a serotonin receptor from humon brain (S-HTlE): a fifth S-HTl-like subtype. Proc. Natn. Acad. Sci. U.S.A. 89: 5.517-5521. MIDDLEMISS, discriminates
D. N. AND FOZARD, J. R. (1983) %Hydroxy-2-(di-n-propylamino) tetralin between subtypes of the S-HT1 recognition site. Eur. J. Pharmacol. 9QlSO-153.
MILLAN, M. J., RIVET, J. -M.. CANTON, H., LEJEUNE, F., GOBERT, A., WIDDOWSON, P., BERVOETS, K., BROCCO, M. AND PEGLION, J. -L. (1993) SlSS3S:a highly selective benzodioxopiperadine S-HTlA receptor ligand which acts as an agonist and an antagonist at presynaptic and postsynaptic sites respectively. Eur. J. Pharmacol. 230: 99- 102. MIR, A. K., HIBERT, M., TRICKLEBANK, M. D., MIDDLEMISS, D. N., KIDD, E. J. AND FOZARD, J. R. (1988) MDL 72832: a potent, selective and stereospecific ligand with mixed
Pharmacological agonist and antagonist properties Pharmacol. 149: 107-120.
profile of
at both central
HT-SOB
and peripheral
1215
S-HTIA
receptors.
Eur. J.
NELSON, D. R. AND THOMAS, D. R. (1989) [3H]-BRL 43694 (Granisetron), a specific ligand for 5-HT3 binding sites in rat brain cortical membranes. Biochem. Pharmac. 18: 1693-1695. NELSON, D. L., HERBET, A., BOURGOIN, S., GLOWINSKI, J. AND HAMON, M. (1978) Characteristics of central 5-HT receptors and their adaptive changes following intracerebral 5. 7dihydroxytryptamine administration in the rat. Mol. Pharmacol. 14: 983-995. NORTH, R. A. AND UCHIMURA, N. (1989) 5-Hydroxytryptamine acts at S-HT2 receptors decrease potassium conductance in rat nucleus accumbens neurones. J. Physiol. 417: I- 12.
to
OKSENBERG, D. AND PEROUTKA, S. J. (1988) Antagonism of S-hydroxytryptaminelA (5. HT 1A) receptor-mediated modulation of adenylate cyclase activity by pindolol and propranolol isomers. Biochem. Pharmac. 37:3429-343.3. PALACIOS, J. M., PAZOS, A. AND HOYER, D. (1987) Characterisation and mapping of S-HTIA sites in the brain of animals and man. In: Brain .5-HTlA Receptors, C. T. Dourish. S. Ahleniui; and P. H. Huston (Eds.), pp 67-8 1, Ellis Horwood Ltd.. Chichester. England. PAZOS, A., HOYER, D. AND PALACIOS, J. M. (1985) Mesulergine. a selective serotonin-2 ligand in the rat cortex, does not label these receptors in porcine and human cortex: evidence for species differences in brain serotonin-2 receptors. Eur. J. Pharmacol. 106: 53 l-538. PEROUTKA, S. J. (1986) Pharmacological differentiation and characterization and 5-HTlC binding sites in rat frontal cortex. J.Neurochem. 37: 529-540.
of 5-HT I A. 5-HT I B.
PEROUTKA, S. J. AND SNYDER, S. H. (1980) Long-term antidepressant spiroperidol-labeled serotonin receptor binding. Science 210: 88-90.
treatment
decreases
RIBLET, L. A., EISON, A. S., EISON, M. S., TAYLOR, D. P., TEMPLE, D. L. AND VANDERMAELEN, C. P. (1984) Neuropharmacology of buspirone. Psychopathology 12: 6978. ROBINSON, D. S., ALMS, D. R., SHROTRIYA, R. C., MESSINA, M. AND WICKRAMARATNE, M. (1989) Serotonergic anxiolytics and treatment of depression. Psychopathol. 22(suppl): 27-36. SALOMON.
Y. (1979) Adenylate cyclase assay. Adv.Cyclic
Nucleotide Res. lo: 35-55
SANGER,G.J. AND NELSON,D.R. (1989) Selective and functional 5-hydroxytryptamine3 antagonism by BRL 43694 (granisetron). Eur. J. Phamacol, 159: 113-124.
receptor
SMITH, L. M. AND PEROUTKA, S. J. (1986) Differential effects S-hydroxytryptaminela drugs on the .5-HT behavioral syndrome. Phamacol. Biochem. Behav. 3: 15 13. I5 19.
selective
SPROUSE, J.S. (1991) Inhibition of dorsal raphe cell firin, a by MDL 7300SEF. a novel S-HT I A receptor ligand. Eur. J. Pharmacol. 201: 163-169. TAYLOR, D. P., ALLEN, L. E., BECKER, J. A., CRANE. M., HYSLOP, D. K. AND RIBLET, L. A. (1984) Changing concepts of the biochemical action of the anxioselective drug,buspirone. Drug Dev. Res. 4: 95-108. TRABER. J. M.. DAVIES, M. A., DOMPERT. W. U., GLASER, T.. SCHUURMAN, T. AND SEIDEL, P. R. (1984) Brain serotonin receptors as a target for the putative anxiolytic TVX Q 782 1. Brain Res. Bull. 12: 74 I-744. TRABER. J. AND GLASER, T. (1987) 5-HTI A receptor-related
anxiolytics.
TIPS 8: 432-437.
TRICKLEBANK, M. D., FORLER, C. AND FOZARD. J. R. (1984) The involvement of subtypes of the 5-HTl receptor and of catecholaminergic systems in the behavioural response to S-hydroxy2(di-n-propyl-amino)tetralin (R-OH-DPAT) by the putative S-HTlA receptor. Eur. J. Pharmacol. 106: 27 l-282. VAN DENHOOFF. P. AND GALVAN. M. (1991) Electrophysiology of the S-HTIA MDL73005EF in the rat hippocampal slice. Eur. I. Pharmacol. 196: 291-298.
llgand
1216
H. Uchida et al
VANDERMAELEN, C. P., WILDERMAN, R. C. AND GEHLBACH, G. (1991) Electrophysiological studies of the effect of buspirone on serotonergic neurons. In: buspirone. Mechanisms and Clinical Aspects, G. Tunnicliff, A. S. Eison and D. P. Taylor (Eds.), pp 215230, Academic Press, Inc., California. VAN WIJNGAARDEN, I., TULP, M. TH. M. AND SOUDIJN, W. (1990) selectivity in 5HT receptor research. Eur.J.Pharmacol. 188: 301-3 12. YAP, C. Y. AND TAYLOR, D. A. (1983) behaviour induced by 5hydroxytryptophan
of
Involvement of 5-HT2 receptors in the wet-dog shake in the rat. Neuropharmacology 2: 801-804.
Inquiries and reprint requests should be addressed to: Haruhisa Uchida Department of Pharmacology Fuji Gotemba Research Labs. Chugai Pharmaceutical Co., Ltd Gotemba-shi, Shizuoka 412, Japan
The concept