Psychopharmacology Department (M.J.M., A.G., F.L., A.N.-T.,
J.-M.R., A.A.), Institut de Recherches Servier, Centre de Recherches de
Croissy, Paris, France; and Medicinal Chemistry B Department (J.-L.P.),
Institut de Recherches Servier, Centre de Recherche de Suresnes,
Paris, France
S33005 displayed marked affinity for native, rat, and cloned
human serotonin (5-HT) transporters (SERT) and less pronounced affinity for norepinephrine (NE) transporters (NET), while its affinity
at dopamine (DA) transporters and >50 other sites was negligible.
Reuptake of 5-HT and (less potently) NE into cerebral synaptosomes was
inhibited by S33005, whereas DA reuptake was little affected. In vivo,
S33005 prevented depletion of cerebral pools of 5-HT by
parachloroamphetamine. Furthermore, it decreased electrical activity of
raphe-localized serotonergic neurones, an action abolished by the
5-HT1A antagonist WAY100,635. At higher doses, S33005
blocked firing of locus ceruleus-localized adrenergic neurones, an
action abolished by the
2-adrenergic antagonist idazoxan. In contrast, S33005 did not inhibit ventrotegmental dopaminergic neurones. In frontal cortex of freely moving rats, S33005
dose dependently elevated dialysate levels of 5-HT, NE, and DA. In hippocampus, levels of 5-HT and NE were
similarly elevated, while in nucleus accumbens and striatum, levels of
5-HT were increased whereas DA was unaffected. Upon chronic (2 weeks)
administration, basal levels of NE were elevated in frontal cortex and,
therein, 5-HT2A receptor density was decreased. Comparative
studies with clinically used antidepressants showed that venlafaxine
possessed a profile similar to S33005 but was less potent. Clomipramine likewise interacted with SERTs and NETs but also with several other
receptors types, while citalopram and reboxetine were preferential ligands of SERTs and NETs, respectively. In conclusion, S33005 interacts potently with SERTs and, less markedly, with NETs. It enhances extracellular levels of 5-HT and NE throughout corticolimbic structures and selectively elevates dialysis levels of DA in frontal cortex versus subcortical regions.
 |
Introduction |
Extensive experimental and
clinical evidence indicates that a perturbation of monoaminergic
transmission is involved in depressive states (Maes and Meltzer, 1995
;
Willner, 1995
; Ressler and Nemeroff, 1999
; Anand and Charney, 2000
).
Correspondingly, currently used antidepressant agents exert their
therapeutic actions via a reinforcement in monoaminergic transmission,
although the relative contribution of serotonergic, adrenergic, and
dopaminergic mechanisms remains to be clarified (Broekkamp et al.,
1995
; Burke and Preskorn, 1995
; Frazer, 1997
; Millan et al., 2000b
).
Monoamine oxidase inhibitors exert antidepressant actions by preventing
the degradation of 5-HT and NE, while mianserin and mirtazapine
display antagonist properties at
2-adrenoceptors (AR) and
5-HT2C receptors inhibitory to serotonergic,
adrenergic, and/or dopaminergic pathways (Burke and Preskorn, 1995
;
Frazer, 1997
; Millan et al., 2000a
). Nevertheless, the majority of
antidepressant agents reinforce monoaminergic transmission by blocking
neuronal uptake of 5-HT and/or NE via actions at SERTs and NETs,
respectively (Barker and Blakely, 1995
; Frazer, 1997
; Sambunaris et
al., 1997
; Goodnick and Goldstein, 1998
; Blakely and Bauman, 2000
).
Despite subtle differences among selective 5-HT reuptake inhibitors
(SSRIs), such as fluoxetine and the highly selective agent citalopram
(Owens et al., 1997
; Sánchez and Meier, 1997
; Tatsumi et al.,
1997
; Goodnick and Goldstein, 1998
; Popik, 1999
), all elevate
extracellular levels of 5-HT in corticolimbic structures upon acute and
chronic administration (Blier and de Montigny, 1994
; Goodnick and
Goldstein, 1998
; Millan et al., 2000b
). On the other hand, the recently
introduced morpholine derivative, reboxetine, interacts preferentially
with NETs versus SERTs in vitro and markedly increases extracellular
levels of NE (Burrows et al., 1998
; Riva et al., 1999
; Sacchetti
et al., 1999
). In this respect, reboxetine resembles the
first-generation, tricylic agent desipramine (Owens et al., 1997
).
However, other tricyclic antidepressants, such as clomipramine,
interact with both NETs and with SERTs (Burke and Preskorn,
1995
; Tatsumi et al., 1997
). This dual activity is likewise displayed
by the cyclohexanol derivative, venlafaxine, which lacks the
undesirable histaminergic, muscarinic, and
1-AR antagonist properties of clomipramine and
other tricyclic agents (Muth et al., 1991
; Schweizer et al., 1997
;
Harvey et al., 2000
). Clinical studies have demonstrated the efficacy
of venlafaxine in major depressive states, including resistant and,
probably, bipolar patients (Amsterdam, 1998
; Poirier and Boyer, 1999
).
It may also possess a rapid onset of activity (Derivan et al., 1995
) and be associated with a high remission rate (Ferrier, 1999
), although
this remains to be confirmed. In addition, venlafaxine improves
generalized anxiety disorders (Rickels et al., 2000
) and social phobia
(Altamura et al., 1999
). These observations have triggered a resurgence
of interest in antidepressants uniting activity at SERTs and
NETs in vivo, with the aim of optimizing clinical efficacy, achieving
more rapid action, and enlarging therapeutic reach (Broekkamp et al.,
1995
; Frazer, 1997
; Sambunaris et al., 1997
).
Venlafaxine, however, displays only modest affinity for SERTs, and its
affinity for NETs is weak (Muth et al., 1991
; Owens et al., 1997
;
Tatsumi et al., 1997
; Béïque et al., 1999
, 2000
). It
would thus be of interest to obtain agents of greater potency. In this
light, we have generated a series of benzocyclobutane derivatives
possessing affinity for both SERTs and NETs. In the present and
accompanying papers, the pharmacological profile of one of these,
S33005 (Fig. 1), is compared with
venlafaxine, citalopram, reboxetine, and clomipramine.
First, we determined their affinities at native, rat, and cloned human
(h) SERTs, NETs, and DA transporters (DAT) and evaluated uptake of
5-HT, NE, and DA into cerebral rat synaptosomes. Second, we examined
the ability of drugs to prevent in vivo depletion of cerebral pools of
5-HT by parachloroamphetamine (PCA), which exerts its actions following
entry into serotonergic terminals via SERTs (Fuller et al., 1991
).
Third, serotonergic perikarya localized in the dorsal raphe nucleus
(DRN) possess both SERTs and inhibitory 5-HT1A
autoreceptors. Blockade of 5-HT uptake therefore suppresses the
electrical activity of serotonergic neurones via activation of
5-HT1A autoreceptors (Blier and de Montigny,
1994
; Gartside et al., 1997
). Thus, we examined the influence of S33005 and other antidepressants on the firing rate of serotonergic neurones. Similarly, we evaluated their actions at adrenergic neurones in the
locus ceruleus (LC), which bear NETs and inhibitory
2-AR autoreceptors (Scuvee-Moreau and Dresse,
1979
; Muth et al., 1991
), and at dopaminergic cell bodies in the
ventrotegmental area, which possess DATs and inhibitory
D2/D3 autoreceptors (Millan
et al., 2000b
). Fourth, the influence of S33005 and the other agents
upon extracellular levels of 5-HT, NE, and DA was simultaneously
quantified in single dialysis samples of the frontal cortex, dorsal
hippocampus, nucleus accumbens, and striatum of freely moving rats.
Finally, several
although not all
classes of antidepressant diminish
the density of cortical 5-HT2A and
-ARs upon
long-term treatment (Okada and Tokumitsu, 1994
; Newman-Tancredi et al.,
1996
; Yatham et al., 1999a
,b
). Thus, the influence of S33005 as
compared with venlafaxine upon frontocortical levels of
5-HT2A and
-ARs, as well as dialysis levels of
5-HT, NE, and DA, was evaluated following their administration for 2 or
3 weeks.
 |
Materials and Methods |
Animals.
These studies used male Wistar rats of 200 to
250 g (Iffa Credo, L'Arbresles, France) housed in sawdust-lined
cages with unrestricted access to standard chow and water. There was a
12-h/12-h light/dark cycle with lights on at 7:30 AM. Laboratory
temperature and humidity were 21 ± 0.5°C and 60 ± 5%,
respectively. Animals were adapted to laboratory conditions for at
least 1 week prior to testing. All animal use procedures conformed to
international European ethical standards (86/609-EEC) and the French
National Committee (décret 87/848) for the care and use of
laboratory animals.
Binding at Native Rat SERTs.
Binding affinity was determined
by competition with [3H]paroxetine (PerkinElmer
Life Sciences, Les Ulis, France). Freshly prepared membranes of
rat frontal cortex were homogenized with a Polytron and then
centrifuged twice at 20,000g. The pellet was resuspended each time in incubation buffer. Membranes were incubated in triplicate with 2 nM [3H]paroxetine and competing ligand
in a final volume of 0.4 ml for 2 h at 25°C. The incubation
buffer contained 50 nM Tris-HCl (pH 7.4), 120 nM NaCl, and 5 mM KCl.
Nonspecific binding was defined with 10 µM citalopram.
Binding at hSERTs.
Binding affinity was determined by
competition with [3H]paroxetine (PerkinElmer
Life Sciences). Membranes prepared from HEK293 cells stably expressing
recombinant hSERTs were purchased from Receptor Biology (Beltsville,
MD) and incubated in triplicate with 2 nM
[3H]paroxetine and competing ligand in a final
volume of 0.4 ml for 1.5 h at 25°C. The incubation buffer
contained 50 mM Tris-HCl (pH 7.4), 120 mM NaCl, and 5 mM KCl.
Nonspecific binding was defined with 10 µM citalopram.
Binding at Native Rat NETs.
Binding affinity was determined
by competition with [3H]nisoxetine (Amersham,
Les Ulis, France). Freshly prepared membranes of rat cortex were
homogenized with a Polytron and then centrifuged twice at
20,000g. The pellet was resuspended each time in incubation buffer. Membranes were incubated in triplicate with 2 nM
[3H]nisoxetine and competing ligand in a final
volume of 0.5 ml for 4 h at 4°C. The incubation buffer contained
50 mM Tris-HCl (pH 7.4), 120 mM NaCl, and 5 mM KCl. Nonspecific binding
was defined with 10 µM desipramine.
Binding at hNETs.
Binding affinity was determined by
competition with [3H]nisoxetine (2.0 nM,
Amersham). Membranes prepared from Madin-Darby canine kidney cells
expressing hNETs were purchased from Receptor Biology and incubated in
triplicate with 2 nM [3H]nisoxetine and
competing ligand in a final volume of 0.5 ml for 1 h at 4°C. The
incubation buffer contained 50 mM Tris-HCl (pH 7.4), 120 mM NaCl, and 5 mM KCl. Nonspecific binding was defined with 10 µM desipramine.
Binding at Native Rat DATs.
Binding affinity was determined
by competition with [3H]GBR12909 (PerkinElmer
Life Sciences). Freshly prepared membranes of rat striata were
homogenized with a Polytron and then centrifuged twice at
20,000g. The pellet was resuspended each time in incubation buffer. Membranes were incubated in triplicate with 2 nM
[3H]GBR12909 and competing ligand in a final
volume of 1 ml for 1 h at 4°C. The incubation buffer contained
50 mM Tris-HCl (pH 7.4), 120 mM NaCl and 4 mM MgCl2.
Nonspecific binding was defined with 10 µM GBR12909.
Binding at hDATs.
Binding affinity was determined by
competition with [3H]GBR12909 (PerkinElmer Life
Sciences). Membranes prepared from CHO cells stably expressing
recombinant hDATs were purchased from Receptor Biology and incubated in
triplicate with 2 nM [3H]GBR12909 and competing
ligand in a final volume of 0.5 ml for 2 h at 4°C. The
incubation buffer contained 50 mM Tris-HCl (pH 7.4) and 120 mM NaCl.
Nonspecific binding was defined with 10 µM GBR12909.
Data Analysis for Binding Studies.
For all of the above
protocols, at the end of the incubation period, membranes were filtered
through Whatman (Packard, Meriden, CT) GF/B filters pretreated
with 0.1% polyethylenimine. Radioactivity retained on the filters was
determined by scintillation counting. Binding isotherms were analyzed
by nonlinear regression using Prism software (GraphPad Software Inc.,
San Diego, CA) to determine IC50 values.
These were converted to inhibition constants
(Ki) by use of the Cheng-Prusoff
equation: Ki = IC50 /
[(L/KD)
1], where
L is the concentration of 3H-labeled
ligand and KD is its dissociation
constant determined in saturation binding experiments. The
KD values were as follows: 0.13 nM for
[3H]paroxetine at both native rat SERTs and
hSERTs; 1.2 and 2.2 nM for [3H]nisoxetine at
native rat NETs and hNETs, respectively; and 1.6 and 1.0 nM for
[3H]GBR12909 at native rat DATs and hDATs, respectively.
Interaction with other Binding Sites.
The potential
interaction of S33005 at diverse (>50) binding sites was evaluated by
using standard procedures (see Results for several key
sites) detailed elsewhere (Millan et al., 2000a
). Inasmuch as S33005
showed negligible (pKi < 5.0)
affinity at all sites examined, these protocols are not further
described herein. Data analysis was as described above.
Influence upon [3H]Monoamine Uptake by Rat Brain
Synaptosomes.
[3H]Monoamine uptake assays
were carried out on synaptosomes prepared from rat cortex
([3H]5-HT), rat hypothalamus
([3H]NE), and rat striatum
([3H]DA), essentially as described previously
(Janowsky et al., 1986
). Synaptosomes were incubated with the
radiolabeled neurotransmitter and drug for 15 min at 37°C before
rapid filtration. [3H]Monoamine uptake into
synaptosomes was determined by liquid scintillation counting.
Influence upon Depletion of Cerebral Pool of 5-HT by PCA.
Levels of 5-HT were determined by high-performance liquid
chromatography and electrochemical detection as previously
described (Millan et al., 2000a
) in the frontal cortex and hippocampus
of rats 60 min after s.c. administration of S33005, vehicle, or other drugs and 30 min after injection of the vehicle or PCA (5.0 mg/kg, i.p.). Data were analyzed by analysis of variance (ANOVA) followed by
Dunnett's test. ID50 values plus 95% confidence
limits (CL) were calculated.
Influence upon the Electrical Activity of Serotonergic,
Adrenergic, and Dopaminergic Cell Bodies.
The influence of S33005
compared with other ligands upon the firing rate of DRN-localized
serotonergic perikarya, LC-localized adrenergic perikarya, and
ventrotegmental area-localized dopaminergic perikarya was determined by
using a procedure described in detail previously (Millan et al.,
2000a
). Briefly, following anesthesia with chloral hydrate (400 mg/kg,
i.p.), rats were placed in a stereotaxic apparatus, and a
tungsten microelectrode was lowered into the DRN, LC, or
ventrotegmental area. Coordinates were as follows: DRN,
AP =
7.8 from bregma, L = 0.0, and H =
5/
6.5 from
dura; LC, AP =
1.2 from zero, L = 1.2, and H =
5.5/
6.5 from dura; and ventrotegmental area, AP =
5.5 from
bregma, L = 0.7, and H =
7/
8.5 from dura. As detailed
elsewhere (Millan et al., 2000a
), serotonergic, adrenergic, and
dopaminergic neurones in the DRN, LC, and ventrotegmental area,
respectively, were recognized by their distinctive waveforms. Following
baseline recording over 5 min, vehicle, S33005, or other agents were
administered i.v. (in a volume of 0.5 ml/kg) in cumulative doses every
2 to 3 min. After vehicle or drug administration, a further injection
of WAY100,635 (0.031 mg/kg, i.v.) or idazoxan (0.063 mg/kg, i.v.) was
made for the DRN and LC, respectively. Drug effects were quantified
over the 60-s bin corresponding to their time of peak action. Spike2 software (CED, Cambridge, UK) was used for data acquisition and analysis. Data are expressed as percentage of change from baseline firing rate (defined as 0%). Data were analyzed by ANOVA followed by
Newman-Keuls test, and ID50 values (95% CL) were calculated.
Influence upon Extracellular Levels of 5-HT, NE, and DA.
Quantification of extracellular levels of 5-HT, NE, and DA in single
dialysate samples of the frontal cortex, dorsal hippocampus (NE and
5-HT), nucleus accumbens (DA and 5-HT), and striatum (DA and 5-HT) was
achieved by using a protocol extensively described previously (Millan
et al., 2000a
). The guide cannulae were implanted under pentobarbital
anesthesia (40.0 mg/kg., i.p.) at the following coordinates 1 week
prior to experimentation: frontal cortex, AP = +2.2 from bregma,
L = ±0.6, and H =
0.2 from dura; dorsal hippocampus, AP =
3.6 from bregma, L = ±1.2, and H =
2.3 from
dura; nucleus accumbens, AP = +0.8 from bregma, L = +0.6, and
H =
4.5 from dura; and striatum, AP = +0.5 from bregma,
L =
2.8, and H =
3.0 from dura. A cuprophane
CMA/11 probe (4 mm in length for the frontal cortex and
striatum, 2 mm in length for the hippocampus and nucleus accumbens,
and, in each case, 0.24-mm outer diameter; Carnegie Medicine,
Stockholm, Sweden) was lowered into position. Three basal samples of 20 min each were taken. Vehicle, S33005, or other drugs were administered
s.c., and samples were taken for a further 3 h. NE, 5-HT, and DA
levels were quantified by high-performance liquid chromatography
followed by coulometric detection as previously described (Millan et
al., 2000a
). The assay limit of sensitivity was 0.1 to 0.2 pg/sample
for 5-HT, NE, and DA in each case. Data were analyzed by ANOVA, with
sampling time as the repeated within-subject factor.
Influence upon DRN Firing Rate and Dialysis Levels of 5-HT, NE,
and DA in the Frontal Cortex: Chronic Administration.
Rats were
treated daily with a single injection of either vehicle, S33005 (10.0 mg/kg, s.c.), or venlafaxine (10.0 mg/kg, s.c.) for 14 days. On the
15th day, rats either received an additional injection of the same drug
or of vehicle. Thereafter, exactly as described above, extracellular
levels of 5-HT, NE, and DA were determined in the frontal cortex of
freely moving rats. In a parallel study, following a chronic 14-day
treatment, the influence of S33005 or venlafaxine (day 15) upon the
electrical activity of serotonergic cell bodies in the DRN was determined.
Influence upon Cortical 5-HT2A Receptors and
-ARs:
Chronic Administration.
Rats were treated with S33005 (10.0 mg/kg,
s.c.), venlafaxine (10.0 mg/kg, s.c.), or vehicle once daily for 2 or 3 weeks. One day following the final injection, as described previously (Newman-Tancredi et al., 1996
), using
[3H]CGP-12177 as a radioligand,
-ARs were
examined in homogenates of cortex. Furthermore, using
[3H]ketanserin as a radioligand,
5-HT2A receptors were examined in frontal cortex.
Bmax and
KD values were determined through conventional procedures (see Newman-Tancredi et al., 1996
).
Drugs.
Actions of drugs in each of the individual studies
described herein were evaluated concurrently. For binding studies,
drugs were dissolved in dimethyl sulfoxide (10
2
M) and dilutions made in the buffer as appropriate. For in vivo studies, drugs were dissolved in sterile water, plus a few drops of
lactic acid if necessary, and pH adjusted to as close to normality (>5.0) as possible. Drug salts and sources were as follows. S33005 HCl
[(
)1-(1-dimethylaminomethyl 5-methoxybenzocyclobutan-1-yl) cyclohexanol], WAY100,635 fumarate
[(N-{2-[4-(2-methoxyphenyl)-1-piperazinyl]ethyl}-N-(2-pyridinyl)cyclohexanecarboxamine], citalopram HCl, idazoxan HCl, reboxetine methane sulfonate, and venlafaxine HCl were all synthesized internally (G. Lavielle and J.-L.
Peglion). GBR12935 2HCl
[1-[2-(diphenylmethoxy)ethyl]-4-(3-phenylpropyl)-piperazine] was
purchased from Sigma/RBI (Natick, MA), and PCA HCl and clomipramine were purchased from Sigma (Saint Quentin-Fallavier, France).
 |
Results |
Characterization of S33005 as Compared with Its Isomer, S33004, and
Racemic S32647.
S33005 is the optically pure (
)-isomer of
racemic (±)-S32647, whereas S33004 is the optically pure (+)-isomer.
S32647 potently interacted with native rat SERTs and, less potently,
with native rat NETs: pKi values
(Ki in nM) = 8.64 ± 0.02 (2.3) and 6.58 ± 0.09 (263), respectively. Similarly, S33005
displayed marked affinity for these sites:
pKi values
(Ki in nM) = 8.71 ± 0.06 (1.9) and 6.75 ± 0.10 (178), respectively. In contrast, S33004
was substantially less active at both SERTs and NETs:
pKi values
(Ki in nM) = 6.36 ± 0.05 (437) and 5.12 ± 0.10 (7586), respectively. These
observations led us to select S33005 for further study.
Interaction of S33005 as Compared with Reference Antidepressant
Agents at Cerebral Rat SERTs, NETs, and DATs.
As mentioned above,
S33005 possessed pronounced affinity for cerebral rat SERTs and less
marked affinity for cerebral rat NETs. A similar pattern of
preferential interaction with native SERTs versus NETs was acquired
with venlafaxine. However, its affinity at these sites was,
respectively, 11- and 6-fold lower than those of S33005
(Fig. 2; Table 1).
Clomipramine also resembled S33005 in its dual interaction at SERTs and
native NETs, with a clear preference for the former. In distinction to
S33005, citalopram revealed pronounced affinity for rat SERTs and
negligible affinity for NETs. On the other hand, reboxetine displayed
an opposite pattern of preference with superior affinity for native
NETs as compared with SERTs. S33005 and all other drugs manifested low affinity for cerebral rat DATs.

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Fig. 2.
Interaction of S33005 as compared with reference
antidepressant agents at native cerebral rat SERTs, NETs, and
DATs. Isotherms are representative of at least three independent
experiments performed in triplicate. All isotherms yielded pseudo-Hill
coefficients not significantly different from unity and corresponding
to pKi values specified in Table 1.
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TABLE 1
Affinities of S33005 as compared with reference antidepressant agents
at monoamine transporters and at monoaminergic receptors known to
modulate cerebral monoaminergic transmission
|
|
Interaction of S33005 as Compared with Reference Antidepressant
Agents at Cloned hSERTs, hNETs, and hDATs.
In analogy to native
rat SERTs, S33005 showed high affinity for heterologously expressed
hSERTs (Table 1). However, its affinity for hNETs was about 10-fold
lower than at native rat NETs. Venlafaxine similarly showed
(~8-fold) lower affinity for hNETs than for native NETs. In addition,
its affinity for hSERTs was somewhat less pronounced than at native
SERTs. In distinction, clomipramine showed more pronounced affinity for
hSERTs versus SERTs and slightly inferior affinity for hNETs versus
native NETs. Citalopram presented ~3-fold lower affinity at hSERTs
versus native SERTs. Its affinity was low at hNETs. Finally, the
affinity of reboxetine for hSERTs was less pronounced than its affinity
at hNETs. At both native DATs and cloned hDATs, the affinity of S33005
and other ligands for hDAT sites was low.
Interaction of S33005 as Compared with Reference Antidepressant
Agents at Additional Sites.
S33005 displayed negligible affinity
(pKi < 5.0) for multiple
monoaminergic receptors, including diverse sites shown in Table 1 that,
as autoreceptors and heteroceptors, modulate corticolimbic serotonergic, adrenergic, and dopaminergic transmission (see Millan et
al., 2000b
). It also showed negligible
(pKi < 5.0) affinity for
histaminergic (H)1 receptors labeled with
[3H]pyralamine (1.0 nM), and for cloned, human,
muscarinic h(M)1, hM2,
hM3, and hM4 receptors
labeled with
[3H]N-methyl-scopolamine (1.0 nM)
(not shown). S33005 did not bind (pKi < 5.0) to either monoamine oxidase A or monoamine oxidase B (not
shown). At a broad diversity of other receptors, enzymes, and ion
channels (>50 sites), S33005 also showed low affinity (pKi < 5.0). S33005 was thus highly
selective for SERTs and NETs. Like S33005, venlafaxine showed low
affinity (pKi < 5.0) for various monoaminergic receptors and H1 and
hM1 receptors. The affinity of clomipramine at
h5-HT1B, h5-HT1D, and
5-HT3 receptors was negligible, although it
showed marked affinity for h5-HT2A and
h5-HT2C receptors, as well as modest affinity for
5-HT3 receptors. At native
1-ARs and cloned
h
1A-ARs, its affinity was marked, whereas its
affinity for native rat
2-ARs and cloned
h
2A-ARs was low. At hD2
and hD3 receptors, the affinity of clomipramine
was modest. Clomipramine also displayed high affinity for both
H1 and hM1 receptors:
pKi values
(Ki in nM), 8.0 (10) and 7.5 (31.6),
respectively. Citalopram manifested negligible affinity for
monoaminergic receptors, with the exception of
h5-HT2C sites for which it showed mild affinity. It showed modest affinity for H1 receptors:
pKi values
(Ki in nM) = 6.3 (501). Finally,
reboxetine showed modest affinity for h5-HT2C
sites but negligible affinity for all other sites.
Influence of S33005 as Compared with Reference Antidepressant
Agents upon Uptake of 5-HT, NE, and DA into Rat Synaptosomes.
In
line with its high affinity at SERTs, S33005 potently and concentration
dependently inhibited the uptake of [3H]5-HT
into cerebral rat synaptosomes (Table 2).
It also inhibited [3H]NE uptake at higher
concentrations, in line with its lower affinity for NETs. In
distinction, only very high concentrations of S33005 modified
[3H]DA uptake. A similar pattern of data was
acquired for venlafaxine although, in line with its lower affinity than
S33005 for native SERTs and NETs, venlafaxine was less potent than
S33005 in inhibiting uptake of [3H]5-HT and
[3H]NE. Clomipramine also preferentially
inhibited [3H]5-HT versus
[3H]NE uptake and weakly inhibited
[3H]DA uptake. In contrast, in line with its
selective interaction with native SERTs versus NETs, citalopram
selectively inhibited synaptosomal uptake of
[3H]5-HT versus [3H]NE
(and [3H]DA). On the other hand, in analogy to
its binding profile, reboxetine behaved as a preferential inhibitor of
[3H]NE versus [3H]5-HT
uptake. It failed to modify [3H]DA uptake.
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TABLE 2
Influence of S33005 as compared with reference antidepressant agents
upon rat cerebral synaptosome uptake of [3H]serotonin (5-HT),
[3H]norepinephrine (NE), and [3H]dopamine (DA)
Data IC50 values are presented as mean ± S.E.M.
pIC50 of three independent determinations, each performed in
triplicate. In addition, equivalent values in nanomolar are indicated
in parentheses: a value of <5 corresponds to an IC50 of
>10,000 nM.
|
|
Influence of S33005 as Compared with Reference Antidepressant
Agents upon Depletion of Cerebral Pools of 5-HT by PCA.
The
administration of PCA (5.0 mg/kg, i.p.) elicited a pronounced reduction
of levels of 5-HT in both the frontal cortex and the hippocampus (Fig.
3): frontal cortex, vehicle/vehicle
(n = 12), 3.61 ± 0.24 ng/mg of protein versus
vehicle/PCA (n = 10), 0.87 ± 0.09, P < 0.001; hippocampus, vehicle/vehicle
(n = 12), 4.23 ± 0.22 ng/mg of protein versus
vehicle/PCA (n = 10), 0.43 ± 0.08, P < 0.001. Preadministration of S33005 dose
dependently, potently, and completely blocked the influence of PCA upon
5-HT levels in both frontal cortex and hippocampus. Administered alone, S33005 did not significantly modify 5-HT levels. For a dose of 10.0 mg/kg, s.c (which abolished the action of PCA): frontal cortex, vehicle/vehicle (n = 12), 3.61 ± 0.24 ng/mg of
protein versus S33005/vehicle (n = 10), 3.76 ± 0.21, P > 0.05; hippocampus, vehicle/vehicle (n = 12), 4.23 ± 0.22 ng/mg of protein versus
S33005/vehicle (n = 10) 4.54 ± 0.2, P > 0.05. Venlafaxine also inhibited the influence of
PCA upon levels of 5-HT in frontal cortex and hippocampus, although it
was less potently active than S33005. Citalopram was also highly
active, whereas clomipramine less potently attenuated the action of
PCA. Reboxetine (10.0 mg/kg, s.c.) did not significantly modify the
action of PCA (not shown). Administered alone, venlafaxine, citalopram,
clomipramine, and reboxetine did not significantly affect basal levels
of 5-HT in either frontal cortex or hippocampus (not shown).

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Fig. 3.
Influence of S33005 as compared with reference
antidepressant agents upon the depletion by PCA of 5-HT levels in
frontal cortex and hippocampus. Data are means ± S.E.M. They are
expressed as percentage blockade of the actions of PCA (5.0 mg/kg,
i.p.) with 0 and 100% values equivalent to no and full blockade,
respectively. Corresponding ID50 values (95% CL) are
indicated in Table 3. ANOVA was as follows. Frontal cortex, S33005,
F(5,56) = 62.1, P < 0.001;
venlafaxine, F(5,57) = 81.2, P < 0.001; clomipramine, F(4,36) = 74.0, P < 0.001; and citalopram,
F(4,28) = 107.7, P < 0.01. Hippocampus, S33005, F(5,50) = 41.1, P < 0.001; venlafaxine,
F(5,57) = 53.3, P < 0.001;
clomipramine, F(4,36) = 36.9, P < 0.001; and citalopram, F(4,28) = 91.5, P < 0.01. Asterisks indicate significance of drug
versus control values in Dunnett's test following ANOVA.
*P < 0.05. VEH, vehicle.
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Influence of S33005 as Compared with Reference Antidepressant
Agents upon the Electrical Activity of Monoaminergic Cell Bodies.
S33005 potently, dose dependently, and completely inhibited the firing
rate of serotonergic perikarya localized in the DRN of anaesthetized
rats (Figs. 4 and 5).
This action was abolished by the selective 5-HT1A
receptor antagonist, WAY100,635. Expressed relative to baseline values
(0%), S33005 (0.125 mg/kg, i.v.) + vehicle =
100.0 ± 0.0% versus S33005 + WAY100,635 (0.031 mg/kg, i.v.) =
1.1 ± 7.6%, P < 0.001. In line with previous work
(Millan et al., 2000b
), WAY100,635 did not modify the electrical
activity of serotonergic neurones alone (not shown). Over a higher dose range, S33005 dose dependently reduced the firing rate of adrenergic neurones of the LC, an action abolished by the
2-AR antagonist idazoxan. S33005 (2.0 mg/kg,
i.v.) + vehicle =
98.8 ± 0.3% versus S33005 + idazoxan
(0.063 mg/kg, i.v.) =
13.3 ± 5.2%, P < 0.001. Administered alone, idazoxan elicited a significant increase in firing rate. Idazoxan (0.063) = +42.0 ± 14.0% versus
vehicle = +1.3 ± 2.2%, P < 0.05. In
contrast to serotonergic and adrenergic neurones, S33005 exerted little
influence upon dopaminergic neurones of the ventrotegmental area. A
slight tendency toward an increase in firing rate was seen at modest
doses, which transformed into a tendency for inhibition at high doses,
but neither effect attained statistical significance. In addition,
S33005 did not modify the firing pattern (regular or burst) of
dopaminergic neurones (not shown). In contrast, as a positive internal
control, the selective DA reuptake inhibitor, GBR12935, dose
dependently inhibited firing of ventrotegmental area dopaminergic
neurones: ID50 = 0.8 (0.6-1.1) mg/kg, i.v. Its
actions were blocked by the
D2/D3 antagonist, haloperidol (0.016 mg/kg, i.v.). Vehicle + GBR12935 (4.0 mg/kg, i.v.) =
83.4 ± 12.5 versus haloperidol + GBR12935 =
2.2 ± 2.2%, P < 0.001.

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Fig. 4.
Dose-dependent influence of S33005 upon the
electrical activity (firing rate) of representative serotonergic,
adrenergic, and dopaminergic neurones localized in the DRN, LC, and
ventrotegmental area (VTA), respectively, of anesthetized rats. S33005
was administered in cumulative doses at 3-min intervals. For the DRN,
this was followed by administration of the selective 5-HT1A
receptor antagonist, WAY100,635, and for the LC, by administration of
the selective 2-adrenoceptor antagonist, idazoxan. Both
WAY100,635 and idazoxan rapidly and completely reversed the inhibitory
influence of S33005 upon DRN and LC neurones, respectively.
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Fig. 5.
Influence of S33005 as compared with reference
antidepressant agents upon the electrical activity (firing rate) of
serotonergic, adrenergic, and dopaminergic cell bodies localized in the
DRN, LC, and VTA, respectively, of anesthetized rats. Data are
means ± S.E.M. of firing rate expressed relative to basal
pretreatment values (defined as 0%). n 5 per
value. Corresponding ID50 values (95% CL) are indicated in
Table 3. ANOVA was as follows. S33005: DRN, F(6,24) = 38.8, P < 0.001; LC, F(6,30) = 72.1, P < 0.001; and VTA,
F(5,25) = 2.3, P > 0.05. Venlafaxine: DRN, F(7,35) = 68.9, P < 0.001; LC, F(5,25) = 57.2, P < 0.001; and VTA, F(6,36) = 2.5, P > 0.05. Clomipramine: DRN,
F(5,30) = 46.8, P < 0.001; LC,
F(5,25) = 5.3, P < 0.01; and
VTA, F(4,20) = 25.5, P < 0.001. Citalopram: DRN, F(5,25) = 113.9, P < 0.001; LC, F(5,25) = 4.1, P < 0.01; and VTA, F(6,30) = 6.2, P < 0.001. Reboxetine: DRN,
F(6,24) = 6.6, P < 0.001; LC,
F(5,25) = 57.2, P < 0.001; and
VTA, F(5,30) = 2.3, P > 0.05. Asterisks indicate significance of drug to control values in
Newman-Keuls test following ANOVA. *P < 0.05. VEH,
vehicle.
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Venlafaxine likewise inhibited both serotonergic and, less potently,
adrenergic cell bodies without affecting dopaminergic neurones.
However, its potency was less pronounced than that of S33005. Its
influence upon serotonergic and adrenergic neurones was reversed by
WAY100,635 and idazoxan, respectively. For DRN, venlafaxine (1.0 mg/kg,
i.v.) + vehicle =
100.0 ± 0.0% versus venlafaxine + WAY100,635 (0.031 mg/kg, i.v.) =
14.3 ± 12.8%, P < 0.01. For LC, venlafaxine (4.0 mg/kg, i.v.) + vehicle =
93.1 ± 4.7% versus venlafaxine + idazoxan
(0.063 mg/kg, i.v.) =
18.7 ± 8.5%, P < 0.001. Clomipramine inhibited serotonergic neurones less potently than
S33005, an action blocked by WAY100,635. It only decreased the firing
rate of adrenergic neurones at a high dose, an effect antagonized by
idazoxan. For DRN, clomipramine (0.5 mg/kg, i.v.) + vehicle =
90.7 ± 6.5% versus clomipramine + WAY100,635 (0.031 mg/kg,
i.v.) =
1.5 ± 11.2%, P < 0.001. For LC,
clomipramine (8.0 mg/kg, i.v.) + vehicle =
63.3 ± 15.5%
versus clomipramine + idazoxan (0.063 mg/kg, i.v.) = +35.1 ± 10.1, P < 0.01. In contrast, citalopram selectively
and WAY100,635-reversibly suppressed the activity of serotonergic
versus adrenergic neurones. For DRN, citalopram (0.5 mg/kg, i.v.) + vehicle =
100.0 ± 0.0% versus citalopram + WAY100,635
(0.031 mg/kg, i.v.) = +58.6 ± 32.5%, P < 0.001. It slightly enhanced the activity of LC adrenergic cell bodies
and marginally excited ventrotegmental area dopaminergic neurones at
high doses. On the other hand, reboxetine selectively and
idazoxan-reversibly inhibited adrenergic neurones. For LC, reboxetine
(0.5 mg/kg, i.v.) + vehicle =
100.0 ± 0.0% versus reboxetine + idazoxan (0.063 mg/kg, i.v.) =
5.8 ± 8.6%,
P < 0.001. Reboxetine significantly
increased the activity of serotonergic neurones, whereas
dopaminergic perikarya were unaffected.
Influence of S33005 as Compared with Reference Antidepressant
Agents upon Extracellular Levels of 5-HT, NE, and DA in Dialysates of
the Frontal Cortex of Freely Moving Rats.
S33005 elicited a rapid,
sustained, and dose-dependent elevation in extracellular levels of 5-HT
in frontal cortex of freely moving rats (Figs.
6
through 8; Table 3). In the same dialysis samples, levels of NE and DA were likewise augmented. Area under the
curve (AUC) analysis revealed that the most pronounced influence of
S33005 was, in fact, upon extracellular levels of NE, with those of
5-HT and DA displaying a less marked increase. A similar pattern of
data was acquired with both venlafaxine and clomipramine, although they
were less potent than S33005. In distinction, citalopram preferentially
elevated levels of 5-HT versus NE and DA and only evoked a slight rise
in levels of NE and DA even at the highest dose tested. On the
contrary, reboxetine potently increased levels of NE and, less
markedly, DA without significantly modifying levels of 5-HT.
Surprisingly, and for reasons remaining to be clarified, the induction
of NE levels by reboxetine was less pronounced at the highest dose
evaluated (40.0 mg/kg, s.c.). Upon i.p. and p.o. administration, S33005
also dose dependently (0.63-40.0 mg/kg, in each case), significantly
(P < 0.01, in each case), and markedly elevated
dialysate levels of 5-HT, NE, and DA in frontal cortex (not shown).

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Fig. 6.
Influence of S33005 as compared with venlafaxine upon
extracellular levels of 5-HT, NE, and DA in dialysates of the frontal
cortex of freely moving rats. Data are means ± S.E.M. of 5-HT,
NE, and DA levels expressed relative to basal pretreatment values
(defined as 100%). These were, representatively (S33005, 10.0 mg/kg,
s.c.), 0.79 ± 0.06, 1.49 ± 0.05, and 1.14 ± 0.06 pg/20 µl of dialysate for 5-HT, NE, and DA, respectively.
n 5 per value. ANOVA was as follows. 5-HT:
S33005, 0.01, F(1,8) = 0.1, P > 0.05; 0.04, F(1,9) = 15.2, P < 0.01; 0.16, F(1,8) = 23.6, P < 0.01; 0.63, F(1,8) = 10.6, P < 0.05; 2.5, F(1,8) = 27.8, P < 0.01; 10.0, F(1,11) = 47.3, P < 0.01; and 40.0, F(1,9) = 100.4, P < 0.01. Venlafaxine, 0.16, F(1,8) = 0.6, P > 0.05; 0.63, F(1,8) = 11.3, P < 0.01; 2.5, F(1,8) = 60.0, P < 0.01; 10.0, F(1,11) = 35.4, P < 0.01; and
40.0, F(1,9) = 125.6, P < 0.01. NE: S33005, 0.01, F(1,8) = 0.3, P > 0.05; 0.04, F(1,9) = 0.3, P > 0.05; 0.16, F(1,8) = 21.3, P < 0.01; 0.63, F(1,8) = 12.5, P < 0.01; 2.5, F(1,9) = 179.9, P < 0.01; 10.0, F(1,10) = 55.5, P < 0.01; and 40.0, F(1,8) = 41.3, P < 0.01. Venlafaxine, 0.16, F(1,8) = 1.7, P > 0.05; 0.63, F(1,8) = 51.4, P < 0.01; 2.5, F(1,8) = 39.6, P < 0.01; 10.0, F(1,11) = 62.8, P < 0.01; and 40.0, F(1,9) = 198.4, P < 0.01. DA:
S33005, 0.01, F(1,8) = 0.1, P > 0.05; 0.04, F(1,9) = 0.1, P > 0.05; 0.16, F(1,8) = 7.1, P < 0.05; 0.63, F(1,8) = 10.1, P < 0.05; 2.5, F(1,9) = 16.7, P < 0.01; 10.0, F(1,11) = 31.6, P < 0.01; and 40.0, F(1,8) = 26.3, P < 0.01. Venlafaxine, 0.16, F(1,8) = 0.1, P > 0.05; 0.63, F(1,8) = 3.8, P > 0.05; 2.5, F(1,8) = 30.0, P < 0.01; 10.0, F(1,11) = 101.8, P < 0.01; and
40.0, F(1,9) = 135.5, P < 0.01. Asterisks indicate significance of drug versus vehicle values.
*P < 0.05.
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Fig. 7.
Influence of citalopram, reboxetine, and clomipramine
upon extracellular levels of 5-HT, NE, and DA in dialysates of the
frontal cortex of freely moving rats. Data are means ± S.E.M. of
5-HT, NE, and DA levels expressed relative to basal pretreatment values
(defined as 100%). For representative basal levels, see legend to Fig.
6. ANOVA was as follows. 5-HT: citalopram, 0.16, F(1,9) = 4.2, P > 0.05; 0.63, F(1,9) = 62.9, P < 0.01; 2.5, F(1,9) = 69.4, P < 0.01; 10.0, F(1,8) = 140.4, P < 0.01; and
40.0, F(1,9) = 107.3, P < 0.01. Clomipramine, 0.63, F(1,8) = 0.1, P > 0.05; 2.5, F(1,8) = 16.3, P < 0.01; 10.0, F(1,10) = 52.0, P < 0.01; and 40.0, F(1,9) = 62.4, P < 0.01. Reboxetine, 0.0025, F(1,8) = 1.3, P > 0.05; 0.04, F(1,8) = 0.1, P > 0.05; 0.63, F(1,8) = 0.1, P > 0.05; 10.0, F(1,12) = 0.3, P > 0.05; and 40.0, F(1,9) = 0.2, P > 0.05. NE: citalopram, 0.16, F(1,9) = 0.8, P > 0.05; 0.63, F(1,9) = 3.8, P > 0.05; 2.5, F(1,9) = 3.3, P > 0.05; 10.0, F(1,8) = 41.2, P < 0.01; and
40.0, F(1,10) = 19.3, P < 0.01. Clomipramine, 0.63, F(1,8) = 1.4, P > 0.05; 2.5, F(1,8) = 64.5, P < 0.01; 10.0, F(1,9) = 76.5, P < 0.01 and 40.0, F(1,9) = 80.5, P < 0.01; Reboxetine, 0.0025, F(1,8) = 3.3, P > 0.05; 0.04, F(1,8) = 88.1, P < 0.01; 0.63, F(1,9) = 44.7, P < 0.01; 10.0, F(1,12) = 46.1, P < 0.01; and
40.0, F(1,9) = 10.9, P < 0.01. DA: citalopram, 0.16, F(1,9) = 0.3, P > 0.05; 0.63, F(1,9) = 1.7, P > 0.05; 2.5, F(1,9) = 0.8, P > 0.05; 10.0, F(1,8) = 1.5, P > 0.05; and 40.0, F(1,10) = 30.5, P < 0.01. Clomipramine, 0.63, F(1,8) = 1.0, P > 0.05; 2.5, F(1,8) = 13.5, P < 0.01; 10.0, F(1,9) = 39.3, P < 0.01; and
40.0, F(1,9) = 120.9, P < 0.01; Reboxetine, 0.0025, F(1,8) = 1.2, P > 0.05; 0.04, F(1,8) = 19.1, P < 0.01; 0.63, F(1,9) = 112.8, P < 0.01; 10.0, F(1,12) = 25.7, P < 0.01; and 40.0, F(1,9) = 155.3, P < 0.01. Asterisks indicate significance of drug versus vehicle values.
*P < 0.05.
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Fig. 8.
Relative potency of S33005 as compared with reference
antidepressant agents upon extracellular levels of 5-HT, NE, and DA in
dialysates of the frontal cortex of freely moving rats. Data
(means ± S.E.M.) are area under the curve analyses based on
individual time courses presented in Figs. 6 to 8. Corresponding
minimal effective doses (P < 0.05 to vehicle) are
indicated in Table 3. VEH, vehicle.
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TABLE 3
Summary and analyses of actions of S33005 as compared with reference
antidepressant agents in functional models reflecting inhibition of
monoamine uptake in vivo
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Influence of S33005 as Compared with Reference Antidepressant
Agents upon Extracellular Levels of 5-HT, NE, and DA in Dialysates of
the Dorsal Hippocampus, Nucleus Accumbens, and Striatum of Freely
Moving Rats.
S33005 elicited a pronounced and sustained elevation
in levels of 5-HT and NE in the dorsal hippocampus
(Fig. 9). For 5-HT AUC analysis,
vehicle = 99.2 ± 2.5% versus S33005 = 207.8 ± 6.1%. For NE AUC analysis, vehicle = 97.8 ± 2.5% versus
S33005 = 231.7 ± 8.1%. Venlafaxine similarly increased
extracellular levels of 5-HT and NE in this structure. For 5-HT AUC
analysis, vehicle = 99.2 ± 2.5% versus venlafaxine = 205.2 ± 10.5%. For NE AUC analysis, vehicle = 97.8 ± 2.5% versus venlafaxine = 204.1 ± 7.0%. Clomipramine also
enhanced levels of both 5-HT and NE (not shown). For 5-HT, F(1,16) = 30.8, P < 0.01; and AUC
analysis, vehicle = 99.2 ± 2.5% versus clomipramine = 165.3 ± 5.9%. For NE, F(1,16) = 34.4, P < 0.01; and AUC analysis, vehicle = 97.8 ± 2.5% versus clomipramine = 189.2 ± 7.4%. In contrast,
citalopram selectively elevated levels of 5-HT as compared with NE (not
shown). For 5-HT, F(1,15) = 87.0, P < 0.01; and AUC analysis, vehicle = 99.2 ± 2.5% versus
citalopram = 193.0 ± 9.0%. For NE, F(1,15) = 0.7, P > 0.05; and AUC analysis, vehicle = 97.8 ± 2.5% versus citalopram = 102.1 ± 3.5%. On the other hand, reboxetine selectively increased levels of NE as compared with 5-HT. For 5-HT, F(1,16) = 0.2, P > 0.05; and AUC analysis, vehicle = 99.2 ± 2.5% versus
reboxetine = 101.5 ± 3.0%. For NE, F(1,16) = 32.6, P < 0.01; and AUC analysis, vehicle = 97.8 ± 2.5% versus reboxetine = 215.0 ± 8.3%.

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Fig. 9.
Influence of S33005 as compared with venlafaxine upon
extracellular levels of 5-HT, NE, and DA in dialysates of the dorsal
hippocampus, nucleus accumbens, and striatum of freely moving rats.
Data are means ± S.E.M. of 5-HT, NE, and DA levels expressed
relative to basal pretreatment values (defined as 100%). These were,
representatively (S33005, 10 mg/kg, s.c.), 0.68 ± 0.05 and
0.83 ± 0.04 pg/20 µl of dialysate for 5-HT and NE,
respectively, in dorsal hippocampus; 0.63 ± 0.04 and 4.8 ± 0.5 pg/20 µl of dialysate for 5-HT and DA, respectively, in nucleus
accumbens; and 0.49 ± 0.03 and 16.8 ± 1.5 pg/20 µl of
dialysate per values for 5-HT and DA, respectively, in the striatum.
n 5 per value. ANOVA was as follows. Dorsal
hippocampus, S33005, 5-HT, F(1,15) = 266.2, P < 0.01 and NE, F(1,15) = 123.3, P < 0.01; and venlafaxine, 5-HT,
F(1,16) = 24.6, P < 0.01; and
NE, F(1,16) = 61.6, P < 0.01. Nucleus accumbens, S33005, 5-HT, F(1,9) = 486.1, P < 0.01 and DA, F(1,9) = 0.5, P > 0.05; and venlafaxine, 5-HT,
F(1,10) = 52.6, P < 0.01 and
DA, F(1,10) = 0.1, P > 0.05. Striatum, S33005, 5-HT, F(1,10) = 137.6, P < 0.01 and DA, F(1,11) = 5.2, P < 0.05; and venlafaxine, 5-HT,
F(1,9) = 107.7, P < 0.01 and
DA, F(1,11) = 3.5, P > 0.05. Asterisks indicate significance of drug versus vehicle values.
*P < 0.05.
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In the nucleus accumbens and striatum, S33005 and venlafaxine likewise
provoked a pronounced increase in dialysis levels of 5-HT, whereas
levels of DA were only marginally affected. Clomipramine and citalopram
likewise significantly elevated levels of 5-HT (not shown). Accumbens,
citalopram, F(1,11) = 22.3, P < 0.01; and AUC analysis, vehicle = 97.7 ± 2.0% versus
citalopram = 178.0 ± 7.3%. Clomipramine,
F(1,10) = 89.9, P < 0.01; and AUC
analysis, vehicle = 99.2 ± 2.5% versus clomipramine = 196.5 ± 7.4%. Striatum, citalopram, F(1,11) = 19.9, P < 0.01; and AUC analysis, vehicle = 102.6 ± 2.9% versus citalopram = 185.9 ± 7.9%.
Clomipramine, F(1,11) = 105.3, P < 0.01; and AUC analysis, vehicle = 102.6 ± 2.9% versus
clomipramine = 191.2 ± 6.0%. However, they did not significantly affect levels of DA (not shown). Accumbens, citalopram, F(1,11) = 0.4, P > 0.05; and AUC
analysis, vehicle = 99.0 ± 1.6% versus citalopram = 101.2 ± 1.3%. Clomipramine, F(1,10) = 1.6, P > 0.05; and AUC analysis, vehicle = 99.0 ± 1.6% versus clomipramine = 108.4 ± 2.9%. Striatum,
citalopram, F(1,12) = 0.1, P > 0.05; and AUC analysis, vehicle = 97.4 ± 1.2% versus
citalopram = 96.3 ± 2.2%. Clomipramine,
F(1,12) = 1.4, P > 0.05; and AUC
analysis, vehicle = 97.4 ± 1.2% versus clomipramine = 106.0 ± 2.4%. Reboxetine did not affect levels of 5-HT and DA in
either the accumbens or the striatum (data not shown).
Used as a positive, internal control, the selective DA uptake
inhibitor, GBR12935, selectively elevated DA as compared with 5-HT
levels in both nucleus accumbens and striatum (not shown). Nucleus
accumbens: for 5-HT, F(1,10) = 1.4, P > 0.05; and AUC analysis, vehicle = 97.7 ± 2.0% versus
GBR12935 = 104.6 ± 2.9%. For DA, F(1,10) = 18.8, P < 0.01; and AUC analysis, vehicle = 99.0 ± 1.6% versus GBR12935 = 326.4 ± 18.6%.
Striatum: for 5-HT, F(1,9) = 0.3, P > 0.05; and AUC analysis, vehicle = 102.6 ± 2.9% versus
GBR12935 = 105.4 ± 2.8%. For DA, F(1,11) = 44.5, P < 0.01; and AUC analysis, vehicle = 97.4 ± 1.2% versus GBR12935 = 254.6 ± 10.5%.
Influence of Chronic Administration of S33005 as Compared
with Venlafaxine upon Extracellular Levels of 5-HT, NE, and DA in
Frontal Cortex and the Electrical Activity of DRN Serotonergic
Neurones.
Following administration of S33005 for 2 weeks, there
was no significant modification in basal dialysate levels of DA in
frontal cortex (Fig. 10). Levels of
5-HT showed a (nonsignificant) tendency for an increase. Furthermore,
there was a significant elevation in basal levels of NE. Expressed
relative to basal levels, an additional injection of S33005 at the dose
used for chronic treatment (10.0 mg/kg, s.c.) evoked an elevation in
frontocortical dialysate levels of 5-HT, NE, and DA similar to that
seen in rats treated chronically with vehicle
(Fig. 11). A comparable pattern of data was obtained for venlafaxine inasmuch as the influence of its acute
administration upon 5-HT, NE, and DA levels in frontal cortex was not
significantly modified by chronic administration (not shown).
Nevertheless, in distinction to S33005, venlafaxine did not show an
increase in basal levels of NE following chronic administration (Fig.
10).

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Fig. 10.
Influence of chronic (2 weeks) administration of
S33005 (10.0 mg/kg, s.c.) and venlafaxine (10.0 mg/kg, s.c.) upon
extracellular levels of 5-HT, NE, and DA in dialysates of the frontal
cortex. Data are means ± S.E.M. n 5 per
value. The asterisk indicates a significant (P < 0.05) difference of chronic S33005 to chronic vehicle values in
Student's t test.
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Fig. 11.
Influence of chronic (2 weeks) administration of
S33005 (10.0 mg/kg, s.c.) upon modulation of extracellular levels of
5-HT, NE, and DA in dialysates of the frontal cortex by additional,
acute administration of S33005 (10.0 mg/kg, s.c.). Dialysate levels are
expressed as a percentage of basal preinjection values, which were
defined as 100% (see Fig. 10). Data are means ± S.E.M.
n 5 per value. There was no significant
difference in ANOVA (P > 0.05) for influence of
S33005 (acute) in rats chronically treated with S33005 as compared with
vehicle (VEH).
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The potency of S33005 for inhibition of the firing rate of DRN
serotonergic neurones was not modified following its administration for
2 weeks: ID50 (mg/kg, i.v.; 95% CL) for chronic
vehicle = 0.18 (0.14-0.21) versus chronic S33005 = 0.18 (0.14-0.20). Similar findings were acquired for venlafaxine: chronic
vehicle = 0.09 (0.6-0.14) versus chronic venlafaxine = 0.07 (0.05-0.10).
Influence Upon Cortical Levels of 5-HT2A Receptors and
-ARs.
Following chronic (2 weeks) administration of S33005,
relative to vehicle treatment (Bmax,
22.9 ± 1.3 fmol/mg of tissue = 100.0 ± 5.5%), there
was a significant (P < 0.05) reduction in the density
(Bmax) of 5-HT2A
receptors in frontal cortex (85.0 ± 2.5%) in the absence of an
alteration of affinity (vehicle, KD = 1.64 ± 0.12 nM versus S33005, KD = 1.39 ± 0.08 nM) (Fig. 12).
Venlafaxine tended to decrease the density of
5-HT2A receptors, although this effect did not
attain statistical significance (92.2 ± 3.7%, P > 0.05); the KD was also not affected
(1.64 ± 0.14 nM). At 3 weeks, relative to vehicle
(Bmax, 28.0 ± 1.4 fmol/mg of
tissue = 100.0 ± 5.0%), both S33005 and venlafaxine yielded
a significant decrease in Bmax:
79.6 ± 2.8 and 77.8 ± 2.8%, respectively,
P < 0.01 in each case.
KD values were unaffected:
vehicle = 2.27 ± 0.11 nM, S33005 = 2.03 ± 0.19 nM, and venlafaxine = 2.18 ± 0.21 nM. Compared with
vehicle-treated controls (Bmax,
5.58 ± 0.27 fmol/mg of tissue = 100.0 ± 4.6%),
following 2 weeks of treatment, S33005 tended to decrease levels of
-ARs in cortex (86.6 ± 4.2%), but this effect just failed to
reach statistical significance (P = 0.056).
Venlafaxine, in distinction, exerted little influence upon
-ARs
(97.7 ± 5.3%). Following 3 weeks of administration, S33005 and
venlafaxine did not significantly modify
-AR density or affinity
(not shown).

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Fig. 12.
Scatchard analysis of the influence of chronic (2 weeks) administration of S33005 upon the density of 5-HT2A
receptors in frontal cortex. A, from representative experiments each
performed in triplicate. For further details, see
Results. B, data are means ± S.E.M.
n 5 per group. Asterisks indicate significance
of differences to vehicle in Student's two-tailed t
test. *P < 0.05.
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Discussion |
Receptor Binding Profile.
S33005 possessed pronounced affinity
for native rat and cloned human SERTs, yielding
pKi values similar to citalopram and clomipramine and superior to venlafaxine and reboxetine (Muth et al.,
1991
; Owens et al., 1997
; Tatsumi et al., 1997
; Béïque et
al., 1999
; Riva et al., 1999
). Citalopram was ~3-fold
less potent at human versus rat SERTs. This observation,
obtained with [3H]paroxetine, may be compared
with ratios of 2- and 10-fold for binding studies performed with
[3H]citalopram at rat SERTs as compared with
[3H]citalopram and
[3H]5-HT, respectively, at hSERTs (Owens et
al., 1997
). On the contrary, clomipramine shows ~10-fold
higher affinity at human versus rat SERTs (Table 1; Owens et
al., 1997
). Such contrasting affinities (Barker and Blakely, 1995
; Sur
et al., 1998
) emphasize the importance of determining drug affinities
in both species.
Although S33005 showed relatively modest affinity at rat NETs, its
actions at these sites are functionally important (see below). Indeed,
this "dual" profile may be distinguished from citalopram
(SERT-selective) and reboxetine (NET-preferential) and resembles
clomipramine and venlafaxine, although the latter was a substantially
weaker ligand. In fact, the affinity of venlafaxine (22 nM) at rat
SERTs corresponds well to the studies of Owens et al. (1997)
,
Béïque et al. (1999)
, and Muth et al. (1986)
. Although
its affinity at rat NETs was only 998 nM, this value is close to those
of 1260 and 1067 reported by Béïque et al. (1999)
and
Owens et al. (1997)
, respectively. Similarly, the affinity of
venlafaxine for hSERTs (76 nM) coincides with previous values (Owens et
al., 1997
; Tatsumi et al., 1997
), while its lower affinity for hNETs
(6310 nM) bears comparison to values of 2269 and 1644 obtained with
[3H]nisoxetine and
[3H]norepinephrine, respectively, by Owens et
al. (1997)
, and of 1060 with [3H]nisoxetine by
Tatsumi et al. (1997)
. Indeed the 83-fold preference of venlafaxine for
hSERTs versus hNETs seen herein corresponds well to values of 283 and
118 for Owens et al. (1997)
and Tatsumi et al. (1997)
, respectively.
While Owens et al. (1997)
found 2-fold lower affinity of venlafaxine at
human versus rat NETs, the ratio was 6-fold herein and 10-fold for
S33005. Venlafaxine and S33005 are chemically related, and this
human/rat difference is a common feature of such derivatives (A. Newman-Tancredi and M. J. Millan, unpublished observations). Apart from methodological aspects (tissue versus cloned sites), species differences or contrasting interactions with
multiple binding sites on NETs may be involved (Barker and Blakely, 1995
; Sur et al., 1998
). In addition, several
pharmacologically distinct isoforms of NETs are differentially
localized in the central nervous system (Hughes and Stanford, 1998
;
Kitayama et al., 1999
). Their existence may also be pertinent to the
disparity between the weak activity of venlafaxine at rat NETs as
compared with its potent influence upon extracellular levels of NE in
vivo (Bolden-Watson and Richelson, 1993
; Hughes and Stanford, 1998
; Béïque et al., 1999
, 2000
) (see below).
The affinity of S33005 for >50 receptors, enzymes, and ion channels
was negligible, supporting attribution of its functional actions to
SERTs and NETs. In corroboration of previous studies, citalopram and
clomipramine displayed modest affinity for
h5-HT2C sites, at which they display
antagonist properties (Pälvimäki et al., 1996
;
Millan et al., 2000a
), and reboxetine also revealed mild affinity for
h5-HT2C receptors. While blockade (or
down-regulation) of 5-HT2C sites favorably
influences mood, it may also elicit hyperphagia (see Millan et al.,
2000b
). Moreover, antagonism by clomipramine of
H1 receptors contributes to weight gain, while its cardiovascular autonomic and sedative side effects reflect actions
at
1-ARs, H1, and
M1 receptors (Burke and Preskorn, 1995
; Owens et
al., 1997
). The absence of affinity of S33005 for these sites is, thus, important.
Prevention of PCA-Induced 5-HT Depletion.
Following access to
serotonergic terminals via SERTs, PCA triggers 5-HT release (Fuller et
al., 1991
). Correspondingly, S33005, venlafaxine, citalopram, and
clomipramine suppressed depletion of cerebral pools of 5-HT by PCA, in
analogy to their attenuation of its behavioral actions (accompanying
paper), whereas reboxetine was inactive. The high potency of S33005
versus venlafaxine in this model underpins in vitro observations
discussed above.
Electrical Activity of Monoaminergic Neurones.
Reflecting its
marked activity at SERTs, S33005 potently and WAY100,635-reversibly
inhibited the firing rate of serotonergic perikarya, and, in line with
its lower activity at NETs, higher doses of S33005 idazoxan-reversibly
inhibited the electrical activity of adrenergic cell bodies. In
correspondence with binding studies