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CNS Drug Reviews

Vol. 8, No.2, pp. 117142


2002 Neva Press, Branford, Connecticut

Pharmacology of Flibanserin
Franco Borsini1, Kennett Evans2, Kathryn Jason3, Frank Rohde1,
Barbara Alexander1, and Stephan Pollentier1
1
2

Boehringer Ingelheim Pharma KG, Biberach an der Riss, Germany;


Boehringer Ingelheim Pharmaceuticals, Burlington, Ontario, Canada;
3
Boehringer Ingelheim Pharmaceuticals, Ridgfield, CT, USA

Key Words: FlibanserinSerotoninDopamineReceptorsFiring rateAdenylyl


cyclaseAnimal modelsAntidepressants.

ABSTRACT
Flibanserin has preferential affinity for serotonin 5-HT1A, dopamine D4, and serotonin
5-HT2A receptors. In vitro and in microiontophoresis, flibanserin behaves as a 5-HT1A
agonist, a very weak partial agonist on dopamine D4 receptors, and a 5-HT2A antagonist.
In vivo flibanserin binds equally to 5-HT1A and 5-HT2A receptors. However, under higher
levels of brain 5-HT (i.e., under stress), flibanserin may occupy 5-HT2A receptors in
higher proportion than 5-HT1A receptors. The effects of flibanserin on adenylyl cyclase
are different from those of buspirone and 8-OH-DPAT, two other purported 5-HT1A receptor agonists. Flibanserin reduces neuronal firing rate in cells of the dorsal raphe, hippocampus, and cortex with the CA1 region being the most sensitive in the brain.
Flibanserin-induced reduction in firing rate in the cortex seems to be mediated through
stimulation of postsynaptic 5-HT1A receptors, whereas the reduction of the number of
active cells seems to be mediated through dopamine D4 receptor stimulation. Flibanserin
quickly desensitizes somatic 5-HT autoreceptors in the dorsal raphe and enhances tonic
activation of postsynaptic 5-HT1A receptors in the CA3 region. Flibanserin preferentially
reduces synthesis and extracellular levels of 5-HT in the cortex, where it enhances
extracellular levels of NE and DA. Flibanserin displays antidepressant-like activity in
most animal models sensitive to antidepressants. Such activity, however, seems qualitatively different from that exerted by other antidepressants. Flibanserin seems to act via
direct or indirect stimulation of 5-HT1A, DA, and opioid receptors in those animal models.
Flibanserin does not display consistent effects in animal models of anxiety and seems to
exert potential antipsychotic effects. Flibanserin may induce some sedation but does not
induce observable toxic effects at pharmacologically relevant doses.
Address correspondence and reprint requests to: Franco Borsini, Boehringer Ingelheim Pharma KG,
Birkendorfer Strasse 65, 88397 Biberach an der Riss, Germany.
Tel.: +49 (7351) 54-7297; Fax: +49 (7351) 54-98647; E-mail: franco.borsini@bc.boehringer-ingelheim.com

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BORSINI ET AL.

BINDING STUDIES
Flibanserin shows the highest affinity for cloned human serotonin 5-HT1A receptors
compared with cloned human D4 (isoforms 4.2, 4.4, and 4.7) and 5-HT2A receptors
(Table 1). This rank of affinity values is still maintained when the affinity for 5-HT1A and
5-HT2A receptors was evaluated in brain tissue. However, flibanserin displays higher affinity for 5-HT1A and 5-HT2A receptors in cloned cells than in cerebral tissue (the affinity
for D4 receptors was not tested in tissues).
Flibanserin also shows some affinity for human D2L and D3 receptors and rat NEalpha1 and 5-HT7 receptors (12). Flibanserin has different affinity for rat (> 10,000 nM)
and human (305785 nM) D2 receptors (12). The affinity for all other receptors, including
the 5-HT transporter (12), varies from low to very low (Table 1).
Flibanserin does not inhibit monoamine oxidase activity (Borsini et al., 1998).

RECEPTOR ACTIVITY
The activity of flibanserin on 5-HT1A receptors was assessed by using biochemical
(forskolin-stimulated adenylyl cyclase in vitro) and electrophysiological (firing rate after
TABLE 1. Binding profile of flibanserin
Receptor
5-HT1A
D4
5-HT2A
5-HT1A
5-HT2A
D2L
D3
NE-alpha1
5-HT7

Species
Human
Human
Human
Human/rat
Human/rat
Human
Human
Rat
Rat

Tissue
CHO
CHO
CHO
Cortex, hippocampus, dorsal raphe
Cortex
HEK293
CHO
Cortex
COS

Ki (nM)
1
424
49
1550
115133
305785
364479
523
990

> 1,000 nM
5-HT1B, 5-HT1D/E , 5-HT2C , 5-HT6, 5-HTT, D1, H1, opioid-mu, opioid-kappa, sigma, Na+ channel/site 2
> 10,000 nM
5-HT3, 5-HT4, NE-alpha2, NE-beta1, NE-beta2, H2, M1, M2, M3, D5, BDZ, NMDA/MK-801, NMDA/
glycine, NMDA/phencyclidine, nicotinic, A1, Na+ channel/site 1, Ca2+ chanel/type L, estrogen, progesterone, testosterone, GABA-A/agonist site, GABA-A/CL channel, insulin, NK-1, opiod-delta, rat D2
CHO, Chinese Hamster Ovary cells; COS, CVI origin CV 40 cells. Where two Ki values are reported,
they represent the lowest and highest Ki values obtained in two or more experiments. See ref. 19 and 50
for methods in human 5-HT2A and 5-HT1A receptors in cells, respectively. These experiments were
conducted by G. B. Schiavi at Boehringer Ingelheim, Italy, and Cerep, France. See references 33, 48,
and 67 for methods in human dopamine receptors. These experiments were conducted by J. Mierau
and G. B. Schiavi of Boehringer Ingelheim. See references 43 and 45 for methods on 5-HT1A and
5-HT2A in human tissues. These experiments were conducted by D. Marazziti and L. Palego of University of Pisa, Italy. See references 52 and 62 for methods on NK1 and glucocorticoid receptors, respectively. These experiments were conducted by Cerep, France. See reference 12 for methods for all
other receptor studies.

CNS Drug Reviews, Vol. 8, No. 2, 2002

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119

microiontophoretic application) indices. Activity at dopamine D4 receptors was investigated using various biochemical parameters (adenylyl cyclase, microphysiometry, and
GTP-shift).
Activity on 5-HT2A receptors was assessed biochemically by determining the phosphatidylinositol turnover.

Activity at 5-HT1A Receptors


Flibanserin behaves as a 5-HT1A agonist in cloned cells and the cortex and hippocampus of human and rat brain (Table 2). In the dorsal raphe, flibanserin behaves as an agonist when firing rate was taken as an index of activity in rats (58) but was devoid of agonist activity when forskolin-stimulated cAMP was measured in human tissue (44).
In contrast to the dorsal raphe nucleus of rats (24), 5-HT1A receptors in the dorsal raphe
nucleus of humans appears to be linked with adenylyl cyclase (44). In human tissues, the
EC50 of flibanserin in reducing forskolin-stimulated cAMP formation is similar to the affinity values for 5-HT1A receptors (Table 1 and 2). In contrast, the EC50 of flibanserin in
reducing forskolin-stimulated cAMP formation in cells or rat tissues is different from the
affinity values for 5-HT1A receptors. The coupling system between the receptor and the
G-proteins is important to demonstrate activity for agonists (55). Thus, it may be that the
relative amount of various G-proteins present in human tissue is different from that
present in CHO cells or rat tissue. Flibanserin binds to only one site in human 5-HT1A receptors (44), whereas it binds to high- and low-affinity sites in rodent 5-HT1A receptors
(12). Thus, a different proportion of high-/low-affinity sites might explain the different
values between 5-HT1A affinity and EC50 on cAMP. Nevertheless, flibanserin is more
potent in reducing cAMP in the hippocampus than in the cortex, both in rats and humans.
In contrast, when electrophysiology is considered, flibanserin is more potent in the cortex
than the hippocampus (Table 2).

TABLE 2. Activity of flibanserin on cAMP in vitro and on firing rates


after microiontophoretical application in vivo
Tissue
CHO cells
Cortex
Hippocampus
Dorsal raphe
Cortex
Hippocampus
Cortex
Hippocampus
Dorsal raphe

Parameter
cAMP
cAMP
cAMP
cAMP
cAMP
cAMP
Firing rate
Firing rate
Firing rate

Species
Human
Human
Human
Human
Rat
Rat
Rat
Rat
Rat

Effect
Full agonism
Agonism
Agonism
No agonism
Full agonism
Full agonism
Full agonism
Partial agonism
Full agonism

EC50 (nM)
457
28
4

913
317

IT50 (nC)

1,260
1,365
260

EC50 indicates the concentration that reduces forskolin-induced cAMP formation by 50% in rat tissue
(12), and in CHO cells (experiment was conducted by E. Giraldo of Boehringer Ingelheim, Italy). See
reference 44 for methods in CHO cells and reference 55 for methods in human tissue. IT50 indicates
the current x seconds that suppresses firing rate by 50% (58).

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The effect of flibanserin in 5-HT1A cloned cells, in human and rat cortex seems to be
mediated by stimulating 5-HT1A receptors (Table 3), as shown by the ability of all the
5-HT1A antagonists we used to reduce the effect of flibanserin. However, it is less clear
whether 5-HT1A receptors mediate the effects of flibanserin in the hippocampus (Table 3),
as suggested by the observation that two out of five 5-HT1A antagonists failed to antagonize flibanserin (17,44,58). Other purported 5-HT1A agonists, such as 8-OH-DPAT and
buspirone, failed to inhibit cAMP in the rat cortex (12) or human hippocampus (44), respectively, so 8-OH-DPAT and buspirone were used as antagonists in these two regions in
our studies.

Activity on D4 Receptors
As far as the effects of flibanserin on dopamine D4 receptors are concerned, these were
only tested in cloned cells (Table 4). Flibanserin behaved as an antagonist of forskolinstimulated cAMP when dopamine D4 receptor density was < 450 fmol/mg protein or as a
full agonist when flibanserin concentrations were very high (> 30 M). Flibanserin behaved as a partial agonist (microphysiometry and GTP shift) when dopamine D4 receptor
density was > 750 fmol/mg protein, which is much higher than that has been reported in
the human brain (0.250 fmol/protein; 49,63). In an attempt to evaluate the affinity/activity of flibanserin for dopamine D4 receptors directly in human brain, the binding of the
suggested dopamine D4 ligand nemonapride was investigated in human postmortem
cortex and striatum. However, no dopamine D4 receptors were detected in the cortex and
caudatum of nonpsychiatric subjects (D. Marazziti, University of Pisa, Italy, personal
communication, January 2001).

Activity at 5-HT2A Receptors


Flibanserin behaves as a 5-HT2A-receptor antagonist. In fact, it does not enhance PI
turnover per se and antagonized 5-HT-stimulated turnover in mouse cortex (12). The affinity values for tissue 5-HT2A receptors (115133 nM) and the Ki in reducing 5-HTstimulated turnover (113 nM) are in good agreement.

Activity at 5-HT7 Receptors


Flibanserin (up to 1 M) did not modify relaxation induced by the 5-HT7 agonist
5-carboxamidotryptamine in ileum that was contracted by administration of substance P
(41). At higher concentrations, flibanserin reduced substance P contractions (41).

IN VIVO BINDING TO 5-HT1A AND 5-HT2A RECEPTORS


Information from in vitro binding studies is limited by the fact that the endogenous
neurotransmitter is not present. The presence in vivo of 5-HT, which has higher affinity for
5-HT1A than 5-HT2A receptors (27), may differently influence the binding of flibanserin to
these receptors. Thus, an experiment was conducted to evaluate the in vivo binding of
flibanserin (Table 5). Flibanserin, in spite of different in vitro affinity values, occupies

CNS Drug Reviews, Vol. 8, No. 2, 2002

Effect of 5-HT1A antagonists


Parameter

Species

WAY100135

CHO cells

cAMP

Human

Antagonism

Cortex

cAMP

Human

Antagonism

Cortex

cAMP

Rat

Cortex

Firing rate

Rat

Hippocampus

cAMP

Human

Hippocampus

cAMP

Rat

Hippocampus

Firing rate

Rat

Tissue

WAY100635

Tertatolol

Pindobind

BMY7378

Buspirone

8-OH-DPAT

FLIBANSERIN

TABLE 3. Effects of various 5-HT1A antagonists on activity of flibanserin on cAMP in vitro


or firing rate after microiontophoretic application in vivo

Antagonism
Antagonism
No antagonism

Antagonism

Antagonism
No antagonism

Antagonism

Data are from references 15, 17, 44, and 58. The data for WAY100135 in CHO cells were obtained by E. Giraldo and R. Targa, Boehringer Ingelheim, Italy, with
a commercially available enzyme immunoassay kit. WAY100635, cyclohexanecarboxamide, N-[2-[4-(2-methoxyphenyl)-1-piperaziyl]ethyl]-N-2-pyridinyl-,
trihdrochloride; BMY 7378, 8-azaspiro[4, 5]decane-7,9-dione, 8 [2-[4-(2-methoxyphenyl]-1-piperazinyl]ethyl]-, dihydrochloride; Pindobind, N1-bromoacetyl-N8-3(4-indolyloxy)-2-hydroxy-propyl-[Z]-1,8-diamino-p-menthane.

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Antagonism
Antagonism

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BORSINI ET AL.

5-HT1A and 5-HT2A receptors in vivo in similar percentages (60). In addition, flibanserin
preferentially occupies 5-HT1A and 5-HT2A receptors in the cortex at doses as low as
1 mg/kg. It has been reported that a full agonist needs to occupy about 20% of receptors to
induce an effect (47,66). In contrast, antagonism is evident with at least 70% of receptor
occupancy (28,38). Thus, flibanserin should be capable of triggering 5-HT1A-mediated

TABLE 4. Effect of flibanserin on cells cloned with human dopamine D4 receptors

Test
Adenylyl
cyclase

Microphysiometry
GTP shift
GTP shift

Receptor density
(cellfmol/mg
protein)

Effect

CHO-446

Flibanserin behaved as an antagonist (Ki = 339 nM) against 1 M dopamine-induced suppression of 30 M forskolin (FSK)-stimulated
cAMP concentration. At the same experimental conditions, clozapine
antagonized dopamine effects (Ki = 115 nM). However, flibanserin behaved as an agonist (about 80% inhibition of FSK-induced cAMP) at
concentrations > 30 M. Dopamine (EC50 = 346 nM) and pramipexole
(EC50 = 34 nM) reduced FSK-induced cAMP by about 80%
CHO-780
Flibanserin behaved as a partial agonist (Ki = 54 nM), displaying only
39% of the efficacy of pramipexole. Flibanserin behaved as an antagonist (IC50 = 83 nM) against pramipexole
CHO-780
Flibanserin behaved as partial agonist; its Ki shifted 6.1 times in the
presence of Gpp(NH)p. At the same experimental conditions, the Ki of
pramipexole shifted 96 times and that of haloperidol 0.6 times
Sf9-860/900 Flibanserin behaved as partial agonist; its Ki shifted 8.5 times in the
presence of Gpp(NH)p. The Ki of pramipexol shifted 25.5 times and that
of haloperidol 0.9 times

The data on cAMP were obtained by E. Giraldo and R. Targa, Boehringer Ingelheimm, Italy (see ref. 2
for methods). Other data were provided by J. Mierau and H. Ensinger of Boehringer Ingelheim,
Germany (see ref. 48 for methods).

TABLE 5. 5-HT1A and 5-HT2A receptor occupancy by flibanserin in rats,


at 30 min after i.p. administration of the drug
Brain region

Receptor occupancy (%)

Dose of flibanserin
(mg/kg)

5-HT1A

5-HT2A

1
10
30
1
10
30
1
10
30

20
53
77
No significant occupancy
60
79
No significant occupancy
44
50

13
42
69
No significant occupancy
50
73
No significant occupancy
53
72

Cortex

Hippocampus

Brainstem

Data, modified from ref. 60.

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123

mechanisms only in the cortex at a dose of 1 mg/kg, while flibanserin doses as high as
30 mg/kg should be necessary to exert antagonist properties on 5-HT2A receptors.
Table 5 refers to an experiment performed in normal rats. In case of stress, 5-HT levels
may increase several fold (39,64), and thus 5-HT may differently compete with flibanserin
for binding to 5-HT1A and 5-HT2A receptors. In order to understand this, a computer simulation was performed in conditions where the concentration of 5-HT varied from 0 to
1 M (Table 6). When 5-HT is present in concentrations between 1 and 10 nM, flibanserin
has higher affinity for 5-HT1A than for 5-HT2A receptors. However, when 5-HT concentrations are between 100 and 1,000 nM, flibanserin should significantly occupy 5-HT2A reTABLE 6. Computer simulations describing the expected behavior of flibanserin in vivo
at various concentrations of 5-HT
Apparent flibanserin affinity values (nM)
5-HT concentration (nM)

Ki for 5-HT1A

Ki for 5-HT2A

0
1
10
100
1000

10
13
43
343
3343

100
100
104
140
500

These simulations with 5-HT1A and 5-HT2A receptors, for which 5-HT has median affinity values (Kd )
of 3 and 250 nM, respectively. Flibanserin was considered to have affinity values (Ki ) of about 10
and 100 nM for 5-HT1A and 5-HT2A receptors, respectively. The apparent Ki values of the drug for the
receptors was calculated by simulating the presence of different 5-HT concentrations (11,000 nM),
according to the following equation (valid for a competitive interaction for the same binding site):
Ki(app) = Ki(1+ [5-HT]/Kd ). This simulation was performed by T. Mennini and M. Gobbi, Mario
Negri Institute, Milan, Italy.

TABLE 7. Plasma concentrations of flibanserin after intraperitoneal administration in rats


Concentration (M) at various times
Dose (mg/kg)

1 hour

3 hours

8 hours

4
1.5
0.8
0.26
16
9.1
6.9
3.0
32
10
7.2
3.5
64
17.6
14.2
10.5
Plasma protein binding of flibanserin in rats is 97%. Thus, the calculated free concentration
of flibanserin is as follows:
Free concentration (nM) at various times
Dose (mg/kg)

1 hour

3 hours

8 hours

4
16
32
64

44
274
300
528

23
206
216
426

8
91
105
314

Values are means of 6 experiments, whose variation coefficient was 3186%.

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BORSINI ET AL.

ceptors at lower concentrations than those that are necessary to occupy 5-HT1A receptors.
According to the hypothesis that 5-HT2A antagonism should be present to favor 5-HT1A
agonism in the cortex (8), such a condition should occur for flibanserin when brain 5-HT
levels exceed 100 nM and if flibanserin concentration is between 104 and 343 nM. As
shown in Table 7, flibanserin can reach sufficient concentrations at doses from 16 mg/kg.

ELECTROPHYSIOLOGICAL EFFECTS
AFTER SYSTEMIC ADMINISTRATION
Electrophysiological data on firing rate were obtained by giving flibanserin intravenously (Table 8). The most striking activity of flibanserin was on firing rate of CA1 hippocampal neurons, where it reduced firing rate by 50% at a dose as low as 3 g/kg (A. Ceci,
Boehringer Ingelheim, data on file). To date, the effects of 5-HT1A antagonists on flibanserin action on CA1 neurons have not been evaluated. Flibanserin reduced firing rate by
50% in the dorsal raphe at 200 g/kg, in CA3 hippocampal neurons at 1.4 mg/kg, and in
cortical neurons at 3 mg/kg (58). The potency difference of flibanserin in reducing the
firing rate of neurons in the CA1 and CA3 regions may not be surprising. In fact, it has
been suggested that the 5-HT1A receptor recognition site may be different in the CA3 and
CA1 areas (3,5). It is interesting to compare these values with those necessary for 50%
5-HT1A receptor occupancy after intraperitoneal (i.p.) administration (Table 5). For this
comparison, one must consider that the bioavailability of flibanserin in rats is 35%. Thus,
the ED50 values (1.4 to 3 mg/kg, i.v.) of flibanserin in reducing the firing rate of neurons
in the CA3 region and cortex may be in line with ED50 values for receptor occupancy
(about 10 mg/kg, i.p., with higher occupancy in hippocampus). In contrast, flibanserin reduced the firing rate of the dorsal raphe neurons by 50% at 0.2 mg/kg, i.v., whereas it occupied 50% of 5-HT1A receptors in the midbrain only at a dose as high as 30 mg/kg, i.p.
However, receptor occupancy in the midbrain may not reflect receptor occupancy in the
dorsal raphe. Nevertheless, flibanserin behaved as a full agonist in the dorsal raphe nucleus and its effect was blocked by 5-HT1A antagonists (Table 9) (58; E. Esposito, Mario
Negri Sud, Italy, personal communication, 1992). The effect of flibanserin in CA3 was

TABLE 8. Effects of intravenous flibanserin on firing rate of neurons


in different brain regions in the rat
Tissue
Dose, mg/kg
0.003
0.01
0.2
0.6
1.4
3
10

Cortex

Hippocampus
CA1: 50% reduction
CA1: 80% reduction

50% reduction
80% reduction

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CA3: 50% reduction


CA3: 80% reduction

Dorsal raphe

50% reduction
100% reduction

FLIBANSERIN

125

also found to be antagonized by 5-HT1A antagonists (58). However, the effect of flibanserin on dorsal raphe and hippocampus is different. The effect on the firing rate of the
dorsal raphe is short lasting. In fact, flibanserin 5 mg/kg, s.c., reduced dorsal firing rate by
only 25% after 2 days and did not reduce it after 7 days, a consequence of receptor desensitization (57). In contrast, the same dose of flibanserin enhanced tonic activation of
postsynaptic 5-HT1A receptors in the CA3 region after 2 days administration as much as
after 7 days (57).
As far as the firing rate of cortical neurons is concerned, flibanserin-induced inhibition
seems to be mediated by 5-HT1A receptors, even if one out of three 5-HT1A antagonists
used in the cortex did not antagonize flibanserin (Table 9). The effect of flibanserin in the
cortex was not blocked by the chemical destruction of 5-HT-containing neurons by
5,7-DHT (15), which significantly depleted brain 5-HT by 80% (NE was reduced by 27%
[not significant]). Thus, it appears that flibanserin does not require 5-HT neurons to exert
its inhibitory effects on the cortex. Once more, the other 5-HT1A agonists 8-OH-DPAT and
buspirone, which were used as comparators, behaved differently from flibanserin. In fact,
in contrast with flibanserin, buspirone excited cortical neurons, and 8-OH-DPAT excited
them at low doses and inhibited them at high doses (15). Additionally, the inhibitory effects of 8-OH-DPAT were completely blocked by 5,7-DHT (15).
In addition to reducing firing rate, flibanserin also decreased the number of spontaneously active cells in the cortex (12). Flibanserin exerted a significant effect already at
2 mg/kg, i.p., and induced the maximal inhibition (about 80%) at 8 mg/kg (Fig. 1). The
maximum effect was at 6 h and a significant reduction was still observed after 24 h
(Fig. 2). When a 5-HT1A antagonist was used to evaluate whether this effect of flibanserin
was mediated by 5-HT1A receptors, the results were unclear. In fact, the 5-HT1A antagonist
N-(1,1-dimethylethyl)-4-(2-methoxyphenyl)-alpha-phenyl-[()WAY100135] (30 mg/kg,
s.c.) antagonized flibanserin only in one of the two experiments performed. The effect of
flibanserin was also antagonized by the D4 antagonist 1H-pyrrolo[2,3-b]pyridine,3-[[4(4-chlorophenyl)piperazin-1-yl]methyl] (L-745, 870) (at 30 mg/kg, i.p., but not at 10 mg/kg).
Thus, the effect of firing rate appears to be mediated by 5-HT1A receptors, whereas the
effect on reduction of spontaneously active cells appear to be mediated by dopamine D4
receptors and probably 5-HT1A receptors (Fig. 3).
Flibanserin, 5 mg/kg, administered s.c. for 2 days, reduced the electrophysiological effects of the 5-HT2 agonist ()-2,5-dimethoxy-4-iodoamphetamine [DOI] by 33% when applied microiontophoretically into the cortex (57). This antagonism towards DOI disappeared
after 7 days. However, it is questionable whether the antagonistic activity of flibanserin

TABLE 9. Effect of various 5-HT1A antagonists, given intravenously, on the reduction


of firing rates induced by intravenous flibanserin
5-HT1A antagonists
Tissue
Cortex
Hippocampus CA3
Dorsal raphe

WAY100135

WAY100635

Tertatolol

Antagonism

Potentiation
Antagonism
Antagonism

Antagonism
Antagonism

Data from references 15 and 58 and from E. Esposito, Mario Negri Sud, Italy, personal communication.

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BORSINI ET AL.

Number of spontaneously active


cells in the cortex

126

12
10

8
6
4

**

2
0

0 mg/kg

0.5 mg/kg

2 mg/kg

8 mg/kg

**

16 mg/kg

Dose

Fig. 1. Effect of flibanserin on the number of spontaneously active cells in the cortex of rats. Columns represent
mean S.E.M. from 4 rats. The method was according to Ceci et al. (22). The effect of flibanserin was observed
6 h after i.p. administration. *P < 0.05; **P < 0.01. The experiment was performed by A. Ceci, Boehringer
Ingelheim, Italy.

Number of spontaneously active cells


in the cortex

12
10

Vehicle

Flibanserin

**
4

**

2
0
0.5 h

3h

6h

Time

24 h

48 h

Fig. 2. Time-course of the effect of flibanserin on the number of spontaneously active cells in the cortex of rats.
Columns represent mean S.E.M. from 4 rats. The method was according to Ceci et al. (22). Flibanserin was
given i.p. at 8 mg/kg. *P < 0.05; **P < 0.01. This experiment was performed by A. Ceci in BI, Italy.

versus DOI is mediated by blockade of 5-HT2A receptors. In fact, consistent 5-HT2A receptor occupancy by flibanserin is obtained after only 30 mg/kg. Furthermore, 5-HT1A receptor stimulation has been reported to reduce 5-HT2-dependent events (26,65). Thus, the antagonism of flibanserin versus DOI may reflect the 5-HT1A agonist activity of flibanserin.

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127

Number of cells per track

12
10

8
6

Vehicle
Flibanserin

4
2
0

Vehicle

WAY100135

L-745,870

Fig. 3. Effects of WAY100135 and L745870 on the flibanserin-induced reduction of spontaneously active cells
in the cortex of rats. Columns represent mean S.E.M. from 4 rats. The method was according to Ceci et al. (22).
Flibanserin was administered i.p. 3 h before recording. WAY 100135 (30 mg/kg) and L645870 (30 m/kg) were
given i.p. 30 min before recording. #P < .01 vs. respective vehicle. *P < .01 two-way ANOVA. This experiment
was performed by A. Ceci, Boehringer Ingelheim, Italy.

EFFECTS ON TURNOVER
AND EXTRACELLULAR LEVELS OF 5-HT
Flibanserin exerts a preferential action in the cortex (Table 10). In fact, flibanserin,
2 mg/kg, reduces 5-HT turnover in the cortex without altering it in hippocampus and
brainstem (18). This preferential activity on the cortex distinguishes flibanserin from the
other 5-HT1A receptor agonists, buspirone and 8-OH-DPAT, which reduce 5-HT turnover
to a similar extent in the cortex, hippocampus, and brainstem (18). The effect of 8-OH-DPAT
and buspirone was interpreted to result from somatic presynaptic 5-HT1A receptor activation, which inhibits 5-HT turnover in all the 5-HT-containing axons departing from the
raphe nuclei. Flibanserin begins to inhibit 5-HT turnover in the hippocampus at 16 mg/kg,
this dose is 8 times higher than the dose necessary to begin to reduce 5-HT turnover in the
cortex. In contrast, flibanserin did not consistently affect 5-HT turnover in the brainstem,
where raphe nuclei are located. Thus, it is not clear why flibanserin reduces 5-HT turnover
in the cortex at doses lower than those required to induce the same effect in the hippocampus. Flibanserin reduced 5-HT turnover in the cortex by 50% at 8 mg/kg i.p., which
fits well with its occupancy of cortical 5-HT1A receptors. 5-HT1A receptors are not located
on cortical or hippocampal nerve endings (35). Thus, it may be that flibanserin stimulates
5-HT1A receptors in the cortex, which in turn trigger a negative feedback on 5-HT release.
Such negative feedback has been shown to occur in the cortex (20,21,34). This may explain the preferential effect of flibanserin only on those neurons that project to the cortex.
Such feedback mechanisms seem to work less efficiently in the hippocampus.
A preferential effect of flibanserin on the cortex was observed also in microdialysis experiments. A reduction in cortical extracellular 5-HT levels was observed already with flibanserin, 3 mg/kg, i.p. (Table 10). Cortical extracellular 5-HT levels were reduced by

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about 50% at a dose of 10 mg/kg, i.p., which fits well with the occupancy of cortical
5-HT1A receptors by flibanserin. Extracellular levels of 5-HT were not changed in the hippocampus by flibanserin at doses that reduced extracellular 5-HT levels in the cortex.
Presynaptic 5-HT1B/D receptors have been reported to inhibit 5-HT release (40). However,
flibanserin has low affinity for these receptors. Thus, the microdialysis experiments also
support the hypothesis that flibanserin may trigger a feedback mechanism on 5-HT cortical neurons through stimulation of cortical postsynaptic 5-HT1A receptors. Electrophysiological experiments showed that very close systemic doses of flibanserin stimulate
5-HT1A receptors in the cortex and the CA3 to a similar extent. Thus, experiments with
microdialysis also support the notion that the negative feedback does not work well in hippocampus. However, another possible hypothesis might be that flibanserin mainly acts on
those 5-HT1A somatic autoreceptors located on dorsal raphe neurons that specifically
project to the cortex. In this case, the occupancy of 5-HT1A receptors in the midbrain (44%
at 10 mg/kg, i.p.) might not reflect the real receptor occupancy in the dorsal raphe. The
same holds for the data on 5-HT synthesis that were obtained from all the brainstem.
When the microdialytic probe was inserted into the dorsal raphe, however, a reduction of
5-HT extracellular concentration was not observed at the dose of 3 mg/kg, which induced
5-HT extracellular reduction in the cortex. Flibanserin significantly reduced 5-HT
extracellular levels in the dorsal raphe only at 10 mg/kg, i.p. Buspirone and 8-OH-DPAT,
which have been reported to be active on somatic 5-HT1A autoreceptors, behave differently from flibanserin. In fact, both buspirone and 8-OH-DPAT reduce 5-HT synthesis and

TABLE 10. Effects on 5-HT turnover and extracellular levels of 5-HT, NE, DA, and GABA
in various brain regions at 45 min after i.p. administration of flibanserin to rats
Brain regions

Dose
(mg/kg)
1
2
4
8
16
32
3

10

5-HT turnover

Microdialysis
5-HT levels
NE levels
DA levels
GABA levels
5HT levels
NE levels
DA levels
GABA levels

Cortex
No significant reduction
20% reduction
28% reduction
50% reduction
69% reduction
68% reduction

Hippocampus
No significant reduction
No significant reduction
No significant reduction
No significant reduction
18% reduction
24% reduction

30% reduction
No effect
30% increase
No significant increase
No effect
50% reduction
No effect
90% increase
100% increase
No effect

Brainstem/dorsal raphe
No significant reduction
No significant reduction
No significant reduction
No significant reduction
38% reduction
No significant reduction
No significant reduction

70% reduction

Data on 5-HT turnover are from ref. 18. Data on microdialysis are from R. Invernizzi, Mario Negri
Institute, Milan, Italy, personal communication. Brainstem was used for the 5-HT turnover, and dorsal
raphe was used for extracellular 5-HT concentrations.

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extracellular levels in the terminal regions and dorsal raphe at the same doses (18,37).
Thus, the hypothesis that flibanserin may primarily interact with dorsal raphe neurons to
exert its neurochemical effects is not so clearly supported by experimental findings.
Flibanserin, at 3 mg/kg, increased the extracellular concentration of norepinephrine
(NE). Since a direct action of flibanserin on noradrenergic receptors may be excluded
(Table 1), it may be that this elevation of NE (and DA, see below) levels is secondary to
the reduced release of 5-HT. In fact, it has been reported that 5-HT may inhibit the release
of NE and DA in the cortex (53,59,61).
The effects of flibanserin at 10 mg/kg in increasing cortical NE and DA are very clear,
as this increase reaches about 100%. However, the increase in DA lasts for a shorter time
than that of NE (Table 11). This increase in DA might explain the dopamine D4-mediated
effects of flibanserin in reducing the number of spontaneously active cells. In fact, these
effects were also exerted to the same extent by the full dopamine D4 agonist pramipexole
(A. Ceci, Boehringer Ingelheim, personal communication, 1998), but the biochemical data
indicated that flibanserin is a partial agonist on dopamine D4 receptors. Thus, it seems
conceivable that flibanserin reduces the number of spontaneously active cells via increased release of DA, which in turn activates dopamine D4 receptors.

SUMMARY OF FUNCTIONAL ASPECTS


The first interaction of flibanserin with brain structures occurs in the hippocampus and
dorsal raphe, as revealed by electrophysiological experiments. The mechanism of this interaction in CA1 has not been explored. In the CA3 and dorsal raphe, flibanserin inhibits
firing rate through stimulation of 5-HT1A receptors. All these events happened at doses
below and around 1 mg/kg. From 1 up to 10 mg/kg, flibanserin interacts with the cortex.
This cortical interaction is evident electrophysiologically and biochemically, and it seems
to be mediated by 5-HT1A receptors, even if dopaminergic D4-mediated effects were also
observed. The interaction with 5-HT2A receptor is not very clear. Above 10 mg/kg, there
is also a biochemical interaction of flibanserin with the hippocampus and dorsal raphe.

TABLE 11. Duration of action of flibanserin


Test
Microdialysis: 5-HT in dorsal raphe
Microdialysis: DA in cortex
Microdialysis: 5-HT in cortex
Microdialysis: NE in cortex
Ex vivo binding in cortex
Ex vivo binding in midbrain
Ex vivo binding in hippocampus
5-HT turnover in cortex
Number of active cells/track

Dose
(mg/kg, i.p.)

Peak time
(minutes)

10
10
10
10
10
10
10
16
8

60
60
60
30
30
30
30
45
360

Duration of action
(hours)
< 1.5
< 1.5
>2
>2
<3
<3
>3
>6
> 24

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Dose (mg/kg)
32
30
16
10

4
3

2
1.4
1
0.2
0.003

68% inhibition of 5-HT synthesis


69% 5-HT2A receptor occupancy
77% 5-HT1A receptor occupancy
69% inhibition in 5-HT synthesis
42% 5-HT2A receptor occupancy
53% 5-HT1A receptor occupancy
50% inhibition of 5-HT extracellular levels
90% increase in NE extracellular levels
100% increase in DA extracellular levels
50% inhibition in 5-HT synthesis
75% reduction of active cells
28% inhibition in 5-HT synthesis
30% increase in NE extracellular levels
30% inhibition of 5-HT extracellular levels
50% inhibition of firing rate
20% inhibition in 5-HT synthesis
22% reduction of active cells

Hippocampus
24% inhibition of 5-HT synthesis
73% 5-HT2A receptor occupancy
79% 5-HT1A receptor occupancy
18% inhibition in 5-HT synthesis
50% 5-HT2A receptor occupancy
60% 5-HT1A receptor occupancy

Midbrain/dorsal raphe
72% 5-HT2A receptor occupancy
50% 5-HT1A receptor occupancy
38% inhibition in 5-HT synthesis
53% 5-HT2A receptor occupancy
44% 5-HT1A receptor occupancy
70% inhibition of 5-HT extracellular levels

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Cortex

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TABLE 12. Summary of the electrophysiological and biochemical effects of intraperitoneal flibanserin

50% inhibition of firing rate in CA3


20% 5-HT2A receptor occupancy
13% 5-HT1A receptor occupancy

50% inhibition of firing rate


50% inhibition of firing rate in CA1

FLIBANSERIN

131

TABLE 13. Comparison of functional effects of flibanserin, buspirone and 8-OH-DPAT


cAMP in rat cortex
cAMP in human cortex
Firing rate in rat cortex
Active cells in rat cortex
cAMP in rat hippocampus
cAMP in human hippocampus
Firing rate in hippocampus
cAMP in human dorsal raphe
Firing rate in rat dorsal raphe

Flibanserin
Inhibits
Inhibits
Inhibits
Inhibits
Inhibits
Inhibits
Inhibition in CA1
higher than in CA3
Inactive
Inhibits

Buspirone
Inactive
Inactive
Excites
Inhibits
Inhibits
Inactive
Inhibits
Inhibits

8-OH-DPAT
Inactive
Inhibits
Excites/inhibits
Inhibits
Inhibition in CA3
higher than in CA1
Inhibits

Thus, flibanserin exerts two different levels of effects in hippocampus and dorsal
raphe. The interaction of flibanserin with these two brain structures is observed electrophysiologically at low doses and biochemically at higher doses (Table 12). In contrast,
flibanserin exerts a consistent activity profile in the cortex, since both electrophysiological
and biochemical techniques reveal effects at similar doses (Table 12). This heterogeneous
picture may reflect the well-known heterogeneous pharmacology of 5-HT1A receptors
(6,7,9,56). In fact, in the dorsal raphe, flibanserin behaves as a full agonist when firing rate
is considered as an index of activity, but it is devoid of stimulant properties when adenylyl
cyclase is taken as a measure of activity. Likewise, in hippocampus, flibanserin behaves as
partial agonist when firing rate is considered, but as a full agonist when adenylyl cyclase
is measured.
Nevertheless, flibanserin seems to exert unique pharmacological properties. In fact, its
effects are different from those of two purported 5-HT1A agonists, buspirone and 8-OH-DPAT
(Table 13). However, it is difficult to understand whether this difference solely depends on
the different drug effects at 5-HT1A receptors or on other factors. In fact, buspirone and
8-OH-DPAT can bind also to dopamine D2 and 5-HT7 receptors, respectively, and flibanserin to dopamine D4 and 5-HT2A receptors.
Finally, the terminal half-life values of flibanserin in plasma were 0.9 h (i.v.) and 1.9 h
(p.o.) in rats. The half-life values are in agreement with the duration of some effects induced by flibanserin, but not with others (Table 11), such as 5-HT turnover in the cortex or
the number of active cells in the cortex. So far, it is unknown whether the metabolites of
flibanserin play a pharmacological role.

BEHAVIORAL EFFECTS
Rats seems more susceptible than mice to the inhibitory effects of flibanserin on motor
activity (14). In fact, reduced motor activity was observed at doses of 8 to 16 mg/kg, i.p.,
in rats, and no reduction of motor activity was seen with doses up to 32 mg/kg, i.p., in mice.
Flibanserin induced failure to grasp the wire in the traction test (R. Cesana, Boehringer
Ingelheim, data on file) in 50% of mice at a dose of about 45 mg/kg, i.p., or 95 mg/kg,

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p.o. At 64 mg/kg, p.o., mice also displayed hypomotility and hypothermia. These effects
were more pronounced at 128 mg/kg. At this high dose, the following additional alterations were also observed: reduced grip strength, ptosis and head weaving, and occasional
catalepsy. Some signs of a weak serotonergic syndrome were observed at 64 mg/kg, i.p.,
in rats (13).

Animal Models Sensitive to Antidepressants


Flibanserin was tested in 13 animal models sensitive to antidepressants. It was active in
the following 10 models (Table 14): learned helplessness in rats (16), bulbectomized rats
(16), chronic mild stress in rats and mice (25), muricidal rats (10), amphetamine withdrawal in rats (31), REM sleep latency reduction (W. Gaida, Boehringer Ingelheim, data
on file), forced swimming test in mice (23), distress calls in chicks (10), and fixed ratio
(FI) schedule in mice (10). In contrast, flibanserin did not show antidepressant-like behavior in the forced swimming test (10) and differential-reinforcement-of-low rate 72 sec
[DRL-72] (16) in rats and tail suspension in mice (10). Active doses ranged between 10
and 32 mg/kg, i.p., except in the chronic mild stress model in mice where activity was
seen after 2.5 and 5 mg/kg, i.p., and in REM sleep latency in rats, where an effect was observed after 1 mg/kg, p.o. In rats, the antidepressant-like effects of flibanserin occur at the
same doses that induce sedation. Thus, sedation may be a possible explanation for the
antimuricidal activity of flibanserin or the effect of flibanserin in bulbectomized rats.
However, there are some tests where flibanserin did not alter rat motor behavior, e.g.,
swimming in the Morris maze (14) or escape behavior in the learned helplessness test
(16). Flibanserin can even increase rat motor activity in amphetamine withdrawal (31).
Thus, it is difficult to ascertain to what extent sedation may be responsible for the effect of
flibanserin in muricidal or bulbectomized rats. However, flibanserin seems to induce se-

TABLE 14. Effects of flibanserin in animal models of depression


Test

Antidepressant-like effects

Rat
Learned helplessness
Bulbectomy
Chronic mild stress
Muricidal behavior
Amphetamine withdrawal
REM sleep latency
Forced swimming test
Differential reinforcement 72 sec

yes
yes
yes
yes
yes
yes
no
no

Mouse
Chronic mild stress
Fixed ratio
Forced swimming test
Tail suspension

yes
yes
yes
no

Chick
Distress-call

yes

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dation in resting animals or in animals whose motivation to explore is not high (as in the
actimeter); at the same time, it increases the drive of animals toward goal-oriented behavior, such as in the learned helplessness test (16) or the fixed ratio operant behavior
(10). Thus, one may understand why there is no motor reduction in swimming to search
for the hidden platform in the Morris maze, or why there is a higher escaping activity in
the learned helplessness test. This might also explain the contrasting finding of depressive-like effects in the forced swimming test in rats and antidepressant-like effects in
mice. Rats have lower motivation to explore the environment, since they are pretested
24 h earlier. Mice, however, are tested in the forced swimming test without pretest and
have, therefore, no familiarity with the environment. Likewise, flibanserin reduced calls in
rat pups that were pretested (54) but increased calls in chicks that were not pretested (10).
The reduction of movement power induced by flibanserin in the tail suspension test may
reflect some effects of flibanserin on spinal 5-HT1A receptors (46), since mice are suspended with the head down in the tail suspension test. However, all these thoughts are
highly speculative, and the failure of flibanserin in the forced swimming test and DRL-72
in rats, as well as in tail suspension in mice, remains difficult to explain. In fact, flibanserin is a 5-HT1A agonist and a 5-HT2 antagonist and one or both these two mechanisms
have been reported to play a role in those tests (see 10,16). This points out, once more, the
difference between flibanserin and the other 5-HT1A agonists.
In summary, flibanserin did not show antidepressant-like activity in all of the tests, and
induced different behavioral changes than those observed with imipramine (Table 15).
Only clinical trials will tell whether flibanserin will be an antidepressant. However, if
flibanserin is an antidepressant, it may be different from the classical antidepressants.
Flibanserins mechanism of action in animal models of depression was investigated
in the forced swimming test in mice (23) and learned helplessness in rats (11). In both
models, the effects of flibanserin were not reduced by brain 5-HT depletion, suggesting
that the effect of flibanserin does not require the integrity of 5-HT containing neurons.
However, whereas the effect of flibanserin seems to be mediated by 5-HT1A receptor stimulation in mice, this does not seem to occur in rats. In both models, dopamine D1 or D2 antagonists reduced the effects of flibanserin. Active doses of flibanserin in these models
ranged from 16 to 32 mg/kg. At this dose range, 5-HT1A and 5-HT2A receptors should be
occupied by approximately 50 to 70% (Table 5). Since flibanserin has lower affinity for
both, rat D1 and D2 receptors (Table 1), it is possible that flibanserin activates dopamine
receptors indirectly, through release of DA. At 10 mg/kg flibanserin has been shown to
increase dopamine release (Table 10). The effect of flibanserin in the learned helplessness

TABLE 15. Behavioral differences between flibanserin and imipramine


Test

Flibanserin

Imipramine/Fluoxetine

Learned helplessness Active after acute administration


Active after repeated administration
in rats
Chronic mild stress
Active after acute administration
Active after repeated administration
in mice
Chick-distress call
Increases calls in the protest-phase Increases calls in the frustrationphase
DRL-72
Inactive
Active after acute administration

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Immobility time (sec)

240

180

*
Vehicle - acute

120

Flibanserin - acute

60

Vehicle - Chronic Flibanserin - chronic


Fig. 4. Force swimming test in mice. Effects of an acute dose of flibanserin, 16 mg/kg, or vehicle in mice repeatedly treated with either flibanserin, 32 mg/kg, or vehicle. Values represent median with interquartile range
from 10 mice. Flibanserin or vehicle was given i.p. Vehicle or flibanserin 32 mg/kg was given from day 1 to 5
and, after 2 days of pause, from day 8 to 10. On day 11, the animals received vehicle or flibanserin 32 mg/kg
only in the morning. The challenging dose of 16 mg/kg was given on day 12. The test, as described in Cesana,
et al. (23), was performed 30 min after the challenging dose of flibanserin. This experiment was conducted by
R. Cesana, Boehringer Ingelheim, Italy. Wilcoxon test: *P < 0.01.

test was also blocked by naloxone (11). Interestingly, like many other antidepressants,
flibanserin activates -opioid receptors without inducing rewarding properties (11).
By repeated daily administration flibanserin did not produce tolerance to its antidepressant-like effects in the chronic mild stress procedure in mice (25), in bulbectomized
rats (16), and in the forced swimming test in mice (Fig. 4). In contrast to imipramine, a
subeffective dose of flibanserin remains inactive even after repeated administrations
(Fig. 5). Thus, it seems that, in contrast to imipramine or fluoxetine, flibanserin has antidepressant-like effects after a single administration and that these effects are maintained
over time. With repeated administration of flibanserin to animals, the effect of the 5-HT1A
agonist 8-OH-DPAT on body temperature is attenuated, whereas the effect on forced
swimming test is still present (Fig. 6). It has been suggested that 8-OH-DPAT induces its
effects in the forced swimming test and on body temperature by acting on 5-HT1A postand presynaptic receptors, respectively (42). Thus, it appears that flibanserin exert its antidepressant-like effect independently from involvement at 5-HT1A presynaptic receptors.
This contrasts with classical antidepressants, the 5-HT postsynaptic activity of which appears to be secondary to the downregulation of 5-HT1A presynaptic receptors (1,4).

Animal Models Sensitive to Anxiolytics


Flibanserin, at 2 to 50 mg/kg, was tested in the elevated plus maze (16), conflict test
(Table 16), and ultrasonic model (54) in rats and in the dark/light exploratory test (16) and
stress-induced hyperthermia (16) in mice. At doses ranging from 5 to 50 mg/kg s.c.,

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135

Immobility time (sec)

240

180
120

After 10 day treatment

After 5 day treatment

60

IM

IM

I+

I+

VE

VE

I
IM
+
H

VE

H
+V

EH

VE

IM

IM

I+

I+

VE

VE

I
IM
+
H

VE

VE

H
+V

EH

Immobility time (sec)

240
180
120

After 10 day treatment

After 5 day treatment

60

I+
FL

I+

VE

VE

I
FL

FL

H
VE

+V
E
H

VE

VE

FL

I+
FL

I+

FL

I
H

FL

H
VE

+V
E
H
VE

Fig. 5. Effect of a repeated administration of an acute inactive dose of flibanserin and imipramine in the forced
swimming test in mice. Values are medians with interquartile range from 10 mice. Drugs were given i.p. Mice
were given flibanserin 8 mg/kg, imipramine 8 mg/kg or vehicle twice daily from days 1 to 4. On day 5, the
forced swimming test, as described by Cesana et al. (23), was performed 30 min after drug or vehicle administration. The animals were again treated in the afternoon. After 2 days pause, the same procedure was followed
from days 8 to 12. This test was performed by R. Cesana, Boehringer Ingelheim, Italy. Wilcoxon test: *P < 0.05.

flibanserin reduced ultrasonic vocalization in rat pups, without altering body temperature
or motor activity. At 8 to 16 mg/kg i.p., flibanserin exerted anxiolytic-like effects in the
two tests in mice. It is worth noting that flibanserin induced strong sedative effects at
16 mg/kg in mice in the dark/light test, which was carried out during the dark phase of
the light/dark cycle. This contrasts with the absence of sedative effects on motor activity
in the actimer when registered during the light phase of the light/dark period. In rats,
flibanserin was devoid of any effect in the elevated plus maze, and exerted anxiogenic-like
effects in the conflict test (Table 16). The possibility that flibanserin can increase pain sensitivity (as the conflict test is based on a weak electrical shock) was evaluated in mice.

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Rectal temperature (C)

39
38.5
38
37.5
37

36.5
36

Vehicle
8-OH-DPAT

35.5
35
34.5
34

Immobility time (sec)

Chronic Vehicle
240

Chronic Flibanserin

220
200
180

Vehicle
8-OH-DPAT

160
140
120
100
Chronic Vehicle

Chronic Flibanserin

Fig. 6. Effects of 8-OH-DPAT on hypothermia and immobility test in mice repeatedly treated with 32 mg/kg
flibanserin. Values are medians with interquartile range from 10 mice. Mice were given vehicle or flibanserin
32 mg/kg i.p. twice daily from days 1 to 5 and, after 2 days pause, from days 8 to 10. On day 11, the animals received vehicle or flibanserin 32 mg/kg only in the morning. On day 12, 8-OH-DPAT 3 mg/kg was administered
s.c., and 30 min later body temperature was measured and, thereafter, the forced swimming test was performed.
This test was performed by R. Cesana, Boehringer Ingelheim, Italy. Wilcoxon test: *P < 0.05 vs. respective
vehicle; #P < 0.01 vs. 8-OH-DPAT in chronic vehicle group.

TABLE 16. Effects of flibanserin in the conflict test in rats


Number of lever presses in 3 min
Drug
Flibanserin
Flibanserin
Flibanserin
Diazepam

Dose (mg/kg p.o.)


3.7
7.4
29.6
10

Predrug session
8.5 (232.8)
19.3 (132.8)
20 (434)
17.5 (0.832.5)

Postdrug session
8.8 (0.824)
1 (0.320)*
0.5 (0.31.5)*
37.5 (16.347)*

Values are median and interquartile range (in bracket) of 17 rats. The operant conflict procedure was
closely related to the original one (30). Predrug session was conducted 1 h after oral vehicle administration and 5 h before drug administration. Postdrug session was conducted 1 h after oral flibanserin.
Sedative effects were observed after 29.6 mg/kg flibanserin. This experiment was performed by
E. Lehr, Boehringer Ingelheim, Germany. Wilcoxon test: *P < 0.01.

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137

Flibanserin exerted no algesic effects. The conflict procedure was based on food pellet delivery, thus a possible effect of flibanserin on food intake cannot be ruled out since it reduces food intake in rats at doses as low as 5 mg/kg (32). The mechanism of action of
flibanserin in animal models of anxiety has not yet been investigated.

Animal Models Sensitive to Antipsychotics


Flibanserin was studied in tests of psychostimulant-induced hypermotility (14). It reduced hypermotility induced by the s agonist (+)SKF-100147 or by d-amphetamine at doses
of 4 and 8 mg/kg, i.p. in mice and at a dose of 8 mg/kg, i.p., in rats. In these experiments,
where the animals were habituated to the actimer, flibanserin did not significantly change
motor activity per se. Flibanserin also reduced d-amphetamine-induced stereotypy at doses
of 8 and 16 mg/kg in rats and apomorphine-induced stereotypy at 16 mg/kg, i.p. (14).

Other Behavioral Effects


5-HT2A antagonism. Flibanserin, 4.1 mg/kg, antagonized head twitches induced by the
5-HT2 agonist DOI in mice (12). However, it is questionable whether this effect depends
on 5-HT2A antagonism or rather on 5-HT1A receptor activation (60).
Analgesic affect. Flibanserin antagonized 50% of phenylquinone-induced writhings in
mice at an oral dose of 10.4 (confidence limit = 2.542.9), indicating that it has a weak analgesic activity (36). Flibanserin displayed antinociceptive effects in the hot plate in mice
only at a dose as high as 128 mg/kg, p.o. At this dose, a clear reduction of motor activity
was observed, which could have affected the motor response of animals in the hot plate
test. Nevertheless, this activity was reduced by naloxone and potentiated by morphine.
Interaction with sedative compounds. At 8 mg/kg i.p., flibanserin potentiated pentobarbital-induced loss of righting reflex in mice (mean duration of the loss of righting reflex
after 40 mg/kg, i.p., pentobarbital: vehicle = 41.3 5 min; flibanserin 8 mg/kg = 69.4 6.4 min,
P < 0.05; flibanserin 32 mg/kg = 119.6 6.9 min, P < 0.01). At 16 mg/kg i.p., but not at
lower doses, flibanserin also potentiated barbital-induced loss of righting reflex in mice
(mean duration of the loss of righting reflex after 180 mg/kg, i.p., barbital: vehicle = 85.1 21.5 min; flibanserin 16 mg/kg = 202.3 28.4 min; P < 0.05; flibanserin
32 mg/kg = 289.6 35.3 min; P < 0.01). At 16 mg/kg, i.p., flibanserin also potentiated
diazepam-induced loss of righting reflex in mice (number of mice out of 10 with loss of
righting reflex after 4 or 8 mg/kg i.p. diazepam: vehicle = 2 and 4, respectively; flibanserin 16 mg/kg = 6 and 10, respectively; P < 0.05). At 100 mg/kg, p.o., but not at lower
doses, flibanserin potentiated ethanol-induced loss of righting reflex in mice (number of
mice out of 10 with loss of righting reflex after 1.6 mg/kg i.p. ethanol: vehicle = 2; flibanserin 100 mg/kg = 8; P < 0.01). However, at this dose, flibanserin did not alter ethanolinduced aerial righting reflex (see ref. 29 for methods).
The fact that flibanserin potentiated either barbital (mainly excreted as unchanged by
kidney) and pentobarbital (mainly metabolized by the liver) seems to exclude a potential
metabolic interference between flibanserin and these two barbiturates. This suggests that
flibanserin may interact with the macromolecular complex of GABA where the two barbiturates act. This hypothesis is supported also by the finding that flibanserin potentiated the

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effects of other drugs acting as agonists on this GABA macromolecular complex, such as
diazepam and ethanol. However, the interaction of flibanserin with this receptor complex
does not seem to be direct, as suggested by the low affinity of flibanserin for the benzodiazepine receptors, the GABA-A agonist site, and GABA-A Cl channel (Table 1). Flibanserin failed to increase extracellular concentrations of GABA (Table 4). Thus, flibanserin
seems to interact with GABAergic transmission indirectly.
In addition, in rats at single doses up to 32 mg/kg i.p., flibanserin does not impair
learning in a water maze (14). At doses up to 64 mg/kg i.p. (maximal dose tested) flibanserin did not produce any relevant changes in blood pressure or heart rate of conscious
rats. At doses up to 100 mg/kg p.o., flibanserin did not injure gastric mucosa or alter gastrointestinal transit in rats. In rats, at doses up to 30 mg/kg i.p., flibanserin did not alter
basal- or cholinergic stimulation-induced salivation; at 30 mg/kg p.o. it had no effect on
urinary volume or electrolytes.

CONCLUSIONS
Flibanserin is capable of modifying animal behavior at two different dosage levels
(Table 17). At doses below 10 mg/kg, it exerts prolongation of REM latency, anti-anhedonic effects, mild analgesia, facilitates exploration in the light/dark test, reduces ultrasound vocalization, antagonizes exploratory activity in bulbectomized rats, reduces psychostimulant-induced hypermotility, and potentiates barbiturate-induced loss of righting
reflex. At these doses, flibanserin seems to exert effects solely by activating 5-HT1A receptors. At higher doses (16 to 40 mg/kg) flibanserin had effects in other tests involving
interactions with other receptors.
The mechanism of action of flibanserin is not the same in all tests. It may directly or indirectly activate 5-HT1A, D1, D2, D4, and -opioid receptors. Flibanserin also increases the
function of GABA multicomplex receptors. The antagonistic activity of flibanserin at
5-HT2A receptors has been observed in biochemical and electrophysiological tests; it is
less evident in behavioral models. In most behavioral tests flibanserin exerts its maximal
activity for a short period of time, which is in agreement with its half-life. This contrasts
with its longer duration of action observed in some biochemical and electrophysiological
parameters. The only exception in behavioral tests is represented by the activity of
flibanserin in the chronic mild test model in mice, where its activity lasted 18 h (25). The
contribution of potential active metabolite(s) cannot be excluded, and this issue needs to
be elucidated.
The effects on motor activity seem to be test-related at low doses, but sedation is
clearly evident at doses above 32 mg/kg. Additionally, flibanserin does not appear to interfere with potential side effects induced by antidepressants, such as serotonergic syndrome (13) and cardiovascular effects (J. Van Ryn, Boehringer Ingelheim, data on file).
The fact that flibanserin, in contrast to other 5-HT1A agonists, reduces the activity of
cortical pyramidal cells makes this compound a unique tool to study the physiopathology
of cortical glutamatergic pyramidal cells. This fact, combined with the findings that flibanserin displays antidepressant-, antipsychotic- and probably anxiolytic-like properties,
indicates that this compound may be an interesting psychotropic drug.

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TABLE 17. The lowest doses of flibanserin that have behavioral effects or the highest doses
free of any behavioral changes
Activity
Rats
Dose Route
3
p.o. REM latency
2.5 i.p. Chronic mild stress
4
i.p.
5
8

Inactivity
Mice

s.c. Ultrasound vocalization


i.p. Reduction in motor activity

Chronic mild stress


Antagonism of amphetamine
hypermotility
Antagonism of SKF hypermotility
Light/dark exploratory test

Antagonism of amph. hypermotility


Antagonism of SKF hypermotility

10

16

Pentobarbital-induced loss of RR
s.c.
Forced swimming test
i.p. Bulbectomized rats
Hypomotility after amph. withdrawal
p.o.
Antagonism of writhings
i.p. Killer rats
Forced swimming test

20
24
30

i.p.
i.p. Learned helplessness
i.p.

32

i.p.

40
100
128

p.o.
p.o.
p.o.
p.o.

Rats or mice

Stress-induced hyperthermia
Diazepam-induced loss of RR
Barbital-induced loss of LRR

FST
FI
Ethanol-induced loss of RR
Antinociception in the hot-plate

FST
reverse

Elevated
plus maze

DRL-72
Tail suspension test
Morris
maze

FST, forced swimming test; SKF, SKF100147; amph., amphetamine; RR, righting reflex.

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