Prolactin blood level and
apomorphine-induced yawning were studied
in rats treated with the substituted benzamide
amisulpride in association with bromocriptine or
carmoxirole; two dopamine D(2) receptor agonists
with high or low propensity to cross the
brain-blood barrier, respectively.
Administration of amisulpride produced a maximum
increase in rat serum prolactin level
(315+/-18%) vs. vehicle-treated animals
(ED(50)=0.25+/-0.017 mg/kg, s.c.). The
concurrent administration of carmoxirole or
bromocriptine completely reversed the
hyperprolactinemia induced by amisulpride (0.5
mg/kg, s.c.) (ID(50)=14.9+/-0.8 mg/kg and
0.81+/-0.03 mg/kg, respectively). Carmoxirole
(15 mg/kg, i.p.) did not affect yawning
induced by apomorphine (0.08 mg/kg, s.c.) nor
amisulpride (0.5 mg/kg, s.c.) blockade of
apomorphine-induced yawning. Conversely,
a significant increase in the number of yawns
was observed when bromocriptine (0.8 mg/kg,
i.p.) was associated with apomorphine in the
absence or presence of amisulpride. These
results suggested that a peripheral dopamine
D(2) receptor agonists could be a useful tool in
alleviating amisulpride-induced
hyperprolactinemia without possibly affecting
its central effect.
1. Introduction
The finding that low doses of amisuipride,
an atypical antipsychotic of the substituted
benzamides class, improved negative symptoms of
schizophrenia (Boyer et al., 1995) led to the
hypothesis that, at small doses, this drug could
be useful in depressive symptoms (Puech et al.,
1981). On such basis, amisulpride has been
largely and successfully employed in dysthymia
and major depression (Lecrubier et al., 1997;
Smeraldi, 1998; Ravizza, 1999). This therapeutic
property of amisulpride has been associated to
its selective antagonism for dopamine D2 and D3
autoreceptors (Perrault et al., 1997). Indeed,
as it has been amply demonstrated, the dopamine
D2-like autoreceptors are involved
in the inhibition of dopamine release,
neuronal firing and tyrosine hydroxylase
activation (Lejeune and Millan, 1995; Saud
Chagny et al., 1991; Walters and Roth, 1976).
The selective antagonism of these autoreceptors
by low doses of amisulpride should then produce
an increase in the dopaminergic neurons firing
(Di Giovanni et al., 1998) and release
(Shoemaker et al., 1997) in the limbic system,
which are accounted for the antidepressant
properties of the drug. In line with this
hypothesis, different studies conducted on
laboratory animals indicated that low doses of
amisulpride selectively block dopamine D2
autoreceptor in vivo. For instance, very low
doses of amisulpride (0.2-0.3 mg/kg) were able
to antagonize rat yawning and
hypomotility induced by pre-synaptic doses of
apomorphine, while only very high doses of
amisuipride (30-100 mg/kg) are needed to reduce
rat hypermotility and stereotypies induced by
post-synaptic doses of apomorphine (Perrault et
al., 1997; Shoemaker et al., 1997).
Although these studies showed that
amisulpride displays a pre-synaptic selectivity,
clinical studies on healthy volunteers
demonstrated that, even at small doses,
amisulpride increases prolactin blood level
(Wetzel et al., 1994), indicating that the same
doses of amisuipride which antagonize mesolimbic
dopamine autoreceptors are able, outside the
brain-blood barrier, to block pituitary dopamine
D2 postsynaptic receptors producing
hyperprolactinemia as a side effect.
The presence of dopamine D2 receptor in the
anterior and intermediate lobes of the pituitary
gland has been extensively demonstrated with
biochemical and pharmacological techniques
(Enjalbert and Bockaert, 1983; Memo et al.,
1986) and the gene encoding for both the
dopamine D25 and D2L receptor isoforms has been
later found in pituitary lactotroph cells (Dal
Toso et al., 1989; Meador-Woodruff et al., 1989;
Bunzow et al., 1988). The stimulation of
pituitary dopamine D2 receptor by dopamine
released from tuberoinfundibular neurons was
proven to reduce prolactin secretion from the
pituitary gland, as confirmed by cell culture
studies on lactotroph cells and by in vivo
evidence (Burns et al., 1991; Ben-Jonathan,
1985). The block of pituitary dopamine D2
receptors by amisulpride should then reduce the
dopaminergic inhibition of the pro lactin
release, leading to hyperprolactinemia in
patients (Wetzel et al., 1994).
The hyperprolactinemia due to different
pathological status is currently treated by
stimulating pituitary dopamine D2 receptor with
dopamine D2 receptor agonists, such as
bromocriptine, that are among the most effective
drugs able to normalize plasma prolactin levels
(Pinzone et al., 2000).
An attempt to reduce the increase in
prolactin secretion induced by benzamides class
of compounds with the use of dopamine D2
receptors agonists was carried out. For
instance, bromocriptine was administered to rat
treated with the benzamide derivative sulpiride
(Hofmann et al., 1979). However, since
bromocriptine readily crosses the brainblood
barrier, it lowered sulpiride-induced
hyperprolactinemia, but it also suppressed the
3,4-dihydroxyphenylacetic acid (DOPAC)
accumulation induced by sulpiride in the nucleus
accumbens, thus interfering with the
sulpirideinduced increase in dopamine synthesis
which is thought to be at the basis of its
action as an antidepressant (Tagliamonte et al.,
1975).
Recently, a new dopamine D2 receptor agonist
carmoxirole has been synthesized (Haase et al.,
1991). Carmoxirole was proven to reduce basal
prolactin blood levels in naive rat and showed
low propensity to cross the blood-brain barrier,
since it exerted no effect on striatal L-DOPA
accumulation up to the dose of 100 mg/kg and it
was able to affect the central levels of
biogenic amine only at high doses (Haase et al.,
1991).
In the present paper, we evaluated the
possibility that carmoxirole may reverse the
amisulpride-induced hyperprolactinemia without
affecting the central effect of this substituted
benzamide. For this purpose, we compared the
effect of carmoxirole and bromocriptine on
prolactin blood level and on apomorphine-induced
yawning in rats acutely treated with low
doses of amisulpride.
4. Discussion
When used in small doses (50-100 mg) the
benzamide derivative amisulpride possesses an
antidepressant effect (Lecrubier et al., 1997;
Ravizza, 1999) and this drug is successfully
used, at these doses, in the treatment of
negative symptoms of schizophrenia and of
dysthymia. However, like other substituted
benzamides, even low doses of amisulpride
elevate plasma prolactin levels in patients,
posing the risk of low compliance (Wetzel et
al., 1994; Von Bahr et al., 1991).
In the present work, using the rat as an
animal model, we confirmed the high propensity
of very small doses of amisulpride to induce
hyperprolactinemia. Furthermore, consistently
with other studies (Scatton et al., 1997;
Perrault et al., 1997), we found that
amisulpride (0.5 mg/kg) was able to completely
reverse apomorphine-induced rat
yawning.
Although some pharmacological or
anatomo-physiological controversies exist
concerning the different brain region involved
(Argiolas and Meus, 1998), the
apomorphineinduced yawning is generally
considered as a centrally mediated effect due to
a direct stimulation of the dopamine D2
receptor. In our condition the same doses of
amisuipride that were able to antagonize this
behavioral effect of apomorphine produced a
maximal increase in prolactin
blood level. Indeed, similarly to what has
been reported in humans (Wetzel et al., 1994),
in the rat we found a doserelated association
between the hyperprolactinemia induced by
amisulpride and the central effect mediated by
the benzamide (i.e. the antidepressant effect in
humans and the antagonism of
apomorphine-mediated yawning in
rats).
The present results indicate that low doses
of bromocriptine decreased amisulpride-induced
hyperprolactinemia, but they also reduced the
effect of amisulpride on apomorphine-induced
yawning. On the other hand, the
peripheral dopamine D2 agonist carmoxirole was
able to reduce the amisulpride-induced
hyperprolactinemia at doses much higher than
bromocriptine, but without affecting the
amisulpride ability to reduce
apomorphine-induced yawning.
The different effect of the two dopamine D2
receptor agonists on amisulpride-induced
hyperprolactinemia reflects their different
affinity for the pituitary dopamine D2
receptors, as observed in our binding study. On
the other side, the lower propensity of
carmoxirole to cross the brain-blood barrier may
explain the different effect, with respect to
bromocriptine, on amisuipride-induced central
effect. Bromocriptine at low doses is able to
reduce cocaine-induced hypermotility and is
known to alter dopamine synthesis and release in
naive rats (Brannan et al., 1993; Jackson et
al., 1995). On the other hand, carmoxirole
(10-100 mg/kg, s.c.) exerts no effect on
hypothalamic L-3,4-dihydroxyphenylalanine
(L-DOPA) accumulation and produces only a small
reduction of striatal DOPAC and homovanillic
acid (HVA) levels, suggesting that carmoxirole
is devoid of central effect at such doses (Haase
et al., 1991). Consistent with this possibility,
we found that carmoxirole did not affect
spontaneous locomotor activity up to the dose of
60 mg/kg.
Interestingly, bromocriptine affected the
apomorphine and apomorphine plus amisulpride
effect on yawning at a dose devoid per se
of any behavioral effects. A peculiarity that
was also observed using apomorphine to
antagonize cocaine behavioral arousal (Campbell
et al., 1989). It is possible that the reported
in vivo slow dissociation of bromocriptine from
the dopamine receptor may account for an
over-effect of the drug when in the presence of
a dopamine receptor stimulation or antagonism
(Bannon et al., 1980). In any case, this
property of bromocriptine is not in favor of any
possible use in reducing hyperprolactinemia
induced by amisulpride in depressive patients,
in spite of its widespread clinical use as an
anti-hyperprolactinemia agent (Pinzone et al.,
2000).
In conclusion, our result indicated that,
even in very small doses, amisuipride is able to
produce hyperprolactinemia and to reverse
centrally mediated apomorphineinduced
yawning in rats. The comparison of the
effects of carmoxirole and bromocriptine on
amisulpride-induced hyperprolactinemia and
yawning indicated that only carmoxirole
was able to restore normal prolactin blood level
without affecting the central activity of
amisulpride in rats. These results might suggest
that, in humans, the combination of carmoxirole
and amisulpride may leave intact the central
effect of this benzamide, which is relevant for
its therapeutic response in affective and
cognitive symptoms of dysthymia and negative
symptoms of schizophrenia, while antagonizing
the endocrine peripheral effect of amisulpride.
However, further studies will be needed in order
to evaluate the other actions of carmoxirole in
the periphery (such as those on blood vessels,
kidney, etc.) and particularly when given in
combination with a substituted benzamide.
