Kuballa G, Nowak P, Labus L, Bortel A,
Dabrowska J, Swoboda M, Kwiecinski A, Kostrzewa
RM, Brus R.
Department of Pharmacology,
Medical University of Silesia, Zabrze,
Poland.
Abstract : The aim of this study was
to examine behavioral and biochemical effects of
nafadotride, the new dopamine D3 receptor
antagonist, and to compare it with haloperidol
(dopamine D2 receptor antagonist) and clozapine
(predominate dopamine D4 receptor antagonist).
Each drug was injected to adult male Wistar rats
intraperitoneally, each at a single dose and for
14 consecutive days. Thirty minutes after single
or last injection of the examined drugs, the
following behavioral parameters were recorded:
yawning,
oral activity, locomotion, exploratory activity,
catalepsy and coordination ability. By HPLC/ED
methods, we determined the effects of the
examined antagonists on the levels of biogenic
amines in striatum and hippocampus: dopamine
(DA), 3,4-dihydroxyphenylacetic acid (DOPAC),
homovanillic acid (HVA), 3-methoxytyramine
(3-MT), 5-hydroxytryptamine (5-HT),
5-hydroxyindoleacetic acid (5-HIAA) and
noradrenaline (NA). Additionally, DA and 5-HT
synthesis rate was determined in striatum and
5-HT in hippocampus. The results of the study
indicate that nafadotride, the dopamine D3
receptor antagonist, has a behavioral and
biochemical profile of action different from
that of haloperidol but partially similar to
that of clozapine.
Introduction
Dopamine (DA) receptors in the central
nervous system attract significant scientific
interest due to their possible involvement in
several psychiatric and neurodegenerative
disorders. Initially, DA receptors were divided
into D1 and D2 subtypes, on the basis of
their
different action on adenylate cyclase
activity. In the 1990s, a third receptor subtype
designated as D3 was cloned and classified as a
subtype of the DA D2 receptor family. The D3
receptor is localized primarily in the limbic
brain structures, including nucleus accumbens
and is expressed both pre- and
post-synaptically. The DA D4 receptor has also
been recently cloned.
DA D2 receptors couple to multiple-effector
systems, including the inhibition of adenyl
cyclase activity, suppression of Ca2+ currents,
and activation of K+ currents. The effector
systems to which D3 and D4 receptors couple,
have not yet been unequivocally defined.
DA D1 and D2 receptors have been implicated
in the pathophysiology of Parkinson's disease
and schizophrenia. A correlation exists between
the average clinical dose of a neuroleptic and
its affinity for brain DA receptors, as
evaluated in the inhibition binding studies with
the D2 antagonist 3H-spiperone. Because
long-term administration of typical neuroleptics
to humans or to experimental animals can lead to
development of extrapyramidal sideeffects
(including parkinsonian-like movement disorders
and tardive dyskinesia), a group of
antipsychotic drugs, referred to as "atypical
neuroleptics", was developed.
The first atypical neuroleptic introduced
into clinical practice was clozapine, which has
higher affinity for the DA D4 receptor vs D2
receptor. Clozapine, in contrast to "typical"
antipsychotics, has low propensity to produce
extrapyramidal side effects.
DA D3 and D4 receptors raised great
interest, because of their distribution in
brain, and because they represent potential
targets for new groups of antipsychotic and
neuroleptic drugs. Among these drugs, several
new DA D3 antagonists were synthesized, like
nafadotride.
The aim of the present study was to examine
behavioral and biochemical effects of the new
central DA D3 receptor antagonist, nafadotride,
and to compare its effects with those of
halopendol (DA D2 receptor antagonist) and
clozapine (predominate DA D4 receptor
antagonist) in rats.
Behavioral study
Yawning behavior
and oral activity : After 30 mm
of acclimation, each rat was observed for the
next 60 min, and numbers of
yawns and
oral movements were counted.
Irritability : After
completing the above-described observations, the
irritability was assessed by a scored test
according to Nakamura and Thoenen.
Locomotor activity : Locomotor
activity was determined on separate groups of
rats (given a single injection or 14-day
treatment). After 30 min of acclimation, each
rat was observed for 10 min to determine the
total time (s) that rats spent walking and
sniffing. Simultaneously, grooming time (s) was
recorded as well as numbers of rearings.
Exploratory activity : After
the 10-min observation of locomotor activity,
each rat was placed individually in the center
of a flat wooden platform, 100 cm square,
surrounded by a 40 cm high fence, to prevent
escape. The platform had 4 rows of 4 holes each,
7 cm in diameter, and 20 cm apart. The number of
times (during a 3-min period) that each rat
stuck its head beneath the intramural line, into
any hole, was counted and recorded.
Locomotor coordination : After
completing 3-min observation of exploratory
activity, each rat was placed on a wooden bar, 3
cm in diameter. The bar rotated longitudinally
at 5 rpm, and the length of time (in seconds)
each rat managed to stay on the rotating bar was
recorded. The maximum time was 300 s. This test
was carried out on each rat three times, with
one-minute intervals between tests, and the mean
time was calculated per rat.
Cataleptogenic activity :
After completing 3-min assessment of locomotor
coordination, each rat was placed on a wire mesh
screen measuring 25 x 50 cm with 1 X 1 cm
squares, and indined by 60° to the
horizontal plane. The time (in seconds) for each
rat to move any paw along at least one screen
division within 60 s (maximal catalepsy time)
was recorded. Measurements were performed 3
times with 10-min intervals. The final number
was the sum of the three measurements.
Each examined group consisted of 8
rats.
Results
Behavioral study : A single
injection of haloperidol, clozapine or
nafadotride did not influence irritability in
rats. Conversely, a 14-day treatment regimen of
haloperidol increased irritability, while a
14-day treatment regimen of nafadotride reduced
irritability, as compared to the control
group.
A single challenge dose of clozapine
significantly reduced the numbers of
yawns, while
haloperidol and nafadotride did not affect
yawning
behavior. The 14day treatment regimen of
haloperidol nonsignificantly reduced the numbers
of yawns,
while the 14-day treatment regimen of
nafadotride greatly increased
yawning
number as compared to the control group.
Oral activity decreased after either single
and multiple injections of clozapine, and
increased after 14 daily injections of
haloperidol, as compared to control group.
Haloperidol at a single dose reduced
locomotor time, while repeated (14 daily
injections) nafadotride increased locomotor
time.
Grooming time decreased after single and
multiple injections of either haloperidol or
clozapine, while nafadotride was without
effect.
Rearings were completely abolished in
animals treated with single or multiple doses of
haloperidol. Conversely, single and multiple
injections of nafatodride increased the number
of rearings, as compared to controls.
Haloperidol injected at a single and
multiple doses reduced the number of "peepings"
in the exploratory test, and the effect of a
single dose was greater than that of multiple
treatments. A single clozapine injection reduced
the numbers of peepings, while repeated
clozapine treatments (daily for 14 days)
increased a number of peepings. Nafadotride was
without effect.
Haloperidol at single and multiple doses
reduced coordination ability of rats (i.e., time
on the rotarod) as compared to controL
Conversely, clozapine increased coordination
ability, while Nafadotride was without effect.
Haloperidol and clozapine, injected once or in a
multiple dose regimen, each produced catalepsy.
Nafadotride only slightly increased catalepsy in
rats after a single treatment, but had no effect
after multiple injections.
Discussion
Nafadotride is a selective antagonist of the
DA D3 receptor, which is presented predominately
in limbic structures, mostly in the nucleus
accumbens. In other brain structures, the
density of the DA D3 receptors is 2-3 times
lower vs D2 receptors. In contrast, DA D2
receptors are situated mainly in the nucleus
accumbens, caudate putamen, olfactory tubercle
and substantia nigra. D4 receptors are situated
mainly in the hippocampus, hypothalamus, frontal
cortex and midbrain and their density is also
much lower than that of DA D2 receptors. It is
of interest that in schizophrenic patients there
is an increased number of DA D3 and D4 receptors
in brain (vs. untreated healthy individuals),
and that the number of D3 and D4 receptors
normalizes when schizophrenics are treated with
antipsychotic drugs.
The DA D3 receptor can be localized
presynaptically (autoreceptor), acting by
autofeedback inhibition to reduce DA exocytosis.
Detailed studies reveal opposite roles for the
DA D2 and D3 receptors in locomotor activity,
learning and memory. The effects depend, in
large part, on the specific agonists or
antagonists used in the studie. Generally,
classic neuroleptics reduce locomotor activity
in mammals, and we confirmed this in the present
study.
Synthesis of 7-OH-DPAT, a selective DA D3
receptor agonist, provides the opportunity to
localize the distribution of D3 receptors in
brain and to determine the function of the D3
receptor. 7-OHDPAT stimulates D3 receptors and
inhibits endogenous DA synthesis. Compared to
the positive locomotor effects of the DA D2
agonist quinpirole, 7-OH-DPAT inhibited
locomotion in rats - in agreement with our prior
results.
Subsequent to cloning of the DA D3 receptor
by Sokoloff et al., numerous antagonists, with
high or low affinity for D3 receptors, were
synthesized and tested. Among these are AJ76,
UH232 and nafadotride, which have high affinity
and high specificity. A major objective is to
identify a new generation antipsychotic drug
with high efficacy.
Nafadotride has 10-20 times higher affinity
for the D3 vs the D2 receptor. At a dose of 1.0
mg/kg, nafadotride selectively blocks the D3
receptor. At a high dose, nafadotride also
blocks DA D2 receptors. Clifford and Waddington
and Sautel et al. found that nafadotride, in
contrast to sulpiride (D2 receptor antagonist),
increased locomotor activity, grooming,
learning, and memory in rats and induced
climbing behavior in mice. Interestingly, a
similar effect on locomotor activity was
confirmed in the present experiment. When
administered daily to rats for 14 consecutive
days, nafadotride increased locomotion and
rearings, but not grooming. At very high doses
(100.0 mg/kg), nafadotride reportedly induced
catalepsy, but we failed to confirm this in our
experiment, either after single or multiple
injections (i.e. in opposition to haloperidol
and clozapine). It must be added that we
examined also the effect of another D3 receptor
antagonist, U-991 94A, on behavior in rats, and
found that U-99 194A blocked locomotor activity
and
yawning
behavior induced by 7-OH-DPAT (D3 receptor
agonist). U-99 194A also induced a moderate
degree of catalepsy and enhanced
haloperidolinduced catalepsy. However, U-99 194A
did not alter DA and DOPAC release in the
striatum of rats, as assessed by in vivo
microdialysis and in vivo voltametry .
Interestingly, in the present experiment,
long-term application of nafadotride induced
yawning
behavior. We interpret this as possible D3
receptor priming, analogous to the observed
increase in oral activity after 14-day
haloperidol treatment. Significantly, longterm
nafadotride treatment failed to induce oral
activity, a characteristic symptom of DA D2
receptor blockade in rats, comparable to
extrapyramidal effects seen in humans treated
prolongably with classic neuroleptics.
Nafadotride increased DA turnover in the
nucleus accumbens, striatum and brain cortex in
the rat, but to a much lesser extent than
haloperidol. Others found that UH232 and AJ76
increased DA and DOPAC levels in microdialysates
of striatum and nucleus accumbens. In the
present experiment, we determined biogenic amine
levels in striatum and hippocampus, as well as,
indirectly, DA and 5-HT turnover in the striatum
and 5-HT in the hippocampus by L-DOPA and 5-HTP
assay, but we did not perform in vivo
microdialysis. Nafadotride applied in single and
multiple doses did not influence DA, DOPAC, HVA
and 5-HT levels in striatum, but increased NA
content of hippocampus, following 14 daily
injections. Nafadotride did not alter L-DOPA
level in the striatum after single and multiple
injections.
In summary, we have compared behavioral and
biochemical effects of nafadotride with
clozapine, a prominent D4 receptor antagonist.
Clozapine, an atypical neuroleptic with 10 times
higher alimity for the DA D4 vs D2 receptor, is
an effective antipsychotic drug which does not
induce extrapyramidal effects. In laboratory
studies on animals, clozapine and several other
D4 antagonists induced moderate catalepsy, as we
confirmed in the present study. Clozapine did
not block amphetamine- and apomorphine-induced
stereotyped behavior, confirming that clozapine
does not alter DA exocytosis in the corpus
striatum. In contrast, clozapine blocked
amphetamine- and apomorphine-induced
hyperlocomotion. However, in DA D4 receptor
knock-out mice, clozapine failed to block
apomorphine-induced hyperlocomotion. Clozapine,
at the doses ranging from 2.0 to 20.0 mg/kg, did
increase DA and DOPAC levels in in vivo
microdialysates of rat brain. Others found no
effect of clozapine on DA release. In the
present experiment, clozapine reduced 5-HTP
level in the striatum after a single injection
only. It appears that the serotonergic system is
involved in the biological actions of
dozapine.
From our experiment, we conclude that the
pharmacological (behavioral and biochemical)
profile of nafadotride action is different from
that of haloperidol, but partially similar to
that of clozapine.
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