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mise à jour du
12 février 2006
Pharmacol Rep
2005; 57; 2;161-169
Central effects of nafadotride,
a dopamine D3 receptor antagonist, in rats.
Comparison with haloperidol and clozapine
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.

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.
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.
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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