Pharmacological Reports Copyright © 2005 2005, 57, 161–169 by Institute of Pharmacology ISSN 1734-1140 Polish Academy of Sciences

Central effects of nafadotride, a D3 receptor antagonist, in rats. Comparison with and *

Grzegorz Kuballa1, Przemys³aw Nowak1, £ukasz Labus1, Aleksandra Bortel1, Joanna D¹browska1, Marek Swoboda1, Adam Kwieciñski1, Richard M. Kostrzewa2, Ryszard Brus1

Department of Pharmacology, Medical University of Silesia, Jordana 38, PL 41-808 Zabrze, Poland

Department of Pharmacology, Quillen College of Medicine, East Tennessee State University, Johnson City, TN 37614, USA

Correspondence: Ryszard Brus, e-mail: [email protected]

Abstract: The aim of this study was to examine behavioral and biochemical effects of nafadotride, the new dopamine D! receptor antagonist, and to compare it with haloperidol (dopamine D receptor antagonist) and clozapine (predominate dopamine D" 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 D! receptor antagonist, has a behavioral and biochemical profile of action different from that of haloperidol but partially similar to that of clozapine.

Key words: nafadotride, haloperidol, clozapine, behavior, brain biogenic amines, rats

different action on adenylate cyclase activity [20]. In Introduction the 1990s, a third receptor subtype designated as D3 was cloned and classified as a subtype of the DA D2 Dopamine (DA) receptors in the central nervous sys- receptor family [48, 49]. The D3 receptor is localized tem attract significant scientific interest due to their primarily in the limbic brain structures, including nu- possible involvement in several psychiatric and neu- cleus accumbens [25, 32, 48, 49], and is expressed rodegenerative disorders. Initially, DA receptors were both pre- and post-synaptically [24, 30]. The DA D4 re- divided into D1 and D2 subtypes, on the basis of their ceptor has also been recently cloned [54].

* Part of this study was presented during Twelth Days of Neuropsychopharmacology (Pol. J. Pharmacol. 2003, 55, 277–278).

Pharmacological Reports, 2005, 57, 161–169 161 DA D2 receptors couple to multiple-effector sys- Chemicals tems, including the inhibition of adenyl cyclase activ- ity, suppression of Ca2+ currents, and activation of K+ The L-aromatic acid decarboxylase inhibitor NSD currents [4]. The effector systems to which D3 and D4 re- 1015 (m-hydroxybenzylhydrazine dichloride) was pur- ceptors couple, have not yet been unequivocally defined. chased from Sigma Chemical Co. (St. Louis, MO, USA). DA D1 and D2 receptors have been implicated in Haloperidol and clozapine were purchased from Polfa the pathophysiology of Parkinson’s disease and Pharmaceutical Company (Warszawa, Poland). Nafado- schizophrenia. A correlation exists between the aver- tride was kindly provided by Dr. P. Sokoloff, Unité de age clinical dose of a neuroleptic and its affinity for Neurobiologie et Pharmacologie, Centre Paul Broca brain DA receptors, as evaluated in the inhibition de L’INSERM, Paris, France. 3 binding studies with the D2 antagonist H- [45]. Because long-term administration of typical neuroleptics to humans or to experimental animals Schedule of drug injections can lead to development of extrapyramidal side- effects (including parkinsonian-like movement disorders and tardive dyskinesia), a group of drugs, Single injection referred to as “atypical neuroleptics”, was developed. The first atypical neuroleptic introduced into clini- Rats were divided into four groups. The first group cal practice was clozapine [8, 19], which has higher (control) was injected with a single dose of saline vs affinity for the DA D4 receptor .D2 receptor. Clo- (0.9% NaCl) at 1.0 ml/kg ip. The second group was zapine, in contrast to “typical” , has low injected with haloperidol at 0.5 mg/kg ip, the third propensity to produce extrapyramidal side effects. group was given nafadotride at 0.25 mg/kg ip, and the DA D3 and D4 receptors raised great interest, be- fourth group received clozapine at 5.0 mg/kg ip.Im- cause of their distribution in brain, and because they mediately after the injection, each rat was individu- represent potential targets for new groups of antipsy- ally placed in a transparent cage in a quiet well- chotic and neuroleptic drugs [39, 41, 48]. Among ventilated and well-illuminated room to acclimatize to these drugs, several new DA D3 antagonists were syn- the laboratory environment. After 30 min, behavioral thesized, like nafadotride [26, 43]. assessment was undertaken. The aim of the present study was to examine be- havioral and biochemical effects of the new central DA D3 receptor antagonist, nafadotride, and to com- Multiple 14-day injections pare its effects with those of haloperidol (DA D2 re- ceptor antagonist) and clozapine (predominate DA D 4 Rats were divided into four groups as above. The first receptor antagonist) in rats. group (control) was injected once daily with saline (0.9% NaCl) at 1.0 ml/kg ip for 14 consecutive days. The second group was injected for 14 consecutive days with haloperidol at 0.05 mg/kg per day ip, the Materials and methods third group was given nafadotride at 0.025 mg/kg per day ip, and the fourth group was administered clozap- Animals ine at 0.5 mg/kg per day ip. All injections were per- formed daily between 9 and 10 a.m. Immediately after Adult 2–3 months old male albino Wistar rats were the last (14th) injection, each rat was placed individu- used in this study. The animals were housed six per ally in a transparent cage in a quiet well-ventilated cage at 22 ± 1oC, with an alternating 12 h/12 h and well-illuminated room to acclimatize to the labo- light/dark cycle. Rats had free access to standard food ratory environment. After 30 min, behavioral assess- pellets (Murigran, Animal Food Works, Motycz, Po- ment was undertaken. land) and filtered tap water. Care of the animals was Doses of haloperidol and clozapine for multiple ap- under the control of the Animal Facility of the Medi- plications approximated that used in humans; for sin- cal University of Silesia. All procedures used in these gle injection, the respective doses were approximately studies were approved by the Local Ethics Committee. 10 times higher.

162 Pharmacological Reports, 2005, 57, 161–169 Nafadotride – central effects in rats Grzegorz Kuballa et al.

Behavioral study measuring 25 × 50 cm with 1×1cmsquares, and in- clined by 60o to the horizontal plane. The time (in sec- Yawning behavior and oral activity onds) for each rat to move any paw along at least one screen division within 60 s (maximal catalepsy time) After 30 min of acclimation, each rat was observed was recorded. Measurements were performed 3 times for the next 60 min, and numbers of yawns and oral with 10-min intervals. The final number was the sum movements were counted [5, 21]. of the three measurements [23]. Each examined group consisted of 8 rats. Irritability Biochemical study After completing the above-described observations, the irritability was assessed by a scored test according Assay of biogenic amines to Nakamura and Thoenen [33]. Separate groups of rats injected once or for 14 days Locomotor activity were used. Thirty minutes after a single or the last in- jection, rats were decapitated by guillotine, and their Locomotor activity was determined on separate brains were immediately excised and placed on ice. groups of rats (given a single injection or 14-day The corpus striatum and hippocampus were separated, treatment). After 30 min of acclimation, each rat was placed on dry ice, weighed, and stored at –70oC, pend- observed for 10 min to determine the total time (s) ing assay of the biogenic amines. Brain specimens that rats spent walking and sniffing. Simultaneously, were homogenized in ice-cold 0.1 M trichloroacetic grooming time (s) was recorded as well as numbers of acid, containing 0.05 mM ascorbic acid. After cen- rearings. trifugation (5000 × g for 5 min), the supernatants were filtered through 0.2 mm cellulose membranes (Titan Exploratory activity MSF Microspin Filters Scientific Resources Inc., Ea- tontown, Great Britain) and the supernatant was After the 10-min observation of locomotor activity, injected onto the HPLC/ED column. Dopamine (DA), each rat was placed individually in the center of a flat 3,4-dihydroxyphenylacetic acid (DOPAC), homova- wooden platform, 100 cm square, surrounded by nillic acid (HVA), 3-methoxytyramine (3-MT), 5-hy- a 40 cm high fence, to prevent escape. The platform droxytryptamine (5-HT), 5-hydroxyindoleacetic acid had 4 rows of 4 holes each, 7 cm in diameter, and (5-HIAA) and noradrenaline (NA) were assayed in the 20 cm apart. The number of times (during a 3-min pe- striatum and hippocampus by an HPLC/ED technique riod) that each rat stuck its head beneath the intramu- [28]. Results are expressed as ng/g of wet tissue. ral line, into any hole, was counted and recorded [14]. Assay of L-dihydroxyphenylalanine (L-DOPA) and Locomotor coordination 5-hydroxytryptophan (5-HTP)

After completing 3-min observation of exploratory This experiment was performed on separate groups of activity, each rat was placed on a wooden bar, 3 cm in rats injected once or for 14 consecutive days with the diameter. The bar rotated longitudinally at 5 rpm, and examined substances. Thirty minutes after a single or the length of time (in seconds) each rat managed to the last injection, rats were injected with the aromatic stay on the rotating bar was recorded. The maximum acid decarboxylase inhibitor, NSD-1015 (100.0 mg/kg time was 300 s. This test was carried out on each rat ip) [7], and decapitated 30 min later for excision of three times, with one-minute intervals between tests, the corpus striatum and hippocampus. Brain speci- and the mean time was calculated per rat. mens were stored at –70oC until assayed. L-DOPA and 5-HTP were assayed in the striatum Cataleptogenic activity and 5-HTP in the hippocampus according to Magnus- son et al. [28]. The level of the above amino acids in After completing 3-min assessment of locomotor co- examined brain’s part expressed indirectly the DA and ordination, each rat was placed on a wire mesh screen 5-HT synthesis rate [7].

Pharmacological Reports, 2005, 57, 161–169 163 Each examined group consisted of 5 rats (tissues). nafadotride did not affect yawning behavior. The 14- day treatment regimen of haloperidol nonsignificantly Statistical analysis reduced the numbers of yawns, while the 14-day treatment regimen of nafadotride greatly increased Analysis of variance (ANOVA) and Student’s t-test yawning number as compared to the control group. were used to evaluate the differences between drug- Oral activity decreased after either single and mul- treated groups and saline-treated groups of rats. tiple 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 Results increased locomotor time. Grooming time decreased after single and multiple Behavioral study (Tab. 1) injections of either haloperidol or clozapine, while na- fadotride was without effect. A single injection of haloperidol, clozapine or nafado- Rearings were completely abolished in animals tride did not influence irritability in rats. Conversely, treated with single or multiple doses of haloperidol. a 14-day treatment regimen of haloperidol increased Conversely, single and multiple injections of nafa- irritability, while a 14-day treatment regimen of nafa- todride increased the number of rearings, as compared dotride reduced irritability, as compared to the control to controls. group. Haloperidol injected at a single and multiple doses A single challenge dose of clozapine significantly reduced the number of “peepings” in the exploratory reduced the numbers of yawns, while haloperidol and test, and the effect of a single dose was greater than

Tab. 1. Effect of single and multiple injections of haloperidol, nafadotride and clozapine on the behavior of rats

No Parameter of behavior Examined substances Saline Haloperidol Nafadotride Clozapine Single Multiple Single Multiple Single Multiple Single Multiple 1 Irritability (scores) 1.00 1.13 0.75 2.25* 1.38 0.63* 1.13 1.63 ± 0.27 ± 0.13 ± 0.25 ± 0.53 ± 0.18 ± 0.18 ± 0.23 ± 0.26 2 Number of yawns 4.13 6.75 2.50 3.38 3.75 14.00* 0.25* 5.13 ± 0.55 ± 1.28 ± 1.22 ± 1.48 ± 1.00 ± 4.39 ± 0.16 ± 1.17 3 Number of oral movements 27.75 23.25 21.25 38.00* 23.63 26.13 11.00* 9.63* ± 5.30 ± 3.90 ± 4.66 ± 6.11 ± 4.11 ± 3.66 ± 1.99 ± 1.14 4 Locomotor activity (seconds) 102.50 62.25 5.00* 81.50 127.00 209.13* 79.00 147.63 ± 38.90 ± 24.78 ± 2.79 ± 34.55 ± 27.52 ± 44.18 ± 44.77 ± 51.06 5 Gromming (seconds) 66.25 103.63 4.25* 10.25* 40.25 80.00 10.00* 46.25 ± 29.44 ± 32.30 ± 3.84 ± 4.91 ± 21.96 ± 41.06 ± 6.44 ± 18.30 6 Number of rearings 0.38 0.25 0.00* 0.00* 1.88 2.50* 0.50 0.63 ± 0.26 ± 0.16 ± 0.00 ± 0.00 ± 0.67 ± 1.05 ± 0.50 ± 0.63 7 Number of peepings 19.25 14.88 1.50* 10.50* 17.50 14.38 11.00* 18.75 ± 3.69 ± 3.20 ± 0.50 ± 1.20 ± 1.65 ± 2.52 ± 2.42 ± 2.55 8 Coordination 226.88 181.25 55.13* 62.00* 177.13 152.63 300.00 266.13 (seconds) ± 47.88 ± 50.35 ± 18.64 ± 28.88 ± 47.32 ± 46.87 ± 0.00 ± 33.88 9 Catalepsy 5.61 5.88 102.50* 26.50* 10.13* 11.25 26.00* 15.13* (seconds) ± 1.51 ± 0.97 ± 20.33 ± 6.56 ± 1.69 ± 2.77 ± 9.25 ± 3.04

Mean SEM, n = 8; * p < 0.05 as compared to the respective control groups (saline)

164 Pharmacological Reports, 2005, 57, 161–169 Nafadotride – central effects in rats Grzegorz Kuballa et al.

Tab. 2. Effect of single and multiple injections of haloperidol, nafadotride and clozapine on the level of biogenic amines (ng/g of wet tissue) in the striatum and hippocampus of rats’ brain

Examined substances Saline Haloperidol Nafadotride Clozapine Single Multiple Single Multiple Single Multiple Single Multiple Striatum DA 8057.519 8353.267 9224.498 7395.502 8665.344 8369.024 9118.352 7232.901 ± 387.673 ± 429.717 ± 416.411 ± 702.551 ± 296.513 ± 541.257 ± 680.276 ± 390.537 DOPAC 852.131 855.807 1148.263* 1198.816* 1017.3696 745.826 1028.402 582.192 ± 30.380 ± 28.917 ± 55.488 ± 36.006 ± 77.0912 ± 35.114 ± 83.040 ± 15.284 HVA 852.473 842.230 1215.898* 1240.399* 940.501 889.816 915.872 848.466 ± 63.545 ± 16.176 ± 64.451 ± 207.670 ± 87.365 ± 32.832 ± 68.840 ± 31.133 5-HT 251.561 372.016 261.248 332.430 283.997 383.172 317.430 362.440 ± 16.623 ± 20.528 ± 17.892 ± 11.076 ± 14.125 ± 24.471 ± 24.074 ± 23.960 5-HIAA 340.479 357.168 418.865 311.511 520.225 299.309 552.092 312.133 ± 11.328 ± 24.245 ± 54.355 ± 15.727 ± 23.717 ± 12.122 ± 51.528 ± 21.721 NA 221.684 268.002 207.677 255.912 266.331 349.315 258.194 437.686 ± 42.647 ± 39.578 ± 21.097 ± 26.118 ± 26.550 ± 85.511 ± 30.365 ± 116.690 Hippocampus 5-HT 262.728 280.430 210.967* 282.005 230.030 330.918 219.469 336.957 ± 25.419 ± 12.324 ± 25.419 ± 16.557 ± 9.695 ± 37.775 ± 10.092 ± 23.021 5-HIAA 217.392 300.341 195.068 299.293 203.590 320.168 198.595 316.036 ± 10.519 ± 23.178 ± 10.340 ± 29.225 ± 14.233 ± 55.491 ± 8.878 ± 15.565 NA 438.358 426.461 406.184 486.900* 431.120 556.318* 382.853 467.866 ± 19.658 ± 9.335 ± 10.031 ± 19.741 ± 29.254 30.183 ± 15.002 ± 36.038

Mean ± SEM, n = 5; * p < 0.05 as compared to the respective control groups (saline)

that of multiple treatments. A single clozapine injec- the control. Nafadotride and clozapine nonsignifi- tion reduced the numbers of peepings, while repeated cantly increased the level of 5-HIAA in the striatum clozapine treatments (daily for 14 days) increased after a single injection. All three examined substances did number of peepings. Nafadotride was without effect. not alter the levels of DA and NA in the striatum after Haloperidol at single and multiple doses reduced coor- a single injection, as compared to the respective controls. dination ability of rats (i.e., time on the rotarod) as com- Fourteen consecutive daily injections of haloperi- pared to control. Conversely, clozapine increased coordi- dol also increased the DOPAC and HVA levels in the nation ability, while nafadotride was without effect. striatum as compared to control. All three examined Haloperidol and clozapine, injected once or in a mul- substances, however, did not alter the striatal tissue tiple dose regimen, each produced catalepsy. Nafado- levels of DA, 5-HT, 5-HIAA and NA after multiple tride only slightly increased catalepsy in rats after a sin- treatments. gle treatment, but had no effect after multiple injections. Haloperidol reduced the 5-HT level in the hippo- campus after a single injection. A single injection of Biochemical study haloperidol, clozapine or nafadotride failed to alter the level of 5-HIAA and NA in the hippocampus. Fourteen consecutive daily injections of nafado- Biogenic amines level (Tab. 2) tride or haloperidol increased the NA level only in the hippocampus, and was without effect on 5-HT and Haloperidol increased the levels of DOPAC and HVA 5-HIAA levels. DA level in the hippocampus was on in the striatum after a single injection, as compared to the border of detection (results were not presented).

Pharmacological Reports, 2005, 57, 161–169 165 Tab. 3. Effect of single and multiple injections of haloperidol, nafadotride and clozapine on L-dihydroxyphenylalanine (L-DOPA) and 5-hydroxytryptophan (ng/g of wet tissue) in the striatum and hippocampus of rats’ brain

Part of the brain Amino acid Examined substances Saline Haloperidol Nafadotride Clozapine Single Multiple Single Multiple Single Multiple Single Multiple Striatum L-DOPA 1200,3 1170.9 3218.9* 2750.4* 1513.1 1098,1 1316.3 1079.1 ± 109.3 ± 98.3 ± 275.7 ± 457.9 ± 140.2 ± 176.0 ± 123.7 ± 120.7 5-HTP 157.2 159.9 87.3* 142.4 160.6 148.2 110.4 160.6 ± 24.1 ± 20.3 ± 16.6 ± 8.6 ± 14.7 ± 20.3 ± 11.9 ± 21.8 Hippocampus 5-HTP 108.2 131.4 101.7 149.3 127.2 127.4 110.1 131.6 ± 5.4 ± 14.5 ± 3.5 ± 23.8 ± 10.4 ± 11.5 ± 12.6 ± 9.8

Mean SEM, n = 5; L-DOPA – L-dihydroxyphenylalanine, 5-HTP – 5-hydroxytryptophane, *p < 0.05 as compared to the respective control groups (saline)

L-dihydroxyphenylalanine (L-DOPA) and The DA D3 receptor can be localized presynapti- 5-hydroxytryptophan (5-HTP) levels (Tab. 3) cally (autoreceptor), acting by autofeedback inhibi- tion to reduce DA exocytosis [44, 55]. Detailed stud- Single and multiple injections of haloperidol in- ies reveal opposite roles for the DA D and D recep- creased L-DOPA level in the striatum of rats. Con- 2 3 tors in locomotor activity, learning and memory. The versely, only a single injection but not multiple treat- effects depend, in large part, on the specific agonists ments with haloperidol reduced the 5-HTP level in or antagonists used in the studies [47, 52, 57]. Gener- striatum. ally, classic neuroleptics reduce locomotor activity in Single or multiple treatments with haloperidol, clo- mammals [50, 51], and we confirmed this in the pres- zapine, or nafadotride did not significantly alter 5-HTP ent study. level in the hippocampus. Synthesis of 7-OH-DPAT, a selective DA D3 recep- tor agonist, provides the opportunity to localize the distribution of D3 receptors in brain [27] and to deter- mine the function of the D3 receptor [12]. 7-OH- Discussion DPAT stimulates D3 receptors and inhibits endoge- nous DA synthesis [15]. Compared to the positive lo- comotor effects of the DA D2 agonist , Nafadotride is a selective antagonist of the DA D3 re- ceptor, which is presented predominately in limbic 7-OH-DPAT inhibited locomotion in rats [52, 56, 57] structures, mostly in the nucleus accumbens [44, 58]. – in agreement with our prior results [36]. Subsequent to cloning of the DA D receptor by In other brain structures, the density of the DA D3 re- 3 vs Sokoloff et al. [48], numerous antagonists, with high ceptors is 2–3 times lower .D2 receptors [11, 16]. In or low affinity for D receptors, were synthesized and contrast, DA D2 receptors are situated mainly in the 3 nucleus accumbens, caudate putamen, olfactory tu- tested. Among these are AJ76, UH232 [53, 56] and nafadotride [43], which have high affinity and high bercle and substantia nigra [11]. D4 receptors are situ- ated mainly in the hippocampus, hypothalamus, fron- specificity. A major objective is to identify a new gen- tal cortex and midbrain [11] and their density is also eration antipsychotic drug with high efficacy. much lower than that of DA D2 receptors. It is of in- Nafadotride has 10–20 times higher affinity for the vs terest that in schizophrenic patients there is an in- D3 . the D2 receptor [17]. At a dose of 1.0 mg/kg, creased number of DA D3 and D4 receptors in brain nafadotride selectively blocks the D3 receptor [47]. At (vs. untreated healthy individuals), and that the a high dose, nafadotride also blocks DA D2 receptors. number of D3 and D4 receptors normalizes when Clifford and Waddington [9] and Sautel et al. [43] found schizophrenics are treated with antipsychotic drugs that nafadotride, in contrast to (D2 receptor [18, 37, 46]. antagonist), increased locomotor activity, grooming,

166 Pharmacological Reports, 2005, 57, 161–169 Nafadotride – central effects in rats Grzegorz Kuballa et al.

learning, and memory in rats; and induced climbing tipsychotic drug which does not induce extrapyrami- behavior in mice. Interestingly, a similar effect on dal effects [34]. In laboratory studies on animals, locomotor activity was confirmed in the present clozapine and several other D4 antagonists induced experiment. When administered daily to rats for 14 moderate catalepsy [34], as we confirmed in the pres- consecutive days, nafadotride increased locomotion ent study. Clozapine did not block amphetamine- and and rearings, but not grooming. At very high doses -induced stereotyped behavior, confirm- (100.0 mg/kg), nafadotride reportedly induced cata- ing that clozapine does not alter DA exocytosis in the lepsy [43], but we failed to confirm this in our experi- corpus striatum. In contrast, clozapine blocked am- ment, either after single or multiple injections (i.e. in phetamine- and apomorphine-induced hyperlocomo- opposition to haloperidol and clozapine). It must be tion [34]. However, in DA D4 receptor knock-out added that we examined also the effect of another D3 mice, clozapine failed to block apomorphine-induced receptor antagonist, U-99194A, on behavior in rats hyperlocomotion [42]. Clozapine, at the doses rang- [6], and found that U-99194A blocked locomotor ac- ing from 2.0 to 20.0 mg/kg, did increase DA and tivity and yawning behavior induced by 7-OH-DPAT DOPAC levels in in vivo microdialysates of rat brain [10, 40]. Others found no effect of clozapine on DA (D3 receptor agonist). U-99194A also induced a mod- erate degree of catalepsy and enhanced haloperidol- release [29, 31, 35, 38]. In the present experiment, induced catalepsy. However, U-99194A did not alter clozapine reduced 5-HTP level in the striatum after DA and DOPAC release in the striatum of rats, as as- a single injection only. It appears that the serotonergic sessed by in vivo microdialysis and in vivo voltametry [6]. system is involved in the biological actions of clo- Interestingly, in the present experiment, long-term zapine [3]. application of nafadotride induced yawning behavior. From our experiment, we conclude that the phar- macological (behavioral and biochemical) profile of We interpret this as possible D3 receptor priming, analogous to the observed increase in oral activity af- nafadotride action is different from that of haloperi- ter 14-day haloperidol treatment. Significantly, long- dol, but partially similar to that of clozapine. term nafadotride treatment failed to induce oral activ- ity, a characteristic symptom of DA D2 receptor Acknowledgments: blockade in rats [22], comparable to extrapyramidal This study was supported by a grant No. NN-4-003/02 from the effects seen in humans treated prolongably with clas- Medical University of Silesia to Ryszard Brus. The excellent technical assistance of Mrs. U. Mikolajun, Mrs. Z. Tramer and sic neuroleptics. Mrs. B. Medrek is highly appreciated. Nafadotride increased DA turnover in the nucleus accumbens, striatum and brain cortex in the rat, but to a much lesser extent than haloperidol [2]. Others found that UH232 and AJ76 increased DA and DO- References: PAC levels in microdialysates of striatum and nucleus accumbens [40, 59]. In the present experiment, we 1. Asghari V, Schoots O, Van Kats S, Ohara K, Javonovic determined biogenic amine levels in striatum and hippo- V, Guan HC, Bunzow JR et al.: Dopamine D" receptor campus, as well as, indirectly, DA and 5-HT turnover repeat: analysis of different native and mutant forms of in the striatum and 5-HT in the hippocampus by the human and rat genes. Mol Pharmacol, 1994, 46, 364–373. L-DOPA and 5-HTP assay, but we did not perform in 2. Audinot V, Newman-Tancredi A, Gobert A, Rivet JM, vivo microdialysis. Nafadotride applied in single and Brocco M, Lejeune F, Deposite I et al.: A comparative multiple doses did not influence DA, DOPAC, HVA in vitro and in vivo pharmacological characterization of and 5-HT levels in striatum, but increased NA content the novel dopamine D! receptor antagonists (+)-S 14297, of hippocampus, following 14 daily injections. Nafa- nafadotride, GR 103,691 and U 99194. J Pharmacol Exp Ther, 1998, 287, 187–197. dotride did not alter L-DOPA level in the striatum af- 3. Baldessarini RJ, Tarazi FI: Drugs and the treatment of ter single and multiple injections. psychiatric disorders. In: The Pharmacological Basis of In summary, we have compared behavioral and Therapeutics. Ed. Hardman JG, Limbird LE, Goodman- biochemical effects of nafadotride with clozapine, Gilman A, McGraw-Hill, 2001, 485–520. 4. Bloom FE: Neurotransmission and the central nervous a prominent D4 receptor antagonist. Clozapine, an system. In: The Pharmacological Basis of Therapeutics. atypical neuroleptic with 10 times higher affinity for Ed. Hardman JG, Limbird LE, Goodman-Gilman A, vs the DA D4 .D2 receptor [1, 13], is an effective an- McGraw-Hill, 1995, 267–293.

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