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Evaluation of Perospirone (SM-9018), a Novel -2 and -2 Receptor Antagonist, and Other in the Conditioned Fear Stress-Induced Freezing Behavior Model in Rats

Kumiko Ishida-Tokuda, Yukihiro Ohno*, Hideki Sakamoto, Tadashi Ishibashi, Junko Wakabayashi, Rie Tojima, Tomoko Morita and Mitsutaka Nakamura

Research Center, Sumitomo Pharmaceuticals Co., Ltd., 3-1-98 Kasugade-naka, Konohana-ku, Osaka 554, Japan

Received March 28, 1996 Accepted July 5, 1996

ABSTRACT-We studied the effects of perospirone (SM-9018), a novel serotonin-2 (5-HT2) and dopa- mine-2 (D2) receptor antagonist (SDA), on conditioned fear stress (CFS)-induced freezing behavior in rats and compared its actions with those of other antipsychotics. Exposure of rats to the environment previously paired with foot shock induced marked freezing behavior, which was reduced by the anxiolytic diazepam (0.1-3 mg/kg, p.o.) or , and (10-100 mg/kg, p.o.). Perospirone at 0.3 - 3 mg/kg, p.o. significantly attenuated the CFS-induced freezing behavior in a dose-dependent manner, while the effect was reduced at the higher dose of 6 mg/kg. Other SDA-type antipsychotics, (1-30 mg/kg, p.o.) and (0.03 -1 mg/kg, p.o.), and selective 5-HT2 antagonists, (0.1-1 mg/kg, p.o.) and (0.3 -1 mg/kg, p.o.), all reduced the freezing behavior with U-shaped dose-response curves. However, neither conventional , (0.1-3 mg/kg, p.o.), (3 -100 mg/kg, p.o.), (3 -100 mg/kg, p.o.), (3-100 mg/kg, p.o.) nor (30-1000 mg/kg, p.o.) reduced the CFS-induced freezing behavior. In addition, subacute treatment of rats with perospirone (1-10 mg/kg/day) or imipramine (30 mg/kg/day) for 2 weeks prevented the induction of the freezing behavior by CFS. These findings suggest that SDA-type antipsy- chotics including perospirone are effective for the treatment of mood disturbances such as anxiety and depressive mood associated with and have a broader efficacy profile as compared with the conventional antipsychotics.

Keywords: Perospirone, Antipsychotic, Conditioned fear stress, Freezing behavior, Schizophrenia

Schizophrenia is a heterogeneous disorder that displays antagonists produce thymosthenic actions and improve diverse symptoms, course, prognosis and probably also the negative symptoms in patients with schizophrenia etiology. The patients show not only positive symptoms (2-4). These agents also reduced the extrapyramidal side (e.g., hallucination, delusion and excitation), but also ex- effects induced by conventional antipsychotics. In addi- hibit negative or deficit symptoms (e.g., flattening affect, tion, clozapine, which possesses both 5-HT2- and D2- apathy and social withdrawal) and dysphoric mood dis- blocking actions, has been shown to improve the negative turbances (e.g., anxiety and depression) (1). The conven- schizophrenic symptoms with a reduced liability for the tional antipsychotics (e.g., haloperidol and chlorproma- extrapyramidal side effects (5). Based on these clinical zine) effectively improve the positive symptoms through findings, several 5-HT2 and D2 antagonists (SDA) are now antagonism of dopamine-2 (D2) receptors. However, being developed as novel type antipsychotics. these drugs are poorly effective against the negative symp- Perospirone (SM-9018; cis-N-(4-(4-(1,2-benzisothiazol- toms and frequently induce extrapyramidal side effects 3-yl)-1-piperazinyl) butyl) cyclohexane-1,2-dicarbox- (e.g., parkinsonism, akathisia and dyskinesia). Previous imide) is a newly developed SDA-type antipsychotic agent studies have shown that the serotonin-2 (5-HT2) receptor that has high affinities for both 5-HT2 and D2 receptors (K;=0.61 and 1.4 nM, respectively) (6-9). Perospirone, * To whom all correspondence should be addressed . like haloperidol, blocks various behaviors induced by dopamine agonists (6). In addition, perospirone, unlike CFS-induced freezing behavior conventional antipsychotics, potently inhibits the 5-HT2 Experiments were carried out according to the method receptor-mediated behavior and only weakly induces of Inoue et al. (23) with slight modifications. On the catalepsy and bradykinesia in rodents (6, 10). Perospi- training day, animals in the conditioning groups were rone was also weaker than haloperidol in inducing the individually placed in the experimental chamber supersensitivity and up-regulation of striatal D2 receptors (30 x 20 x 30 cm) with a grid floor and subjected to an after its repeated administration (11, 12). These findings unconditioned stimulus (US) (2 mA of scramble foot suggest that perospirone belongs to the class of atypical shock) for 30 min (Scrambler SGS-004; Tokai Irika, antipsychotics with fewer extrapyramidal side effects in Tokyo). Animals in the no-conditioning group (No-cond) humans. Furthermore, a recent clinical study has shown were also placed individually in the chamber for 30 min, that perospirone improves not only the positive symp- but received no US. On the testing day, scheduled at 24 hr toms, but also the negative symptoms and anxiety- after the training session, the rats were again placed in the depression in schizophrenia (13). identical chamber, in which US was previously applied. Despite a relatively broad spectrum of clinical actions The CFS-induced freezing behavior of the animals was of SDA-type antipsychotics (e.g., clozapine, risperidone measured for 5 min in the absence of US using a time- and perospirone) (4, 5, 13, 14), their action on the mood sampling procedure (test session). Each rat was rated as disturbances and the negative symptoms have not been either being frozen or active every 10 sec in a total of 30 well-documented in experimental animals due to the lack sampling periods (5 min). Freezing behavior was defined of appropriate animal models. Nevertheless, several stud- as the complete lack of all observable movements of the ies have shown that some of the SDA-type antipsychot- body and vibrissae except those related to respiration, ics (e.g., clozapine) are effective in the models of mood and the animal was judged as being frozen if it kept the disorders (e.g., punished responding and forced swim- behavioral status of freezing for a whole 10-sec period in ming test), suggesting their potential anxiolytic and anti- each sampling period. All other behavior seen in the depressant activities (15, 16). It is therefore of interest to sampling period was judged as active. The freezing score clarify the differences in actions between the SDA-type of each rat was calculated as the sum of the number of and the conventional antipsychotics in the animal model sampling periods in which the animal was rated as being of mood disorders. Fanselow demonstrated that con- frozen. Perospirone and all other test drugs were ad- ditioned fear stress (CFS), exposure of animals to the ministered 60 min before the test session. environment previously paired with electrical foot shock, induced a marked freezing behavior in rats (17). CFS Subacute treatment is considered to be a psychological stress without physical Perospirone (1, 3 and 10 mg/kg) and imipramine (30 stimuli and an animal model of anxiety, fear and/or mg/kg) were orally administered to the rats once a day depressive mood because the CFS-induced freezing be- (between 9 AM-11 AM) for 14 days. The training session havior could be ameliorated by several types of anxiolytic with US was carried out 60 min before the last drug ad- agents (18-20) and antidepressants (21, 22). In the ministration, and the CFS-induced freezing behavior was present study, we examined the effects of perospirone on recorded in the same manner as in the acute experiment the CFS-induced freezing behavior in rats and compared 24 hr after the conditioning session. its actions with those of other SDA-type and conven- tional antipsychotics. The effects of selective 5-HT2 Drugs antagonists were also examined to explore the role Perospirone hydrochloride, haloperidol, clozapine, of the 5-HT2 antagonism in the antipsychotics' actions risperidone, diazepam, mosapramine dihydrochloride, in the CFS model. thioridazine and tiapride were synthesized in our labo- ratory. Chlorpromazine hydrochloride, imipramine hydrochloride and desipramine hydrochloride were pur- chased from Sigma Chem. Co. (St. Louis, MO, USA), Animals and ketanserin tartrate and ritanserin were from Research Male Sprague-Dawley rats (Nihon SLC, Inc., Shizuoka) Biochem., Inc. (Natick, MA, USA). All drugs were sus- each weighing 175-255 g were used. The rats were housed pended in 0.5% methylcellulose. in a temperature (23±2'C)-, and humidity (55_h10%)- controlled room under the standard lighting condition Data analyses (light/dark: 8 AM/8 PM) for at least one week before The data are expressed as the mean±S.E.M. Differ- being tested. They were given tap water and standard rat ences in the freezing scores among multiple treatment chow ad libitum. groups were analyzed by the non-parametric Kruskal- Wallis test followed by Steel's test. Comparisons be- tween two groups were made by the Mann-Whitney U- test. Percent changes in the CFS-induced freezing behav- ior was also obtained by the following equation: (the mean score of test group - the mean score of No-cond group) / (the mean score of vehicle-treated group - the mean score of No-cond group) x 100. The apparent dosage of a drug that reverses the CFS-induced freezing behavior by 50% (ED50) was determined by the method of Litchfield and Wilcoxon.

Effects of perospirone and other antipsychotics on CFS- induced freezing behavior The No-cond group normally exhibited a low freezing score of 6.19 ± 0.80 (N = 36), probably due to the habitu- ation to the test environment (Fig. 1). CFS (an exposure to the same environment previously paired with foot shock) induced marked freezing behavior in rats, which was characterized by a complete suppression of motility Fig. 1. Effectsof perospirone on the conditioned fear stress (CFS)- with occasional defecation and urination. The freezing induced freezing behavior in rats. Each column shows the behavior score was usually increased to about 19 to 22 by mean±S.E.M. of 6 or 12 animals except for the no-conditioning the CFS in most experiments. (No-cond)group (N=36). Perospirone was orally administered 1 hr before observation of the freezing behavior. The dashed line shows Oral administration of perospirone at 0.3-6 mg/kg the freezing score of the No-cond group. ##P<0.01, significantly significantly attenuated the CFS-induced freezing behav- different from the value of the No-cond group (Mann-Whitney U- ior without affecting the activity in the No-cond group test). **P<0.01, significantly different from the control value (Fig. 1). The inhibitory action of perospirone appeared in (Kruskal-Wallistest followed by Steel's test). a dose-dependent manner at doses of 0.3 - 3 mg/kg, while it tended to be reduced at a higher dose (6 mg/kg). The maximal inhibition rate of the CFS-induced freezing by

Fig. 2. Effects of clozapine and risperidone on the conditioned fear stress (CFS)-induced freezing behavior in rats. Each column shows the mean ±S.E.M. of 6 to 18 animals. Drugs were orally administered 1 hr before observation of the freezing behavior. The freezing score in the no-conditioning group is shown as a dashed line for comparison. *P<0.05, **P<0.01, significantly different from the control values (Kruskal-Wallis test followed by Steel's test). perospirone was 79.8010 at 3 mg/kg, and the apparent dose (ED50 value) that reversed the freezing by 50010was 1.3 mg/kg. Similarly, other SDA-type antipsychotics, clozapine (1-30 mg/kg, p.o.) and risperidone (0.03-1 mg/kg, p.o.), also reduced the incidence of the CFS- induced freezing with U-shaped dose-response curves (Fig. 2). The maximal inhibition rates of freezing behav- ior by clozapine and risperidone were 46.7% and 61.4010, respectively, and the ED50 value of risperidone was 0.13 mg/kg. In contrast, none of the administered conven- tional antipsychotics, haloperidol (0.1-3 mg/kg, p.o.), chlorpromazine (3 - 100 mg/kg, p.o.), thioridazine (3 - 100 mg/kg, p.o.), mosapramine (3 - 100 mg/kg, p.o.) and tiapride (30-100 mg/kg, p.o.), significantly affected the CFS-induced freezing behavior (Table 1). Converse- ly, high doses of haloperidol and chlorpromazine rather augmented the CFS-induced freezing behavior (Table 1).

Effects of other CNS drugs and 5-HT2 antagonists on CFS-induced freezing behavior We next examined the effects of the anxiolytic diaz- epam, the antidepressants imipramine and desipramine, and the selective 5-HT2 antagonists ritanserin and ketan- serin on the CFS-induced freezing behavior. As shown in Fig. 3, the administration of diazepam (0.3 - 3 mg/kg, p.o.) significantly reduced the CFS-induced freezing be- havior in a dose-dependent manner, while the effect was Each value shows the mean±S.E.M. of 6 rats. Drugs were orally administered 1 hr before observation of the freezing behavior.

Fig. 3. Effects of diazepam, desipramine and imipramine on the conditioned fear stress (CFS)-induced freezing behavior in rats. Each column shows the mean ±S.E.M. of 6-12 animals. Drugs were orally administered 1 hr before observation of the freezing behavior. The dashed line shows the freezing score in the no-conditioning group for comparison. *P<0.05, sig- nificantly different from the control values (Kruskal-Wallis test followed by Steel's test). Fig. 4. Effects of ketanserin and ritanserin on the conditioned fear stress (CFS)-induced freezing behavior in rats. Each column shows the mean ±S.E.M. of 6-9 animals. Drugs were orally administered 1 hr before observation of the freezing be- havior. The dashed line shows the freezing score in the no-conditioning group for comparison. *P<0.05, significantly different from the control values (Kruskal-Wallis test followed by Steel's test).

diminished at a dose of 10 mg/kg. Imipramine (10-100 mg/kg, p.o.) and desipramine (10- 100 mg/kg, p.o.) also reduced the incidence of CFS-induced freezing with the maximum inhibition rates of 58.9% and 52.4% (at 100 mg/kg), respectively (Fig. 3). On the other hand, both 5-HT2 antagonists, ritanserin (0.1-1 mg/kg, p.o.) and ketanserin (0.3 -1 mg/kg, p.o.), reduced the CFS-induced freezing behavior with U-shaped dose response curves (Fig. 4). The maximal inhibition rates of ritanserin and ketanserin were 60010 to 65010, and apparent ED50 values were 0.28 and 0.54 mg/kg, respectively.

Subacute effects of perospirone on CFS-induced freezing behavior The animals were treated once a day with an oral dose of perospirone (1-3 mg/kg/day), imipramine (30 mg/kg/day) or vehicle for 2 weeks. The training session was carried out 60 min before the last treatment and the CFS-induced freezing behavior was observed after a 24-hr withdrawal. Under these conditions, CFS induced a marked freezing behavior in control animals treated with the vehicle alone (Fig. 5). However, the induction of the CFS-induced freezing behavior was attenuated by the subacute perospirone treatment in a dose-dependent Fig. 5. Effects of subacute treatments with perospirone and im- manner (Fig. 5). The perospirone treatment at 3 or 10 ipramine on induction of the freezing behavior by the conditioned mg/kg/day reduced the CFS-induced freezing scores by fear stress (CFS) in rats. Each column shows the mean ±S.E.M. of 7 about 75%, the scores not being significantly different or 8 animals. The rats were treated with an oral dose of perospirone, from that in the No-cond group. A similar attenuation in imipramine or vehicle for 2 weeks, and the CFS-induced freezing behavior was observed 24 hr after the last treatment. The dashed line the induction of freezing behavior by the CFS was also shows the freezing score in the no-conditioning (No-cond) group. obtained by the subacute treatment with imipramine (30 **P<0 .01, significantly different from the control values (Mann- mg/kg/day) (Fig. 5). Whitney U-test). DISCUSSION 3H- (D2) binding with a potency similar to that of haloperidol (K, value =1.4 and 1.8 nM, respectively), The present study demonstrated that the exposure of whereas it inhibits the cortical 3H-ketanserin (5-HT2) rats to a distinctive environment previously paired with binding with more than 100 times the potency of foot shock (US) induces a marked freezing behavior haloperidol (K; value= 0.61 and 116 nM, respectively) (6). characterized by the complete suppression of motility These findings suggest that the blockade of 5-HT2 recep- with occasional defecation and urination. The CFS-in- tors contributes to the inhibition of the CFS-induced duced freezing behavior was significantly ameliorated by freezing behavior by the SDA-type antipsychotics. In this administration of the anxiolytic diazepam. In addition, study, the selective 5-HT2 antagonist ritanserin sig- single and repeated administrations of the anti- nificantly reduced the CFS-induced freezing behavior. depressants, desipramine or imipramine, reduced the Ketanserin, which has 5-HT2- and al-blocking activities, incidence of the CFS-induced freezing behavior. These also tended to reduce the freezing behavior. Thus the 5- findings are consistent with previous studies (18-21) and HT2 receptor blockade might be at least partly involved in suggest that the CFS-induced freezing responses in rats the amelioration of the CFS-induced freezing behavior by are closely associated with the emotional states of anxi- the SDA-type antipsychotics. Our findings are in agree- ety, fear and/or depressive mood in humans. ment with the previous neurochemical studies (23, 24) In this study, the newly developed antipsychotic that showed that an increased activity of the perospirone at moderate doses significantly improved the system is involved in the freezing behavior induced by CFS-induced freezing with an ED50 value of 1.3 mg/kg. psychological stress (e.g., CFS). The reason why most of The freezing score was reduced to a level similar to that the drugs (i.e., the SDA-type antipsychotics, the 5-HT2 of the No-cond group by the administration of 3 mg/kg antagonists and diazepam) inhibited the freezing behavior perospirone, while its action tended to be reduced with with the common characteristic of U-shaped dose- increasing dosage (6 mg/kg). Perospirone has been response curves is presently uncertain. In the rat open- shown to exert anti- actions (e.g., the an- field test, we observed that the antipsychotics perospi- tagonisms for the methamphetamine-induced hyperactiv- rone, clozapine, risperidone, haloperidol and chlor- ity and -induced climbing behavior) with inhibited the line-crossing activity with ED50 ED50 values of 2.2 to 3.5 mg/kg (6). Thus the present values of 5.9, 21, 0.59, 0.48 and 9.1 mg/kg (p.o.), findings suggest that perospirone at clinical doses is effec- respectively (unpublished observation). These doses were tive for mood disturbances (i.e., anxiety and depression) relatively consistent with the dose range for the efficacy- associated with schizophrenia. The anxiolytic potential of decline of the former three drugs and for the freezing- perospirone was also observed in our recent study using potentiation of the latter two drugs in the CFS model. the rat social interaction test, where perospirone, like Thus, the inhibitory effects of the SDA-type antipsychot- diazepam, significantly increased the time spent in active ics on locomotor activity might inhibit or mask their social interaction by the pair of Lister-hooded rats ameliorative actions in the CFS-induced freezing. (unpublished observation). In addition, a recent clinical We have recently demonstrated that the subacute treat- study revealed that perospirone effectively improved not ment of rats with perospirone (10 mg/kg/day, for 2 only the positive symptoms but also the negative symp- weeks) produced a significant reduction (about 20%) toms and anxiety-depression in patients with schizo- in the density (Bm,,) of the cortical 5-HT2 receptors (12). phrenia (13). However, such changes were not observed in animals Among the antipsychotics examined in this study, only treated with haloperidol. Antidepressants also produce the SDA-type antipsychotics with combined 5-HT2- and the down-regulation of 5-HT2 receptors after repeated D2-blocking actions improved the CFS-induced freezing administration, which has been suggested to be one of behavior. Clozapine and risperidone, like perospirone, the possible mechanisms of the action significantly attenuated the freezing behavior with U- (25 -27). We therefore compared the subacute effects of shaped dose-response curves. In contrast, administration perospirone and imipramine on the CFS-induced freezing of any of the conventional antipsychotics, haloperidol, behavior model. The animals were treated with perospi- chlorpromazine, thioridazine, mosapramine or tiapride, rone (1 - 10 mg/kg/day) or imipramine (30 mg/kg/day) failed to improve the CFS-induced freezing behavior. The for 2 weeks, and the CFS-induced freezing behavior was former two drugs rather potentiated the freezing at higher observed 24 hr after the last administration of each drug. doses. The SDA-type antipsychotics commonly exhibit a Under these conditions, both perospirone and imipra- higher affinity for 5-HT2 receptors than D2 receptors, and mine prevented the induction of the freezing behavior by they are expected to block the 5-HT2 receptors at clinical CFS. Interestingly, the dose-response curve of pero- doses (4). For example, perospirone inhibited the striatal spirone was no longer U-shaped at these doses. These findings suggest that perospirone at clinical doses exerts 6 Hirose A, Kato T, Ohno Y, Shimizu H, Tanaka H, Nakamura imipramine-like mood stabilizing actions under repeated M and Katsube, J: Pharmacological actions of SM-9018, a new administration. Since perospirone down-regulated the neuroleptic drug with both potent 5-hydroxytryptamine2 and dopamine2 antagonistic actions. Jpn J Pharmacol 53, 321-329 cortical 5-HT2 receptors under the same treatment as in the present study (12), the 5-HT2 receptor changes might (1990) 7 Maruoka Y, Ohno Y, Kato T, Hirose A, Tatsuno T and contribute to the ameliorative effects of subacute pero- Nakamura M: Effects of SM-9018, a potential atypical neuro- spirone on the CFS-induced freezing behavior. leptic, on the central system in rats. Jpn J It is of interest that the 5-HT2 antagonists and SDA- Pharmacol 62, 419-422 (1993) type antipsychotics, but not conventional ones, improved 8 Ishida K, Ohno Y, Ishibashi T and Nakamura M: Effects of the CFS-induced freezing behavior. Previous studies have SM-9018, a novel 5-HT2 and D2 antagonist, on electrically- shown that SDA-type antipsychotics including perospi- evoked [3H]acetylcholine release from rat striatal slices. Gen Pharmacol (1996) (in press) rone, clozapine and risperidone effectively improved the 9 Ishibashi T, Ikeda K, Ishida K, Yasui J, Tojima R, Nakamura negative symptoms of schizophrenia, which do not M and Ohno Y: Contrasting effects of SM-9018, a potential respond well to the conventional antipsychotics (1, 4, 13, , and haloperidol on c-fos mRNA expres- 14). In addition, selective 5-HT2 antagonists (e.g., ritan- sion in the rat striatum. Eur J Pharmacol 303, 247-251 (1996) serin and ) have also been shown to produce 10 Ohno Y, Ishida K, Ikeda K, Ishibashi T, Okada K and the thymosthenic effects in schizophrenia patients and Nakamura M: Evaluation of bradykinesia induction by SM- improve the negative symptoms (2, 3). Thus, the differen- 9018, a novel 5-HT2 and D2 receptor antagonist, using the tial suppression of the CFS-induced freezing behavior by mouse pole test. Pharmacol Biochem Behav 49, 19-23 (1994) 11 Ohno Y, Ishida K, Ishibashi T, Ikeda K, Kato T and Nakamura the SDA-type antipsychotics may reflect their efficacy for M: Effects of chronic treatments with SM-9018, a potential the negative schizophrenic symptoms. There are so far atypical neuroleptic, on behavioral dopaminergic and only a few animal models proposed that can predict the serotonergic sensitivity in rats. Gen Pharmacol 26, 489-494 efficacy of antipsychotics for the negative symptoms (28). (1995) Although the pathophysiological mechanisms of the 12 Ohno Y, Ishibashi T, Okada K, Ishida K and Nakamura M: negative symptoms are still unknown, the CFS-induced Effects of subchronic treatments with SM-9018, a novel 5-HT2 freezing behavior in rats may serve as a useful model that and D2 antagonist, on dopamine and 5-HT receptors in rats. 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