D3 partial agonist and 5-HT1A agonist actions. II. Preclinical behavioral effects

Neuropharmacology 39 (2000) 1211–1221 www.elsevier.com/locate/neuropharm

PD 158771, a potential antipsychotic agent with D2/D3 partial agonist and 5-HT1A agonist actions. II. Preclinical behavioral effects A.E. Corbin a

a,*

, L.T. Meltzer a, F.W. Ninteman a, J.N. Wiley a, C.L. Christoffersen a, D.J. Wustrow b, L.D. Wise b, T.A. Pugsley a, T.G. Heffner a

Neuroscience Therapeutics, Parke-Davis Pharmaceutical Research, Division of Warner-Lambert Company, Ann Arbor, MI 48105, USA Neuroscience Chemistry, Parke-Davis Pharmaceutical Research, Division of Warner-Lambert Company, Ann Arbor, MI 48105, USA

b

Accepted 20 October 1999

Abstract PD 158771 has been described in receptor binding and biochemical tests as a partial agonist at dopamine (DA) D2 and D3 receptors as well as an agonist at serotonin (5-HT)1A receptors. The present studies describe the profile of PD 158771 in rodent and primate behavioral tests. PD 158771 reduced spontaneous locomotor activity in mice (ED50=0.38 mg/kg, i.p.) and rats (ED50=1.2 mg/kg, i.p. and 0.16 mg/kg, s.c.), and reduced amphetamine-stimulated locomotion in mice (ED50=0.13 mg/kg, i.p.). At relatively higher doses up to 3 mg/kg, s.c. in rats, PD 158771 did not produce locomotor stimulation or induce stereotypy, indicating a lack of postsynaptic DA agonist activity. PD 158771 reduced apomorphine stimulated locomotion in rats at a dose 4.6-fold greater than those that reduced spontaneous locomotor activity, indicating weak postsynaptic DA antagonist actions; results consistent with a partial agonist profile. PD 158771 produced anxiolytic-like effects in the water-lick (Vogel) conflict test, effects possibly due to the 5-HT1A activity. However, PD 158771 was inactive in the water wheel behavioral despair model in rats, indicating lack of antidepressant properties. Similar to known antipsychotics, PD 158771 produced a potent and long-lasting inhibition of conditioned avoidance responding in squirrel monkeys. In contrast to standard antipsychotics, and similar to clozapine, PD 158771 did not cause catalepsy in rats at a dose 20-fold higher than the ED50 dose for locomotor inhibition. PD 158771 also had a somewhat lower liability than haloperidol to produce extrapyramidal dysfunction in squirrel and cebus monkeys sensitized to the dystonic effects of haloperidol. The data indicate that PD 158771 is a DA partial agonist with weak intrinsic activity that selectively activates brain DA autoreceptors. PD 158771 produced behavioral effects consistent with potential antipsychotic and anxiolytic efficacy, and has an improved profile in the extrapyramidal side effect model when compared to certain currently available antipsychotic agents.  2000 Elsevier Science Ltd. All rights reserved. Keywords: Partial dopamine agonist; 5-HT1A agonist; Antipsychotic; Locomotor activity; Avoidance responding; Extrapyramidal dysfunction

1. Introduction The limited efficacy of typical antipsychotic drugs for improvement of both the positive and negative sympAbbreviations: B-HT 920 (talipexole)=2-amino-6-allyl-5,6,7,8-tetrahydro-4H-thiazolo-(4,5-d)-azepine; CI-1007 (PD 143188-6614)=R(+)-1,2,3,6-tetrahydro-4-phenyl-1[(3-phenyl-3-cyclohexen-1-yl)methyl] pyridine maleate; DA=dopamine; EPD=extrapyramidal dysfunction; EPS=extrapyramidal side effects; 5-HT=5-hydroxytryptamine (serotonin); LMA=locomotor activity; 8-OH-DPAT=8-hydroxy-2-(din-propylamino)tetralin; 3-PPP=3-(3-hydroxyphenyl)-N-n-propylpiperidine; SDZ 208-912=N-[(8-α)-2-chloro-6-methylergoline-8-yl]-2,2dimethylpropanamide. * Corresponding author. Tel.: +1-734-622-7030; fax: +1-734-622-1437. E-mail address: [email protected] (A.E. Corbin).

toms of schizophrenia, in addition to the movement disorders associated with long-term treatment of antipsychotic drugs, has driven the development of an improved antipsychotic agent. Blockade of dopamine (DA) receptors in the limbic forebrain has been postulated to be the mechanism by which antipsychotic drugs produce their therapeutic effects (Carlsson, 1978; Meltzer and Stahl, 1976). However, antipsychotic drugs with prominent DA D2 antagonist effects cause tardive dyskinesia and extrapyramidal side effects (EPS) in humans, presumably through concomitant blockade of striatal DA receptors (Baldessarini and Tarsy, 1980). One of the current approaches to the development of improved antipsychotic agents involves reducing the amount of dopamine neurotransmission with partial DA

0028-3908/00/$ - see front matter  2000 Elsevier Science Ltd. All rights reserved. PII: S 0 0 2 8 - 3 9 0 8 ( 9 9 ) 0 0 2 1 4 - 2

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D2 agonists or DA autoreceptor agonists. Such an agonist, which preferentially acts at presynaptic DA D2 or D3 autoreceptors and modulates the activity of DA neurons (Roth 1979, 1984; Tang et al., 1994) as opposed to blocking postsynaptic DA receptors in the nucleus accumbens and striatum, may have less propensity to produce motor side effects. The DA D3 receptor, preferentially distributed in the limbic vs. striatal regions of brain (Landwehrmeyer et al., 1993), has been identified as a novel target for an improved antipsychotic drug. Blockade of these receptors may retain antipsychotic efficacy with reduced liability for extrapyramidal side effects (Sokoloff et al., 1990; Bouthenet et al., 1991; Le´vesque et al., 1992). In addition, antipsychotic drugs are currently being developed which antagonize both DA and serotonin (5-HT2A) receptors in brain; such compounds may have improved efficacy and less severe side effects than currently available drugs (Gerlach and Peacock, 1995; Piercey et al., 1994; Moore et al., 1994; Perregaard et al., 1992; Seeger et al., 1995). The blockade of 5-HT2A receptors has been implicated in both the improved efficacy against the negative symptoms of schizophrenia and the improved extrapyramidal syndrome (EPS) profile of certain antipsychotic drugs (Meltzer, 1989; Meltzer, 1995b; Carpenter, 1995; Curtis and Kerwin, 1995; Abi-Dargham et al., 1996). The synthesis and release of 5-HT can also be modulated by the action of 5-HT1A agonists and partial agonists on somatodendritic neurons (DeVry, 1995). 5-HT1A receptor agonists are thought to attenuate certain D2 receptormediated side effects in rodents and monkeys (McMillen et al., 1988; Neal-Beliveau et al., 1993; Casey, 1993; Wadenberg et al., 1994). Additionally, the 5-HT1A system has been postulated to be involved in the etiology of anxiety and depression (Taylor, 1990; Lucki et al., 1994). The novel antipsychotic, clozapine, has been shown to be a 5-HT2A antagonist and partial agonist at 5-HT1A receptors in vitro and possibly in vivo (NewmanTancredi et al., 1996; Rollema et al., 1997), which may contribute to its decreased EPS liability and its efficacy against negative symptoms. Thus, a compound with partial agonist activity at DA D2 and D3 receptors, in addition to agonist actions at 5-HT1A receptors and potential 5-HT2A receptor antagonist actions, may retain antipsychotic efficacy, have reduced liability for the severe movement disorders that develop upon acute and long term treatment, and could potentially alleviate certain negative symptoms associated with schizophrenia, in addition to potential anxiolytic and/or antidepressant effects. PD 158771 (trans-{4-[2-(4-phenyl-piperazin-1-yl)ethyl]-cyclohexyl}-pyrimidin-2-yl-amine) was identified (Wustrow et al., 1998) and has been characterized in the preceding paper as a DA autoreceptor agonist and a partial DA D2 agonist (accompanying publication by Akunne et al. 1999, 1996; Zoski et al., 1996). In

addition, PD 158771 also acts as a weak partial agonist at DA D3 receptors, as an antagonist at DA D4 receptors, and as an agonist at 5-HT1A receptors (accompanying publication; Zoski et al., 1996). This paper describes the effects of PD 158771 in behavioral tests predictive of antipsychotic, anxiolytic and antidepressant activity, including locomotor activity, water wheel behavioral despair, water-lick (Vogel) conflict and catalepsy studies in rodents, as well as conditioned avoidance responding and extrapyramidal dysfunction tests (EPD) in monkeys.

2. Methods 2.1. Subjects Male CD-1 mice (25–35 g) from Charles River Labs, housed 10 per cage, were used for the spontaneous locomotor activity and amphetamine-stimulated locomotor activity tests. Male Sprague–Dawley rats (180–250 g, housed five per cage) from Harlan Labs Inc. (Indianapolis, IN) were used for spontaneous locomotor activity, apomorphine-stimulated locomotor activity, and catalepsy tests. Male Wistar rats (Hilltop Laboratories) were used for the water-lick (Vogel) conflict test, while male Wistar–Kyoto rats (Charles River Laboratories) were used for the water wheel behavioral despair model. Adult male squirrel monkeys (Saimiri scureus; 0.8–1.0 kg) were used for the conditioned avoidance tests. Adult male squirrel and cebus monkeys (Cebus apella; 2.0– 4.0 kg) were used for extrapyramidal dysfunction (EPD) tests. Squirrel monkeys were pair-housed, while cebus monkeys were singly-housed. Animals were maintained on a 12/12 h light/dark cycle (lights on between 06.00 and 18.00 h) with free access to standard chow and water. Rats and monkeys dosed p.o. were fasted overnight. Rats used in the water-lick conflict test were deprived of water, as described below in Methods. Animal care was in accordance with guidelines established by the National Institutes of Health and the American Association for the Accreditation of Laboratory Animal Care. All experimental protocols were approved by an Animal Care and Use Committee. 2.2. Spontaneous locomotor activity (LMA) in mice and rats Locomotor activity (LMA) was measured in groups of three mice (n=3–4 groups) immediately after i.p. dosing for 1 h in darkened cylindrical photobeam chambers (35 cm diameter × 20 cm deep). Individual rats (n=4– 7) dosed p.o. or i.p. were allowed a 1 h drug absorption period prior to a 30 min test in the photobeam chambers. Rats dosed s.c. or i.v. were placed immediately into chambers for a 30 min test. Each LMA count represented

A.E. Corbin et al. / Neuropharmacology 39 (2000) 1211–1221

six consecutive interruptions of the radially arranged photobeams. Data were expressed relative to vehicletreated controls tested concurrently. ED50 values and 95% confidence limits were calculated using linear regression analysis; significant changes in locomotor activity were calculated by grouped t-test. (Tallarida and Murray, 1986). 2.3. Amphetamine-stimulated locomotor activity in mice Groups of three mice (3–4 groups/dose) were dosed i.p. with saline or 0.5 mg/kg d-amphetamine and placed into the circular photobeam chambers for a 20 min dark acclimation/drug absorption period. Mice were removed and injected i.p. with saline or PD 158771 and returned to the respective chamber for a 1 h locomotor activity test. Data were expressed as a percent of amphetaminetreated control value. Blockade of amphetamine ED50 values and 95% confidence limits were calculated from individual percent reversal of amphetamine stimulation at each dose using linear regression analysis; significant changes in activity were calculated by grouped t-test (Tallarida and Murray, 1986). 2.4. Apomorphine-stimulated locomotor activity in rats Rats (n=4–10) were dosed s.c. with saline or apomorphine (0.3 mg/kg) and placed in the circular photobeam chambers for a 15 min dark acclimation/drug absorption period. Rats were removed from the chambers and dosed with saline or test compound and returned to the chamber for a 30 min locomotor activity test. Data were expressed as percent reversal of apomorphine-stimulated activity at each dose; reversal ED50 values and 95% confidence limits were calculated with linear regression analysis. Significant changes in activity level were calculated by grouped t-test (Tallarida and Murray, 1986). 2.5. Catalepsy in rats Rats (n=6) were dosed with saline or test drug and tested for catalepsy at intervals of 1, 2 and 4 h after dosing. The rat’s forepaws were placed on a horizontal bar elevated 12 cm above the tabletop. All rats were given three trials at each time interval. Each trial was scored as follows: 0 for holding the position ⬍30 s, 1 for holding for 30–59 s, or a maximum score of 2 for staying on the bar for 60 or more seconds. The overall catalepsy score was the mean of the three trials at each interval (the maximum mean score=2.0). Significant changes from control were calculated using the nonparametric Wilcoxon Rank-Sum Test.

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2.6. Water lick (Vogel) conflict test in rats Male Wistar rats (n=8) were deprived of water for 24 h, then allowed to drink from a drinking tube in a test cage for 10 min or until 5 ml water was consumed. Rats not drinking in the 10 min period were not used for the study. Rats were then dosed with vehicle or test drug and deprived of water for an additional 24 h. Chlordiazepoxide was tested concurrently as the reference anxiolytic. On the test day, rats were again dosed with the test drug and 30 min later placed into test cages for a 10 min test. For each 10 licks of water rats received a 1 s shock (1 mA) through the drinking tube. Significant increases in number of shocks taken were determined by grouped t-test (Tallarida and Murray, 1986). 2.7. Water wheel behavioral despair test in rats Wistar–Kyoto rats (n=12) were placed into water filled tanks, each containing a wire mesh wheel mounted at water level. Rats rotated the wheel as they attempted to escape from the water. After a 15 min session in the tanks, rats were dosed with vehicle or test drug and returned to home cages. Twenty-four hours later, rats were again dosed with their respective treatments, and were tested in the tanks 1 h later for a 5 min test period. Significant increases in wheel rotations above control were determined by grouped t-test (Tallarida and Murray, 1986). 2.8. Conditioned avoidance test in monkeys Male squirrel monkeys (n=6) were trained in a modified Sidman avoidance procedure (Heffner et al., 1989) to press a lever to avoid electric shock (2.0 mA; 0.75 s duration) delivered through the grid of the test chamber. During a response–shock component, shock was delivered when 20 s elapsed without a response, and this switched the schedule to a shock–shock component during which time shocks were delivered every 10 s until a lever press terminated the shock delivery and reinstated the response–shock component. After training, the monkeys were tested once a week with vehicle or test drug administered p.o. immediately prior to the start of a 6 h testing period. Avoidance responding was expressed as the percent of possible shocks avoided (180 per hour). Each monkey served as its own control, and percent changes relative to control were calculated for avoidance at each hour of the test. ED50 values and 95% confidence limits for inhibition of avoidance were calculated using regression analysis from dose–effect curves with data for the hour of peak activity. 2.9. Extrapyramidal dysfunction (EPD) in monkeys Squirrel (n=4–5) and cebus monkeys (n=2–4) were sensitized to the dystonia-producing effects of haloperi-

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dol once a week (3 mg/kg p.o. for squirrel monkeys; 1 mg/kg p.o. for cebus monkeys) (Heffner et al., 1989). Signs of EPD included involuntary twisting of the neck or torso, protrusion of the tongue, tonic extension of the limbs and pressing of the body against cage walls. After EPD was established, the monkeys showed acute dystonias after administration of lower doses of haloperidol or other DA antagonists that produce EPS in man. Test agents were administered orally once a week to sensitized monkeys and rated by experienced, blinded observers every hour over a 6 h period for the appearance of EPD. A subjective rating scale was used to estimate the intensity of the EPD when present. Individual signs of EPD were rated as being absent (0), weak (1) or strong (2). At each time-point for each individual monkey, the scores for all signs present were totaled, and these were totaled over the 6 h observation period to give a cumulative score. Group means were derived for all monkeys tested at each dose. 2.10. Drugs PD 158771 was dissolved in 0.9% saline for i.p., i.v. or s.c. administration in rats or mice. PD 158771 was dissolved in water for p.o. administration (by gavage) in rats and monkeys. d-Amphetamine and apomorphine (Sigma Chemical Co., St Louis, MO) were dissolved in 0.9% saline. Haloperidol (Research Biochemicals International, Natick, MA) was dissolved in acetic acid and 0.9% saline. CI-1007, EMD 38362 and chlordiazepoxide were synthesized at Parke-Davis Pharmaceutical Research Division (Ann Arbor, MI). Clozapine, B-HT 920, (+)-3-PPP, (⫺)-3-PPP and 8-OH-DPAT were purchased from Research Biochemicals International (Natick, MA). Imipramine and fluoxetine were purchased from Sigma Chemical Co. (St Louis, MO). SDZ 208-912 was gratefully supplied by Sandoz Pharmaceuticals (Basel, Switzerland); terguride was supplied from Schering Corp. (Kenilworth, NJ). Gepirone was supplied by Bristol Myers Co (Wallingford, CT). When necessary, 1 N HCl was used to facilitate dissolution of insoluble compounds, and the pH was titrated back to 5.0– 7.0 with 1 N NaOH. The surfactant, Emulphor, was also added in small amounts to facilitate dissolution when necessary. Doses represent the active moiety. Drugs and the appropriate vehicle solutions were administered to mice i.p. in a volume of 10 ml/kg, to rats p.o. and i.p. in a volume of 5 ml/kg, to rats s.c. in a volume of 2 ml/kg, and to monkeys p.o. and rats i.v. in a volume of 1 ml/kg body weight. 3. Results 3.1. Effects on spontaneous locomotor activity (LMA) in mice and rats PD 158771 dose-dependently reduced spontaneous locomotion in mice dosed i.p. The spontaneous loco-

motor activity (LMA) ED50 was 0.38 mg/kg (Fig. 1). No stimulation of LMA was observed at doses 100-fold higher than the ED50 for LMA inhibition in mice. In rats, PD 158771 produced dose-related inhibition of spontaneous LMA by the i.v., s.c., i.p. and p.o. routes of administration (Fig. 2) with varying potencies. The p.o. ED50 of 4.6 mg/kg suggests low bioavailability compared to the i.v. and s.c. ED50 values of 0.07 and 0.16 mg/kg, respectively. No stimulation of spontaneous LMA was observed at relatively high doses in rats by any of the routes tested. 3.2. Effects on amphetamine-stimulated activity in mice PD 158771 reversed the hyperlocomotion produced by 0.5 mg/kg i.p. amphetamine in mice in a dose-related manner with an ED50 of 0.13 mg/kg (Fig. 1), similar to the potency observed in the spontaneous LMA test. 3.3. Effects on apomorphine-stimulated activity in rats PD 158771 reversed the locomotor stimulation produced by apomorphine (0.3 mg/kg, s.c.) in a dose dependent manner (Fig. 3). Thus, PD 158771 was slightly more potent (4.6-fold) in reducing spontaneous locomotion than apomorphine-stimulated locomotion (s.c.

Fig. 1. Dose–response curves for the effect of PD 158771 on spontaneous locomotor activity (Spont. LMA) and amphetamine-stimulated (Amph.) locomotor activity in mice dosed i.p. Each point is the mean±S.E.M. from 3 to 4 groups of three mice. Spontaneous locomotion was measured for 1 h immediately after i.p. dosing. For the amphetamine study, d-amphetamine (0.5 mg/kg) was given i.p. 20 min prior to i.p. PD 158771, after which locomotor activity was monitored for 1 h. Control activity counts in the spontaneous LMA study were 613±133; control activity counts in the amphetamine-stimulated LMA study were 430±56. *P⬍0.05 vs control.

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haloperidol and PD 158771 are shown in Table 1. The ratios calculated for each compound were rank ordered as a behavioral estimate of intrinsic agonist activity at DA receptors. Thus, PD 158771 had a profile in this procedure similar to other partial agonists (ie. CI-1007, (⫺)-3-PPP and terguride). PD 158771 had a higher ratio than the weak partial DA agonist SDZ 208-912 and the DA antagonist haloperidol, and a lower ratio than EMD 38362, (+)-3-PPP and B-HT 920. 3.4. Catalepsy in rats

Fig. 2. Dose–response curves for the effect of PD 158771 on spontaneous locomotor activity in rats by the i.v., s.c., i.p. and p.o. routes of administration. Each point is the mean±S.E.M. of 4–7 rats. Locomotor activity was measured for 30 min. Rats dosed p.o. and i.p. were tested 1 h after dosing; rats dosed i.v. or s.c. were tested immediately after dosing. Control activity counts for the i.v., s.c., i.p. and p.o. routes of administration were 220±24, 137±37, 154±20, and 196±21, respectively. *P⬍0.05 vs control.

No catalepsy was observed in rats at doses equivalent to 10 and 20 times (13 and 26 mg/kg, i.p.) the rat LMA ED50 of 1.3 mg/kg, i.p. (Table 2). In contrast, the DA antagonist antipsychotic haloperidol induced catalepsy at 5 and 10 times its rat LMA ED50. For comparison, clozapine (5× its LMA ED50) and CI-1007 (10× its p.o. LMA ED50) also did not induce catalepsy (Table 2). 3.5. Effects in the water lick (Vogel) conflict test in rats PD 158771 dose-dependently antagonized shockinduced inhibition of drinking behavior (Fig. 4). Significant increases in shocks taken occurred at the 1 and 2 mg/kg i.p. doses. The anxiolytic standard chlordiaTable 1 Effect of PD 158771 and standard DA agents in locomotor activity studiesa

Fig. 3. Effect of PD 158771 on apomorphine (Apo)-stimulated (0.3 mg/kg, s.c.) locomotion in rats. Each vertical bar is the mean±S.E.M. of 4–10 rats. Apomorphine is dosed s.c. 15 min prior to s.c. PD 158771. Locomotor activity was then measured for 30 min. Control activity counts were 106±27. *P⬍0.05 from control, #P⬍0.05 vs apomorphine control.

ED50 values=0.16 vs 0.74 mg/kg, respectively). Several other partial DA agonists were tested for reversal of apomorphine-stimulated LMA. To attempt to correct for potency differences, the reversal of apomorphine-stimulated LMA ED50 was divided by the spontaneous LMA ED50. The locomotor activity ED50 values and the ratios calculated from these values for other partial agonists,

Compound

Spontaneous LMA ED50 (mg/kg, s.c)

Apomorphinestim. LMA ED50 (mg/kg, s.c.)

Ratio (apo-stim LMA/spont LMA ED50s)

Haloperidol

0.5 (0.34;0.72)

0.1

SDZ 208-912 Clozapine (⫺)-3-PPP Terguride

0.05 (0.03; 0.09) 1.3 (0.76; 2.3) 6.5 (4.1; 10.2) 2.5 (1.5; 4.1) 0.31 (0.18; 0.55) 15.4 (8.0; 29.5) 0.74 (0.36; 1.5)

5.6 (3.8; 8.1) 8.1 (3.4; 19.3) 2.1 (1.3; 3.5) 0.17 (0.09; 0.33) 5.2 (2.5; 10.7) 0.16 (0.11; 0.24) 0.27 (0.17; 5.9 (3.3; 10.5) 0.44) 0.54 (0.29; 1.0) ⬎30 0.024 (0.016; ⬎3.0 0.035)

CI-1007 PD 158771 EMD 38362 (+)-3-PPP B-HT 920

0.2 0.8 1.2 1.8 3.0 4.6 22 ⬎56 ⬎125

a Comparison of effects of PD 158771 with other antipsychotic agents, partial DA agonists and full DA agonists to inhibit spontaneous and apomorphine-stimulated (0.3 mg/kg, s.c.) locomotor activity in rats. Locomotor activity ED50 values and 95% confidence limits were calculated using regression analysis. The ratio of ED50 values for reversal of apomorphine-stimulated locomotor activity/spontaneous locomotor activity estimates the intrinsic activity at DA receptors. n=4–15 rats per dose, 3–5 doses per drug tested.

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Table 2 Effect of PD 158771 and other antipsychotics in the rat catalepsy testa Compound

Vehicle PD 158771 PD 158771 Haloperidol Haloperidol Clozapine CI-1007

Table 3 Effect of PD 158771 and standard agents in the rat water wheel behavioral despair testa

Dose Mean catalepsy score (max. 2.0) (mg/kg, i.p.)

0 13 26 1.0 2.0 36.0 30.0 (p.o.)

1h

2h

4 h postdose

0 0 0 0.5±0.1 1.2±0.2* 0 0

0 0 0 1.5±0.2* 1.7±0.1* 0 0

0 0 0 1.3±0.3* 1.9±0.1* 0 0

a Each score is the mean±S.E.M. of 6 rats. PD 158771 was tested at 10 and 20× the i.p. LMA ED50, haloperidol at 5 and 10×, clozapine at 5×, and CI-1007 at 10× the respective LMA ED50s. The maximum possible score is 2.0. *P⬍0.05 vs control.

Wheel rotations (5 min test) Compound

Dose Control Test drug (mg/kg, i.p.) (Mean±S.E.M.) (Mean±S.E.M.) 24 and 1 h pre-test

PD 158771

0.06 0.12 0.25 0.5 1.0 2.0 30.0 40.0 15.0 1.0 0.031 0.062 0.125 2.5 5.0 10.0 0.625 1.25 2.5

Imipramine Fluoxetine Gepirone 8-OH-DPAT Haloperidol (s.c.)

Clozapine

CI-1007

3.4±1.2 3.4±1.2 3.4±1.2 1.5±0.3 1.5±0.3 4.9±0.7 1.5±0.3 4.9±0.7 4.9±0.7 2.1±0.5 1.8±0.5 1.8±0.5 1.8±0.5 2.8±0.6 2.8±0.6 2.8±0.6 4.3±0.8 4.3±0.8 4.3±0.8

2.8±1.0 2.6±0.7 2.8±0.9 1.3±0.3 1.3±0.2 3.6±0.7 26.7±7.4** 13.1±1.1** 8.7±1.5* 32.0±5.0** 0.8±0.4 0.9±0.2 0.5±0.3 4.8±1.6 14.4±4.3* 22.3±3.3** 3.8±0.8 2.1±0.4* 1.8±0.4*

a Each value is the mean±S.E.M. of 12 rats. Rats were dosed with test compound at 24 and 1 h prior to test. *P⬍0.05, **P⬍0.01 vs corresponding control.

Fig. 4. Effect of PD 158771 on water lick conflict behavior. Each vertical bar is the mean±S.E.M. of 8 rats. Rats were dosed i.p. with drug 24 h prior and again at 30 min prior to a 10 min test. Chlordiazepoxide (CDZP) was included as a positive control. *P⬍0.05 vs control.

zepoxide (10 mg/kg, i.p. ), tested alongside PD 158771, also significantly increased the number of shocks taken, and is included in Fig. 4. For comparison, haloperidol, clozapine and CI-1007 did not significantly increase shocks taken at doses up to 0.25, 10 and 10, i.p., respectively (Ninteman et al., 1996). 3.6. Effects in the water wheel behavioral despair test in rats PD 158771 did not significantly increase wheel rotation over control levels during the 5 min test in rats (Table 3). These effects are different than those of the antidepressant agents imipramine and fluoxetine, and the 5-HT1A agonists gepirone and 8-OH-DPAT, all of which significantly increased wheel rotations over control level (Table 3). For comparison, haloperidol and CI-1007 also did not increase wheel rotations at doses up to 0.125 and

2.5, respectively, while clozapine produced significant, dose-dependent increases in wheel rotations up to 10 mg/kg, i.p. 3.7. Inhibition of avoidance responding in squirrel monkeys PD 158771 caused a steep, dose-related inhibition of conditioned avoidance responding in squirrel monkeys over a 6 h test period (Fig. 5). No psychomotor stimulation was observed with higher doses of PD 158771, indicating lack of postsynaptic DA receptor stimulation that has been observed with other partial agonists with higher levels of intrinsic activity. The ED50 for PD 158771 at the hour of peak activity (hour 3) was 0.42 mg/kg, p.o. (95%C.L.=0.39; 0.45). ED50 values for all compounds were calculated at the hour of peak activity. Although there are potency differences, the effects of PD 158771 are similar to haloperidol, clozapine, CI-1007 and SDZ 208-912 (Table 4). 3.8. Extrapyramidal dysfunction (EPD) in primates PD 158771 did not produce EPD in haloperidol-sensitized cebus monkeys at 0.3 mg/kg, p.o., a dose similar to the conditioned avoidance ED50 of 0.42 mg/kg in

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Table 5 EPD liability of PD 158771 and other antipsychotics in cebus monkeys sensitized to the dystonic effects of haloperidola Compound

Dose (mg/kg, p.o.)

Multiple of avoidance ED50

# with Cumulative EPS/# tested score

PD 158771

0.3 1.5 3.0 0.3 0.5 0.2 0.4 2.5 5.0

0.7× 3.7× 7× 0.5× 1× 1× 2× 4× 8×

0/2 3/3 2/2 3/4 4/4 3/4 3/3 0/4 2/4

Haloperidol SDZ 208-912 CI-1007

0 26.7±0.9 28.5±5.5 14.5±5.6 24.5±1.9 6.5±3.4 25.7±6.8 0 4.3±2.7

a Test compounds were administered p.o. to haloperidol-sensitized cebus monkeys. Monkeys were rated by trained observers for signs of EPD for 6-h postinjection (each score is the mean±S.E.M. of 2– 5 monkeys).

Fig. 5. Dose– and time–effect curves for the effects of PD 158771 dosed orally on conditioned avoidance responding in squirrel monkeys over a 6 h period. Each point is the mean±S.E.M. of six monkeys.

Table 6 EPD liability of PD 158771 and other antipsychotics in squirrel monkeys sensitized to the dystonic effects of haloperidola

Table 4 PD 158771 and other antipsychotics in the monkey conditioned avoidance testa

Compound

Dose (mg/kg p.o.)

Multiple of avoidance ED50

# with Cumulative EPS/# tested score

Compound

Avoidance ED50 at hour of peak activity (mg/kg, p.o.+95% C.L.)

PD 158771

SDZ 208-912 PD 158771 Haloperidol CI-1007 Clozapine

0.21 (0.12; 0.36) 0.42 (0.39; 0.45) 0.52 (0.50; 0.53) 0.6 (0.46; 0.87) 19.6 (15.3; 25.0)

1.5 3.0 1.5 2.0 3.0 0.4 1.0 2.5 5.0

3.7× 7× 3× 4× 6× 2× 5× 4× 8×

2/4b 4/4c 5/5 5/5 5/5 5/5 5/5 0/3 1/3

Haloperidol

a ED50s and 95% C.L. were calculated using regression analysis at the hour of peak avoidance activity during the 6 h test. Drugs were administered orally immediately before the test session. Three to four doses tested per compound. n=6 squirrel monkeys/dose.

squirrel monkeys (Table 5), yet higher doses produced EPD. Doses 3.7 times that of the conditioned avoidance ED50 of PD 158771 were required to achieve the same intensity of EPD as was produced by haloperidol (1×) or SDZ 208-912 (2× the avoidance ED50). CI-1007 produced no significant extrapyramidal side effects in cebus monkeys (Table 5). In squirrel monkeys, PD 158771 produced some EPD at 1.5 (in 2/4 monkeys) and 3.0 (in 4/4 monkeys) mg/kg, p.o. (Table 6), doses equivalent to 3.7 and 7 times the conditioned avoidance ED50. Haloperidol and SDZ 208-912 produced EPD in all monkeys at all doses tested, and even produced EPD at doses less than the avoidance ED50 value. CI-1007 produced no EPD at 4 times, and EPD in 2 of 4 cebus monkeys at 8 times the avoidance ED50. However, emesis occurred in two to three of the squirrel monkeys tested with PD 158771, making comparison to these agents difficult.

SDZ 208-912 CI-1007

2.8±2.4 9.0±1.2 8.0±1.9 17.6±4.0 23.8±2.9 16.4±3.3 14.8±4.7 0 3.0±3.0

a Test compounds were administered p.o. to haloperidol-sensitized squirrel monkeys. Monkeys were rated by trained observers for signs of EPD for 6-h postinjection (each score is the mean±S.E.M. of 2– 5 monkeys). b emesis in 3 of 4 monkeys. c emesis in 2 of 4 monkeys.

4. Discussion The present behavioral data, as well as the biochemical data in the accompanying paper (Akunne et al., 1999), indicate that PD 158771 is a D2/D3 partial agonist with 5-HT1A agonist actions that is active in preclinical tests predictive of antipsychotic activity. Several DA D2 partial agonists are known to produce antipsychotic-like effects in behavioral models of antipsychotic activity, yet little is known about the effects of DA D3 partial agonists in these models. It is unclear whether DA D3 receptors function as autoreceptors (Damsma et al., 1993; Meller et al., 1993), postsynaptic receptors (Svensson et al., 1994; Waters et al., 1993), or both (for a review, see Wustrow and Wise, 1997). DA

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D3 receptors have been proposed to be involved in the control of locomotor activity (Daly and Waddington, 1993; Svensson et al., 1994; Waters et al., 1993). DA D3 receptor agonists have been shown to reduce spontaneous locomotor activity and amphetamine-stimulated locomotion in rodents (Pugsley et al., 1995a; Svensson et al., 1994); however due to the lack of selectivity of these compounds, the contribution of DA D3 receptors remains unclear. It remains to be determined which, if any, of the behavioral effects of PD 158771 are due to specific actions at DA D3 receptors. ED50 values were compared for spontaneous LMA vs apomorphine-stimulated LMA as a measure of DA partial agonist/postsynaptic antagonist activity. Apomorphine and certain other DA agonists have been shown to reduce locomotor activity at lower doses (a presynaptic action) while stimulating activity at higher doses (a postsynaptic action). A dose of 0.3 mg/kg apomorphine was selected for these studies as this was the lowest dose that consistently increased locomotor activity in rats that have been acclimated to the test chambers. PD 158771 reduced apomorphine stimulated LMA in rats, an effect consistent with other partial DA agonists and with DA antagonists. The ratio for PD 158771 was only slightly higher than the ratio for the partial DA agonists (⫺)-3PPP, terguride and CI-1007. CI-1007 has been characterized as an autoreceptor agonist and partial DA agonist antipsychotic (Pugsley et al., 1995b; Meltzer et al., 1995), and is currently in development for the treatment of schizophrenia. As expected, the ratio for PD 158771 was also higher than the ratio calculated for the DA antagonists clozapine and haloperidol, and the weak partial DA agonist SDZ 208-912, which has been shown to produce behavioral effects similar to haloperidol (Clark et al., 1991). Conversely, PD 158771 is less agonist-like than EMD 38362 (HCl salt of roxindole), (+)-3-PPP and the full DA agonist B-HT 920, compounds with higher intrinsic DA agonist activity (Wiedemann et al., 1992). Similar potency and relative intrinsic activity profiles have been reported previously for SDZ 208-912, (⫺) 3PPP and (+)-3-PPP (Arnt and Hyttel, 1990; Clark et al., 1991; Svensson et al., 1991). Given the behaviorallyderived estimate of intrinsic activity for PD 158771 as a weak partial agonist, one may predict that PD 158771 may produce sufficient attenuation of excessive DA neurotransmission to produce an antipsychotic-like effect without causing a complete blockade of DA neurotransmission, and its accompanying neurological side effects, with chronic treatment. Moreover, PD 158771 like other DA autoreceptor agonists, caused a monophasic decline in locomotor activity and in reversing amphetamine-stimulated behavior in mice. These effects of PD 158771 appear to mainly reflect activation of functionally-defined presynaptic DA receptors. Similar potency and efficacy were observed in rats with PD 158771 in locomotor activity

studies by several routes of administration, with a rank order of potency i.v.⬎s.c.⬎i.p.⬎p.o. Given the wide range between the efficacious dose i.v. vs p.o. in rats, however, one might suspect low bioavailability with PD 158771. Consistent with its antipsychotic profile in rodents, PD 158771 also inhibited conditioned avoidance responding in monkeys, a learned behavior dependent upon intact central DA function. Known antipsychotics have been shown to possess potent DA D2 receptor antagonist actions in this model, and the effects of PD 158771 are similar to the effects of the potent DA antagonist antipsychotic haloperidol and the atypical antipsychotic clozapine. Given the antipsychotic-like partial DA agonist profile of PD 158771, it’s efficacy in this model, and the correlation observed between inhibition of conditioned avoidance in monkeys and antipsychotic efficacy (Heffner et al., 1989), one might predict clinical efficacy against the positive symptoms of schizophrenia. The lack of locomotor stimulation at the highest doses of PD 158771 tested in both mice and rats suggests a lack of postsynaptic DA receptor agonist effects which can exacerbate the positive symptoms of schizophrenia in man. The lack of stimulant effects with PD 158771 in monkeys at higher doses also suggests that it should lack the psychomotor agitation observed clinically with higher intrinsic activity DA agonists (such as Wiedemann et al., 1992). It has been postulated that 5-HT1A activity may influence both the therapeutic and extrapyramidal side effect liability of antipsychotic agents (Wadenberg et al., 1994; Meltzer, 1995a,b; Christoffersen and Meltzer, 1998). PD 158771 produced a flattened posture and hypothermia at efficacious doses in rodents (unpublished data), effects consistent with 5-HT1A agonism, although it is difficult to determine whether the hypothermia was due to the DA partial agonist profile, the 5-HT1A agonist profile, or some combination of both. 5-HT1A agonists have also been demonstrated to produce anxiolytic-like and antidepressant-like activity in preclinical tests, thus the 5-HT1A activity of PD 158771 may contribute to its anxiolyticlike effects in the Vogel conflict model. The increase in shocks taken with PD 158771 was similar to that of the anxiolytic chlordiazepoxide, and similar to the effects seen with diazepam, oxazepam and buspirone, yet different from the profile of haloperidol, clozapine and CI1007 (Ninteman et al., 1996). Although anxiolytic-like effects were not observed with clozapine in the Vogel conflict test, clozapine has been shown to have anxiolytic-like activity in other conflict models, possibly linked to the 5-HT1A partial agonist profile of clozapine. One may have predicted potential antidepressant activity in the water wheel behavioral despair model with PD 158771, given its binding activity at 5-HT1A receptors, and the fact that 5-HT1A agonists gepirone and 8-OH-DPAT increased wheel rotations in this model

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(Ninteman et al., 1995). Perhaps the negative result with PD 158771 indicates that a threshold level of intrinsic activity at 5-HT1A receptors may be necessary for antidepressant-like effects to be evident in behavioral models of depression. Interestingly, clozapine increased wheel rotations in this model, consistent with antidepressantlike activity, which may be explained by the 5-HT1A partial agonist profile of clozapine, while haloperidol and the DA partial agonist CI-1007 did not. Besides its affinity for DA D2 and 5-HT1A receptors, PD 158771 had moderate affinity for 5-HT2A receptors. Administration of direct 5-HT2A agonists such as quipazine elicits a characteristic head shaking behavior (head twitch) in mice, the pharmacology of which has been attributed to activation of 5-HT2A receptors. In our studies, no head twitches were observed, indicating that the moderate affinity of PD 158771 for 5-HT2A receptors does not suggest 5-HT2A agonist actions. Additionally, preliminary PanLab studies indicated that PD 158771 acted as a 5-HT2A antagonist in a rat aorta contraction assay. Certain atypical antipsychotic agents such as clozapine and risperidone have greater affinity for 5-HT2A receptors relative to their DA D2 affinities. Although the causal mechanism is not clear, antagonism of both DA D2 and 5-HT2A receptors is believed to reduce the motor side effects induced by DA D2 receptor antagonism in the basal ganglia. Since 5-HT2A antagonism together with DA D2 antagonism is thought to contribute to atypical antipsychotics, it is possible that the weaker affinity of PD 158771 for the 5-HT2A receptor together with its partial D2/D3 autoreceptor agonist and 5-HT1A agonist actions may also contribute to the atypical profile of PD 158771. The lack of catalepsy in rats at 10 and 20 times the i.p. LMA ED50 indicated that PD 158771 does not have a side effect profile similar to the typical DA antagonist haloperidol. Higher doses of PD 158771 were not tested, yet the apparent lack of effect of PD 158771 in this test is more consistent with that observed with the atypical antipsychotic, clozapine, which does not produce EPS in man, and the partial DA agonist antipsychotic CI-1007. The lack of catalepsy could be due to the 5-HT1A agonist actions and/or the possible 5-HT2A antagonist effects of PD 158771. One might postulate from these data that PD 158771 does not have the propensity to produce dystonias in man that have been associated with the blockade of DA receptors. The 5-HT1A and 5-HT2A activity of PD 158771 may also contribute to its improved profile compared to standard antipsychotics in EPD studies. However, in contrast to the rat catalepsy study, PD 158771 did produce EPD in haloperidol-sensitized cebus monkeys comparable in intensity to that produced by haloperidol and the weak partial DA agonist SDZ 208-912. However, it is important to note that PD 158771 may have a lower liability for producing EPS in humans than haloperidol

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because a higher multiple of the conditioned avoidance ED50 of PD 158771 is required (3.7×) to achieve a similar magitude of EPD produced by haloperidol (1×) and SDZ-208-912 (2×) in cebus monkeys. The catalepsy model is performed in experimentally naive rats, whereas the EPD model utilizes monkeys that have been continually sensitized to the dystonic effects of the DA antagonist haloperidol. Therefore, there can be discrepancies between the catalepsy and EPD data, as the EPD model is a much more sensitive model for predicting side effects of potential antipsychotic agents. The efficacy in the conditioned avoidance test and EPD model in sensitized monkeys are thought to reflect clinical efficacy and the propensity to induce EPS in schizophrenic patients. In addition, it should be pointed out that the dose–effect curves for the conditioned avoidance and EPD tests in monkeys are quite steep, accounting for the large changes in activity associated with small increments in dosage. Haloperidol is active in conditioned avoidance and EPD tests in monkeys at about equivalent doses, and this reflects well its clinical profile, where efficacy and EPS are observed at similar doses. In comparison, although PD 158771 induced EPD in monkeys equivalent to that of haloperidol, it was at a dose 3-fold higher than the dose active in conditioned avoidance responding in monkeys. Thus, it is possible that PD 158771 at clinically effective doses will have a lower propensity to induce EPS in schizophrenic patients than classical antipsychotics like haloperidol. The partial agonist nature of PD 158771 at DA D2 receptors in addition to it possessing 5-HT1A and 5-HT2A affinities would certainly support such a prediction. However, only clinical trials will determine whether PD 158771 is both efficacious and has a lower incidence of EPS than haloperidol. Lower liability for EPS in humans would also be predicted for CI-1007, like PD 158771, because higher multiples of the avoidance ED50 are required to produce EPD in cebus monkeys. The only antipsychotic with essentially no effect in the cebus EPD model is clozapine (Casey, 1991), which produces essentially no EPS in humans (Kane et al., 1988; Meltzer, 1989; Gerlach et al., 1996). Clozapine showed essentially no EPD in either the cebus or squirrel monkey models (data not shown), however only doses less than or equivalent to the avoidance ED50 (20 mg/kg) were tested, as higher doses tend to produce toxicity. Because PD 158771 produced emesis of unknown origin in the squirrel monkeys at both doses tested, it is difficult to compare it with other reference agents included in Table 6. Nevertheless, the data is included for reference comparison. Interestingly, the slightly reduced liability to induce EPD with PD 158771 in cebus monkeys vs haloperidol and SDZ 208-912 (Table 5) was similar to the profile observed with these compounds in the reversal of apomorphinestimulated locomotor activity model in rats (Table 1).

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Specifically, PD 158771 does not have a low ratio like haloperidol and SDZ 208-912, compounds which produce EPS in man (Naber et al., 1992) and EPD in monkeys at lower multiples of the avoidance ED50 than PD 158771. Based on the correlation between EPD liability in monkeys and EPS in man (Casey, 1991; Heffner et al., 1989), coupled with the lack of catalepsy observed in rats, PD 158771 may have a reduced liability for induction of neurological side effects in man compared to typical DA antagonist antipsychotics. In conclusion, the 5-HT1A agonist and DA D2/D3 partial agonist, PD 158771, resembles clinically effective antipsychotic drugs in its effects on rodent locomotor and monkey conditioned avoidance tests. In addition, the 5-HT1A agonist (anxiolytic-like) properties of PD 158771 may improve antipsychotic efficacy relative to the classical antipsychotics by alleviating stress effects believed to exacerbate symptoms of schizophrenia (van Kammen et al., 1989). While PD 158771 caused some EPD in haloperidol-sensitized cebus monkeys, its apparent lack of activity in the catalepsy test, along with its 5-HT1A agonist properties and possible 5-HT2A receptor antagonist effects, suggest that it may show an improved EPS profile in humans compared to typical DA antagonist antipsychotics that lack serotonergic affinity. These data suggest that PD 158771 may be a potent and efficacious antipsychotic agent with reduced liability for EPS in man than currently available DA antagonist antipsychotic agents.

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