Potential antidepressant properties of SR 57746A, a novel compound with selectivity and high affinity for 5-HT1A receptors

ejp ELSEVIER

European Journal of Pharmacology253 (1994) 139-147

Potential antidepressant properties of SR 57746A, a novel compound with selectivity and high affinity for 5-HTIA receptors Luigi Cervo, Caterina Bendotti, Gianluca Tarizzo, Alfredo Cagnotto, Malgorzata Skorupska, Tiziana Mennini, Rosario Samanin * Istituto di Ricerche Farmacologiche 'M. Negri', Via Eritrea 62, 20157 Milan, Italy

(Received 9 August 1993; revised MS received 3 December 1993; accepted 7 December 1993)

Abstract

SR 57746A, 4-(3-trifluoromethylphenyl)-N-[2-(naphth-2-yl)ethyl]-l,2,3,6-tetrahydropyridine HCI, was studied for its specific 5-HTIA receptor agonist action and antidepressant-like effects in the rat. The compound showed a high affinity for 5-HTIA specific binding sites in the rat hippocampus (IC50 3 nM), moderate affinity ( 1 0 - 7 - 1 0 - 6 M) for dopamine D 2 receptor, 5-HT uptake, 5-HT 2 and al-adrenoceptor binding sites and practically no effect on binding sites of monoamine, GABAA, benzodiazepine and histamine receptors. It inhibited forskolin-stimulated adenylate c3zclase activity in rat hippocampal membranes at concentrations of 10 - 6 and 10 -5 M. The effect of 10 - 6 M SR 57746A on forskolin-stimulated adenylate cyclase activity was completely antagonized by 10 -6 M (-)-propranolol. Administered per os as a three-dose course to rats, SR 57746A significantly increased struggling in the forced swimming test at doses from 0.3 to 3 mg/kg. Single doses had no such effect. The effect of a three-dose course with 1 mg/kg SR 57746A on rats' struggling was antagonized by pretreatment with 5 mg/kg i.p. metergoline, a non-selective 5-HT receptor antagonist, and by 20 mg/kg i.p. (-)-propranolol, an antagonist at 5-HT 1 receptors. Three oral doses of 100 mg/kg parachlorophenylalanine, an inhibitor of 5-HT synthesis, and 100 mg/kg i.p. (+)-sulpiride, an antagonist at dopamine D 2 receptors, also antagonized the effect of SR 57746A in the forced swimming test. The results show that SR 57746A has selectivity and high affinity for 5-HT1A receptors. The compound is a full agonist at this receptor and shows an antidepressant-like effect in the forced swimming test by a mechanism which seems to involve presynaptic 5-HTIA receptors and a permissive role of brain dopamine D 2 receptors. Key words: SR 57746A (4-(3-triflu•r•methy•pheny•)-N-[2-(naphth-2-y•)ethy•]-1•2•3•6-tetrahydr•pyridine

HCI); 5-HTIA receptor;

Adenylate cyclase activity; Forced swimming test; Antidepressant activity

1. Introduction

Considerable evidence suggests that brain serotonin (5-HT) is involved in the antidepressant activity, but it is not clear whether an increase or reduction in 5-HT transmission is important for this therapeutic effect (see Garattini and Samanin, 1988). The recent finding that 8-hydroxy-2-(di-n-propylamino)tetralin (8-OHDPAT), an agonist at 5-HT1A receptors (Middlemiss and Fozard, 1983), shows antidepressant-like effects in the forced swimming test (Cervo and Samanin, 1987a) and in the learned helplessness model (Giral et al., 1988) has opened up new prospects on the role of

* Corresponding author. Tel. (02) 390141, telex 331268 NEGRII. 0014-2999/94/$07.00 © 1994 Elsevier Science B.V. All rights reserved SSDI 0014-2999(93)E0892-V

5-HT receptor types in the aetiology of depression and in antidepressant activity. Buspirone and gepirone, two 5-HT1A receptor agonists, were recently found to have favourable effects in the treatment of depression (Robinson et al., 1990; Jenkins et al., 1990). Preliminary studies on the specific binding of radiolabelled ligands to various neurotransmitter receptors suggested that SR 57746A, 4-(3-trifluoromethylphenyl)N-(2-(naphth-2-yl)ethyl]- 1,2,3,6-tetrahydropyridine HCI (Bianchetti et al., 1990), has high affinity and specificity for 5-HT1A binding sites and is therefore of potential use in the treatment of depression. We studied the ability of SR 57746A to inhibit the specific binding of radiolabelled ligands to various neurotransmitter receptors. To ascertain whether the compound showed 5-HTIA receptor agonist activity we

140

L. Cervo et al. / European Journal of Pharmacology 253 (1994) 139-147

studied its effect, in comparison with that of 8-OHD P A T , on forskolin-activated adenylate cyclase activity in rat hippocampal membranes. In another series of experiments we examined the effect of various doses o f S R 57746A on the behaviour of rats in the forced swimming test. T h e role of 5-HT1A receptors was assessed by studying the effect of SR 57746A in rats treated with metergoline, a non-selective 5 - H T receptor antagonist, (Hoyer, 1988) and ( - ) propranolol, an antagonist at 5 - H T 1 receptors (Hoyer, 1988). Parachlorophenylalanine, an inhibitor of 5 - H T synthesis, was used to establish w h e t h e r 5-HT-containing neurons were involved. Parachlorophenylalanine was recently f o u n d to block the effect of 8 - O H - D P A T on the struggling of rats in the forced swimming test (Cervo and Samanin, 1991). Since it has b e e n suggested that brain d o p a m i n e plays a permissive role in the antidepressant effect of various drugs, including 8 - O H - D P A T (Borsini et al., 1984, 1985; Cervo and Samanin 1987a,b, 1988; Cervo et al., 1990), the effect of S R 57746A was also studied in

Table 1 Conditions for binding assays Receptor and brain a r e a 5-HTIA Hippocampus 5-HTIB Striatum 5-HTlc Pig frontal cortex 5-HT2 Prefrontal cortex 5-HT3 Cortex Benzodiazepine Hippocampus Benzodiazepine 'Peripheral' hippocampus Serotonin uptake site Cortex aI-NA Total brain minus cerebellum a 2-NA Total brain minus cerebellum /31-NA Cortex /32-NA Cerebellum D1 Striatum D2 Striatum GABAA Cortex H 1

Total brain minus cerebellum

3H ligand (nM) [ 3H]8-OH-DPAT (1) [3H]Serotonin (2) [3H]Mesulergine (1) [3H]Ketanserin (1) [3H]BRIA3694 (1) [3H]Flunitrazepam (1) [ 3H]PK 11195 (1) [3H]Paroxetine (0.1) [3H]Prazosin (0.2) [3H]Clonidine (1) [3H]Dihydroalprenolol (1) [ 3H]DHA (1) [3H]SCH 2 3 3 9 0 (0.4) [3H]Spiperone (0.2) [3H]Muscimol (2) [3H]Mepyramine (2)

rats treated with sulpiride, a selective d o p a m i n e receptor blocker (Jenner and Marsden, 1981).

2. Materials and methods 2.1. Animals Male C D - C O B S rats (Charles River, Italy) were used. T h e animals were h o u s e d four to a cage at constant r o o m t e m p e r a t u r e (21 + I°C) and relative humidity (60%), with water and food ad libitum. 2.2. Biochemical experiments 2.2.1. Binding assay Rats, weighing about 150 g, were killed by decapitation and their brains were quickly removed. T h e various brain regions were dissected according to Glowinski and Iversen (1966) and Invernizzi et al. (1991). Pig brains were obtained from a local slaughterhouse and placed on ice immediately after death. Frontal cerebral

Non-specific binding (/zM) Serotonin (1) Serotonin (10) Mesulergine (10) Methysergide (1) GR38032 (10) Clonazepam (1) PK 11195 (1) Fiuoxetine (10) Phentolamine (3) ( - )-Noradrenaline (100) (+)-Propranolol (1) ( + )-Propranolol (1) (-)-c/s-Flupentixol (10) ( - )-Sulpiride (100) GABA (20) Mepyramine (1)

Incubation

Reference

30 min, 25°C

Hall et al., 1985

30 min, 25°C

Blurton and Wood, 1986

30 min, 370C

Pazos et al., 1984

15 min, 37°C

Leysen et al., 1982

30 min, 25°C

Nelson and Thomas, 1989

90 min, 0°C

Ehlert et al., 1981

120 min, 4°C

Benavides et al., 1983

60 min, 22°C

Habert et al., 1985

30 min, 25°C

Greengrass and Bremner, 1979

20 min, 25°C

Greenberg et al., 1976

20 rain, 250C

Bylund and Snyder, 1976

20 rain, 25°C

Bylund and Snyder, 1976

15 min, 370C

Billard et al., 1984

15 min, 37°C

Creese et al., 1978

30 min, 0°C

Herschel and Baldessarini, 1979

30 min, 25°C

Tan Tran et al., 1978

L. Cervo et al. / European Journal of Pharmacology 253 (1994) 139-147

cortices were dissected and stored at - 80°C until used. The conditions used for the binding assay are summarized in Table 1. Samples were filtered through Whatman G F / B glass fibre filters using a Brandel apparatus (rood 48RP) and washed 3 times with 4 ml of ice-cold buffer. The radioactivity trapped on the filters was counted in 8 ml of Filter Count (Packard) in an LKB 1214 liquid scintillation spectrometer, with a counting efficiency of 57%. For inhibition experiments, the drugs were added to the binding mixture at 5-7 different concentrations. Inhibition curves were analysed using the 'Allfit' program (De Lean et al., 1978) running on an IBM AT-PC.

2.2.2. Adenylate cyclase assay Adenylate cyclase was assayed as described by Salomon et al. (1974). Freshly dissected hippocampi were homogenized with a Teflon-glass pestle with 10 volumes of 5 mM Tris-HC1 pH 7.4 containing 10% sucrose, 1 mM ethyleneglycol-bis-(/3-aminoethyl ether)N,N'-tetraacetic acid (EGTA), 1 mM dithiothreitol. The homogenate was centrifuged at 900 x g for 10 min and the supernatant was then centrifuged at 9000 x g for 20 min. The pellet from the second centrifugation was resuspended in 20 volumes of dithiothreitol and recentrifuged at 9000 X g for 20 min. The final pellet containing the crude membrane preparation was resuspended in 30 volumes of dithiothreitol and the suspension was kept on ice until used in the assay. Adenylate cyclase activity was assayed in an incubation mixture of 120/zl containing 75 mM Tris-HCl pH 7.4, 0.33 mM EGTA, 0.5 mM isobutylmethyl-xanthine, 1 mM cyclic AMP, 0.35 mM dithiothreitol, 0.1 mM GTP, 2 mM MgCI2, 0.1 mM unlabelled ATP, 1 /zCi [a-32p]ATP, 5 mM creatine phosphate, 5 U creatine phosphokinase, 50/zg bovine serum albumin, 100 mM NaCI, 10 /zM forskolin and various concentrations (10 - 9 to 10 - 5 M ) o f 8-OH-DPAT or SR 57746A. In another experiment ( - )-propranolol 10 ~ M plus 1/zM 8-OH-DPAT or plus 1 /zM SR 57746A were added to the incubation mixture. The enzymatic reaction was started by adding an aliquot of the crude membrane suspension at a final protein concentration of about 1 mg/ml. The mixture was incubated at 30°C for 5 min and the reaction was terminated by addition to each tube of 200 /zl of a solution containing 2% sodium dodecyl sulphate, 45 mM ATP, and 1.3 mM cAMP, followed by heating of the sample for 3-5 min at 90°C. The [32p]cAMP formed was purified by chromatography on a Dowex AG 50WX4 (200-400 mesh, chloride form, 1 ml) and alumina column (AG7 Bio-Rad). cAMP recovery was monitored by adding 2.5 nCi (5000 cpm) of [3H]cAMP to each sample. Each experiment was done in triplicate. The protein content was measured spectrophotometrically according to Lowry et al. (1951).

141

2.3. Behavioural experiments in rats 2.3.1. Swimming behaviour and open field activity Rats, weighing 220-250 g, were placed individually in Plexiglas cylinders (height 40 cm, diameter 18 cm) containing 17 cm of water at 25°C, and 15 min later they were removed to a 30°C drying room for 30 min (pre-test). For drug testing animals were replaced in the cylinders 24 h later and their behaviour during a 5-min test period was quantified by measuring the time they spent struggling or floating. Struggling was defined as the amount of time the animal spent making vigorous attempts to escape, so that all four limbs were moving, generally against the wall of the cylinder, with the front limbs breaking through the surface of the water. The time spent diving, searching for an underwater way out of the cylinder, was included in this behaviour. Floating was defined as the amount of time the animal remained passively in the water. During this period the rat rests, keeping its head at the surface of the water, in a horizontal or vertical posture with all four limbs motionless, without sinking. The remaining activity, consisting mainly of treading water, was not recorded; during this period the animal maintained a vertical position with the head at the surface of the water by an action as if walking upstairs, or 'dog-paddling' around in the cylinder; sinking below the water was also observed. Behavioural responses were timed by two observers who did not know which treatment the rats had received. In order to see whether changes in immobility were associated with changes in motor activity, naive animals treated with SR 57746A were tested for activity in an open field. The apparatus was a square box (100 × 100 cm) with 40 cm high plastic walls. The floor consisted of a sheet of white plastic painted with a black grid dividing the field into 25 (5 x 5) equal squares. Illumination was identical to that used for the immobility test and the animals underwent the same treatment schedules. One hour after SR 57746A the animals were placed in a comer of the apparatus and the number of times each rat entered a square with all four paws in 5 min was recorded by an observer who was unaware of the treatments. 2. 4. Drug treatments 2.4.1. Forced swimming In the first experiment, two groups of 32 animals underwent different treatment schedules with SR 57746A (Research Center Sanofi, Midy, Milan, Italy). For the single administrations, different groups (n = 8) received a single oral dose of SR 57746A (0.3-3.0 mg/kg) or vehicle 1 h before testing. For the triple

L. Cervo et aL / European Journal of Pharmacology 253 (1994) 139-147

142

administrations, different groups (n = 8) received various doses of SR 57746A (0.3-3.0 mg/kg) 23.5, 5 and 1 h before the test, with the first dose given 30 min after the pre-test. In the second experiment, the effect of a three-dose treatment with 1 mg/kg SR 57746A in 24 h, as stated above, was evaluated in separate groups (n = 8) of naive rats that had received different treatments. The doses, routes and pretreatment times before the last dose of SR 57746A were those reported to have significant effects on 5-HT or dopamine mechanisms (the appropriate references for each compound are given in brackets): (-)-propranolol hydrochloride (Icpharma, Milan, Italy) 20 mg/kg s.c., 30 min (Middlemiss et al., 1977; Nahorski and Willcocks, 1983); metergoline maleate (Farmitalia-Carlo Erba, Milan, Italy) 5 mg/kg i.p., 180 min (Romandini and Samanin, 1984); (+)sulpiride, Dobren (Ravizza, Milan, Italy) 100 mg/kg i.p., 30 min (Ljungberg and Ungerstedt, 1978). In the last experiment the effect of three doses of SR 57746A in 24 h was studied in 32 animals given 100 mg/kg parachlorophenylalanine (Janssen, Beerse, Belgium) orally once daily for 3 days, the last dose being

given 48 h before testing. Thirty-two rats that had received the vehicle (0.5% carboxymethylcellulose) were used as controls. Two hours after testing, vehicle- and parachlorophenylalanine-treated animals were killed by decapitation for biochemical assay of 5-HT. The brains were rapidly removed, the cerebella were discarded, and the remaining tissues were frozen on dry ice and stored at -20°C until used in the assay. 5-HT was measured by high-performance liquid chromatography with electrochemical detection according to Invernizzi et al. (1989). 2. 4.2. Open field Three groups of eight naive rats received orally 1.0 or 3.0 mg/kg SR 57746A or vehicle as a three-dose course 23.5, 5 and 1 h before the 5-min test. 2.5. Animal care Procedures involving animals and their care were conducted in conformity with the institutional guidelines that are in compliance with national and international laws and policies (EEC Council Directive

5HT 2 Receptors

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log [drug] Fig. 1. Inhibition curves for SR 57746A at 5-HT1A, 5-HT 2, 5-HT uptake and al-adrenoceptor sites, x axis = log [M] of SR 57746A. y axis = cpm 3H ligand bound per sample. Each point is the m e a n of triplicate samples. Curves were fitted to the experimental points by non-linear regression, using the 'Allfit' program which gives m a x i m u m and m i n i m u m effects, slopes and drug concentrations with half-maximal effect (ICs0). The ICs0 _+ S.E. were as follows: 5-HT1A = 3.3 _+ 0.28 nM; 5-HT 2 = 697 _+ 256 nM; 5-HT uptake = 389 _+ 95 riM; a l - N A = 634 + 208 nM.

L. Cervo et aL / European Journal of Pharmacology 253 (1994) 139-147

143

86/609, OJL 358, 1, December 12, 1987; NIH Guide for the Care and Use of Laboratory Animals, NIH Publication No. 85-23, 1985).

Table 2 Inhibitory effect of 8-OH-DPAT and SR 57746A on forskolin-stimulated adenylate cyclase activity in rat hippocampal membranes: antagonism by (-)-propranolol

2.6. Statistics

Drugs

(mean+ S.E.)

The adenylate cyclase activity data were analysed by analysis of variance with randomized blocks design followed by Dunnett's test and by factorial analysis of variance within subjects followed by Tukey's test for multiple comparisons. The effects of SR 57746A in the forced swimming test were analysed by one-way analysis of variance, and post-hoc comparisons with the vehicle-treated group were made with Dunnett's test (two-tailed). The data from interaction experiments were analysed by two-way analysis of variance (ANOVA). Multiple post-hoc comparisons between groups were made with Tukey's test for unconfounded means. Statistical analysis of the data from motor activity experiments was done by non-parametric analysis of variance (Kruskall-Wallis test). Post-hoc comparisons with the control group (saline treated) were made using Ryan's procedure. The biochemical data were analysed by Student's t-test.

3. Results

3.1. Effects on receptor binding sites Fig. 1 shows the inhibition curves of SR 57746A for 5-HTIA, 5-HT2, 5-HT uptake sites and ~l-adrenoceptors. SR 57746A had an IC50 value of 3 nM at the 5-HTIA sites defined by the specific binding of [3H]8OH-DPAT (1 nM) in hippocampal membranes whereas

Ao =

.~~

.~

110] 90-t

=

80

kS

=="2 60

Forskolin 10/z M Forskolin 10/zM + 8-OH-DPAT 1 p.M Forskolin 10/zM +SR 57746A 1/~M

Control

( - )-Propranolol

61.4 + 2.8

58.7 + 2.9

51.3+2.1 a

58.1 +2.7

52.2+2.2 a

57.8-1-2.7

The data points represent the means + S.E. of four separate experiments, each done in triplicate. Basal levels were calculated to be 12.6+ 1.4 pmol c A M P / m i n / m g protein. A concentration of 10 ~M (-)-propranolol was used. Data were analysed by factorial analysis of variance within subjects followed by Tukey's test for multiple comparisons. Fint (d.f. 2,6) = 94.37, P < 0.01. a p < 0.01, Tukey's test compared to forskolin.

4 x 10 -7, 7 x 10 -7 and 6 x 10 -7 M were required to achieve half-maximal inhibition of the specific binding of 3H ligands to 5-HT uptake, 5-HT z and al-adrenergic binding sites. SR 57746A at 10 -5 M had no appreciable affinity for any of the other receptor binding sites. It was noted that the curve for inhibition by SR 57746A of the specific binding of [3H]spiperone had a slope significantly lower than 1 and that maximal inhibition of total [3H]spiperone binding by SR 57746A was considerably lower than that exerted by sulpiride (43 and 68% respectively). Thus, depending on whether the IC50 was calculated on the basis of specific binding defined by sulpiride or the drug concentration giving half-maximal effect, a value of 1.2 + 0.6 ~M or 263 + 29 nM was obtained. 8-OH-DPAT instead had the same slope and maximal inhibition of [3H]spiperone binding as sulpiride, with an IC50 of 1.9 + 0.3 x 10 -5 M.

3.2. Effects on adenylate cyclase activity

100]

~

cAMP p m o l / m i n / m g protein

&

-

, -7

, -6

, -5

Log[Drug](M) Fig. 2. Effect of SR 57746A and 8-OH-DPAT on forskolin-stimulated adenylate cyclase activity in rat hippocampal membranes.The data points represent the means + S.E.M. of three separate experiments each done in triplicate. ( o ) 8-OH-DPAT, ( 0 ) SR 57746A. Forskolin-stimulated activity and basal activity were calculated to be 51.0+3.0 pmol and 9.7+0.4 pmol of c A M P / m i n / m g of protein, respectively, a p < 0.01, Dunnett's multiple comparisons with forskolin stimulation in the absence of drugs.

As shown in Fig. 2, SR 57746A was less potent than 8-OH-DPAT in inhibiting forskolin-stimulated adenylate cyclase activity in rat hippocampal membranes. Only concentrations of 10 -6 and 10 -5 M of the compound produced a significant effect, whereas 8-OHDPAT was also effective at the concentration of 10 -7 M. The effect of 1 tzM SR 57746A and 8-OH-DPAT was completely antagonized by 10/~ M (-)-propranolol (Table 2). (-)-Propranolol by itself did not modify forskolin-stimulated adenylate cyclase activity.

3.3. Effects in the forced swimming test Table 3 shows the effect of SR 57746A on the rats' behaviour in the swimming test and their open field activity. Single doses of 0.3-3 mg/kg SR 57746A did

144

L. Cervo et al. / European Journal of Pharmacology 253 (1994) 139-147

not modify the time the animals treated with the vehicle spent in struggling (F(3,28) = 1.2, P > 0.05) whereas a significant reduction of floating (F(3,28 = 4.2, P < 0.05) was found with the highest dose ( P < 0.05, Dunnett's t-test). Administered as a three-dose treatment, SR 57746A significantly increased the time the animals spent struggling (F(3,28) = 6.1, P < 0.01). Post-hoc comparison with the vehicle-treated group showed the difference was more significant with 3 mg than with 1 or 0.3 mg ( P < 0.01 and P < 0.05 respectively, Dunnett's t-test). The compound also reduced the time the animals spent floating (F(3,28) = 6.1, P < 0.01). Posthoc comparison with the vehicle-treated group showed the difference was significant with 1 and 3 mg ( P < 0.01, Dunnett's t-test), while the lower dose had no such effect ( P > 0.05, Dunnett's t-test). The animals' activity in the open field was not significantly modified by SR 57746A. Table 4 shows the effect of a three-dose treatment with 1 m g / k g SR 57746A on struggling and floating by rats that had received various treatments aimed at impairing serotonin transmission, or ( _+)-sulpiride. Metergoline 5 m g / k g completely antagonized the effect of 1 m g / k g SR 57746A, administered as a three-dose treatment, on the time the animals spent struggling (F(1,28)SR 57746A~-- 26.1, P < 0.01, F(1,28)metergolin e = 2.3, P > 0.05, F(1,28)SR 5 7 7 4 6 x m e t e r g o l i n e ~--- 9.3, P < 0.01, A N O V A 2 X 2). The effect of SR 57746A was also antagonized by 20 m g / k g ( - ) - p r o p r a n o l o l (F(1,28)SR 57746A = 11.0, P < 0.01, F(1,28)(_)_propranoloI = 3.2, P > 0.05, F(1,28)SR 5 7 7 4 6 A × ( - ) - l a r o p r a n o l o l = 7.2, P < 0.05, A N O V A 2 X 2), 100 X 3 m g / k g parachlorophenyl-

Table 4 Effects of various treatments on the forced swimming behaviour of rats treated with SR 57746A Treatment

Struggling (s)

Floating (s)

O p e n field (total counts)

Single Vehicle 0.3 1.0 3.0

49.3 -+ 7.8 69.3 + 8.6 76.4_+ 10.0 63.3+14.1

133.9 + 12.8 136.3 + 16.5 132.6 _+16.9 73.3+12.7 b

45.7+ 6.7 81.3÷ 10.8 b 85.7_+ 7.1 b 100.15:11.7 a

122.9+12.3 80.45:18.1 40.7+14.1 a 39.35:13.2 a

Three doses in 24 h Vehicle 0.3 1.0 3.0

58.7_+ 7.3 73.0 _+12.6 60.3 -+ 7.8

Values are the means-+ S.E. of 8 rats. The single and last doses of SR 57746A were administered orally 1 h before the 5 min test. T h e forced swimming results were analysed by analysis of variance and post-hoc comparison with the control group using D u n n e t t ' s test. Data from the open field experiment were analysed by non-parametric analysis of variance (Kruskall-WaUis test). Post-hoc comparisons with the control (vehicle) condition were made according to Ryan's procedure. a p < 0.01, b p < 0.05 vs. respective vehicle, D u n n e t t ' s test.

(s)

51.6_+ 105.0461.6+ 75.0+

Vehicle + vehicle Vehicle + SR 57746A ( - )-Propranolol + vehicle ( - )-Propranolol + SR 57746A

36.1-+ 6.2 161.6+15.2 102.8+12.5 a 59.5-+13.6 a 46.0_+ 12.6 134.6 + 14.0 53.0-+11.8 d 115;9-+19.3 d

6.9 164.0_+16.4 8.1 a 56.9+10.3 a 7.1 110.0-+14.7 2.6 c 40.1+ 7.6 a

46.5-1- 8.0 189.0-+ 9.3 Vehicle + vehicle 108.5_+13.5 a 89.2_+17.3 a Vehicle + SR 57746A 52.0+ 6.4 174.7_+16.3 Parachlorophenylalanine + vehicle Parachlorophenylalanine + SR 57746A 55.2_+ 7.1c 39.4_+ 7.9 a Vehicle + vehicle Vehicle + SR 57746A ( -+ )-Sulpiride + vehicle ( -+ )-Sulpiride + SR 57746A

45.9_+ 6.7 145.4_+ 8.7 105.1 _+ 13.4 a 61.7_+ 10.3 a 34.4-1- 6.5 162.2_+15.2 51.0_+ 7.3 d 105.4+22.1 b

Values are means_+ S.E. of 8 rats. Three doses of 1.0 m g / k g SR 57746A suspended in 0.5% carboxymethylcellulose were administered orally 23.5, 5 and 1 h before the test. Injection times before the test were 240 min for metergoline (5 m g / k g ) and 90 rain for ( - ) - p r o p r a n o l o l (20 m g / k g ) and (_+)-sulpiride (100 m g / k g ) . Parachlorophenylalanine 100 m g / k g or vehicle (0.5% carboxymethylcellulose) was administered orally once daily for 3 days. T h e last dose of parachlorophenylalanine was given 48 h before the test. The data were analyzed by factorial analysis of variance ( A N O V A 2 x 2 ) followed by Tukey's test. a p < 0.01, b p < 0.05 vs. respective vehicle, Tukey's test. e p < 0.01, dp < 0.05, A N O V A 2 X 2. See Results for details.

alanine

Treatment

Floating

Vehicle + vehicle Vehicle + SR 57746A Metergoline + vehicle Metergoline + SR 57746A

(F(1,28)SR

57746A = 12.6, P < 0.01, 6.7, P < 0 . 0 5 , F ( 1 , 2 8 ) S R 57746Axparachlorophenylalanine = 10.2, P < 0.01, A N O V A 2 x 2) and 100 m g / k g (_+)-sulpiride (F(1,28)sa 57746A= 17.9, P < 0.01, F(1,28)(±)_sulpiride = 13.4, P < 0.01, F(1,28)SR 5 7 7 4 6 A X ( z t z ) - s u l p i r i d e ~-- 5 . 6 , P < 0.05, A N O V A 2 x 2). As previously found with parachlorophenylalanine and 8-OH-DPAT, the effect on floating was not antagonized by any treatment, with the exception of ( - ) propranolol (F(1,28)SR 57746A= 14.8, P < 0.01, F (l,28)(_)_propranolo I = 0.9, P > 0.05, F(1,28)SR 57746A×(-)-propranolol= 7 . 0 , P < 0.05, A N O V A 2 X 2). Brain levels of 5-HT were markedly reduced in parachlorophenylalanine-treated rats. Levels of 5-HT ( n g / g S.E.) were controls 271 + 8.2 and parachlorophenylalanine-treated rats 25 + 2, P < 0.01 (Student's t-test). None of the treatments induced any overt behavioural change or motor dysfunction which could have interfered with the effect of SR 57746A on struggling. This was borne out by the fact that the time spent struggling or floating by animals that had received the various treatments was not significantly different from that of controls.

F(1,28)parachlorophenylalanine = Table 3 Effects of SR 57746A on forced swimming behaviour and open field activity of rats

Struggling

(s)

L. Cervo et al. / European Journal of Pharmacology 253 (1994) 139-147

4. Discussion

SR 57746A showed high affinity for the 5-HT1A specific binding sites in the rat hippocampus (IC50 = 3 nM), moderate affinity (10-7-10 -6 M) for 5-HT uptake, 5-HT 2 and al-adrenoceptor binding sites and practically no effect on the other binding sites examined. The receptor binding profile of SR 57746A has some similarities with and differences from those of other compounds having high affinity for 5-HT1A receptor binding. At 5-HT uptake sites, SR 57746A had an affinity similar to that of 8-OH-DPAT (Van Wijngaarden et al., 1990), while flesinoxan, ipsapirone and buspirone were not active. Unlike 8-OH-DPAT (Van Wijngaarden et al., 1990), SR 57746A was not active on aE-adrenoceptor binding. SR 57746A had an affinity for dopamine D E binding sites similar to that of flesinoxan and buspirone but higher than that of ipsapirone and 8-OH-DPAT (Van Wijngaarden et al., 1990; Neale et al., 1987). The same occurred for the al-adrenoceptor site, with SR 57746A and 8-OH-DPAT having the highest and lowest affinity respectively. SR 57746A showed an affinity for the 5-HT 2 binding sites higher than that of the other compounds, with 8-OHDPAT being the least active (Van Wijngaarden et al., 1990). Although SR 57746A had moderate affinity for dopamine D 2 receptors, 5-HT uptake sites, 5-HT 2 and al-adrenoceptors, the compound had a high degree of selectivity for 5-HT1A receptors, as shown by the 5HT1a/dopamine D E (0.013), 5-HTIA/5-HT uptake (0.008), 5-HT1A/5-HT 2 (0.005) and 5-HT1g/al-adrenoceptor (0.005) ratios. The fact that SR 57746A, like various 5-HTIA receptor agonists (De Vivo and Maayani, 1986), inhibited forskolin-stimulated adenylate cyclase activity in rat hippocampal membranes and that this effect was antagonized by (-)-propranolol, an antagonist at 5-HT 1 receptors (Hoyer, 1988), suggests that the compound is an agonist at 5-HT1A receptors. As previously reported for 8-OH-DPAT (Cervo and Samanin, 1987a, 1991), SR 57746A significantly increased the time the animals spent attempting to escape (struggling). The effect of SR 57746A, like that of other antidepressant drugs (Porsolt et al., 1978), was seen only after repeated doses and in the absence of any stimulatory effect on locomotor activity, as assessed by the rats' activity in the open field. Metergoline, a non-selective 5-HT receptor antagonist (Hoyer, 1988), antagonized the effect of SR 57746A on struggling, suggesting that 5-HT receptors are involved. In order to clarify the 5-HT receptor types mediating the effect of SR 57746A we studied its ability to increase struggling in animals treated with (-)-propranolol. The fact that (-)-propranolol completely antagonized the effect of SR 57746A, together with the compound's lack of affinity for 5-HT m and

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5-HTIc binding sites, clearly suggests that 5-HTIA receptors are involved. It has been suggested that the effect of 8-OH-DPAT on the behaviour of rats in the forced swimming test depends on the stimulation of presynaptic 5-HT1A receptors in the raphe area since it was abolished in animals depleted of brain 5-HT by intracerebroventricularly injected 5,7-dihydroxytryptamine and in animals that had received three oral doses of parachlorophenylalanine (Cervo and Samanin, 1987a, 1991). Moreover, the effect could be produced by direct injection of 8-OH-DPAT in the raphe (Cervo et al., 1988; Cervo and Samanin, 1991). We therefore used parachlorophenylalanine to assess whether the effect of SR 57746A depended on intact serotoninergic neurons. Parachlorophenylalanine completely prevented this effect on struggling, suggesting that presynaptic 5-HT1A receptors are involved. Brain dopamine, particularly dopamine D E receptors, appears to have a permissive role in the effect of various antidepressant drugs, including 8-OH-DPAT, in the forced swimming test (Borsini et al., 1984, 1985, 1988; Cervo and Samanin, 1987a,b, 1988; Cervo et al., 1990). This suggestion is based on the finding that sulpiride, a selective dopamine D 2 receptor antagonist, prevented the effects of the drugs in this test. Dopamine D 2 receptors also seem to be involved in the effect of SR 57746A on struggling since sulpiride completely antagonized its effect. In view of the low affinity of 8-OH-DPAT for dopamine D E receptors (Neale et al., 1987) and the fact that facilitation of dopamine-mediated behaviour may be consequent to a reduced activity of 5-HT neurons in raphe nuclei (Fink and Oelssner, 1981), it was suggested (Cervo and Samanin, 1987a) that facilitation of dopamine transmission by 8-OH-DPAT could be secondary to its ability to reduce the activity of 5-HT cells in the raphe area (Sprouse and Aghajanian, 1986). This could also be true for SR 57746A, although a direct effect of high concentrations of SR 57746A on dopamine O 2 receptors cannot be excluded with certainty. The fact however that SR 57746A caused no locomotor stimulation in rats makes it unlikely that it really stimulated central dopamine D E receptors at the doses used in the present study. Locomotor stimulation is typically seen with drugs which directly or indirectly activate dopamine D E receptors in the rat brain (McDevitt and Setler, 1981). Like 8-OH-DPAT (Cervo and Samanin, 1991), SR 57746A, administered as a three-dose treatment in 24 h, significantly reduced the time the animals spent floating. Desipramine instead increased struggling while having no effect on floating (Armario et al., 1988). That floating is not simply the reciprocal of struggling is clearly shown by the fact that metergoline, parachlorophenylalanine and sulpiride antagonized the

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effect o f S R 57746A on struggling but n o t on floating. Similar results w e r e f o u n d in a p r e v i o u s study with p a r a c h l o r o p h e n y l a l a n i n e and 8 - O H - D P A T ( C e r v o an d S a m a n i n , 1991). T h e results with S R 57746A f u r t h e r suggest that struggling is a m o r e reliable m e a s u r e o f a n t i d e p r e s s a n t activity t h a n immobility in th e f o r c e d s w i m m i n g test ( A r m a r i o et al., 1988; C e r v o an d S a m a n i n , 1991), particularly w h e n drugs with high affinity for 5-HTIA r e c e p t o r s a r e used. In conclusion, the study s h o w e d that S R 57746A has selectivity an d high affinity for 5-HTIA receptors. Exp e r i m e n t s with a d e n y l a t e cyclase activity in h i p p o c a m pal m e m b r a n e s suggest that t h e c o m p o u n d is an agonist at this r e c e p t o r . S R 57746A h a d an a n t i d e p r e s sant-like effect in t h e f o r c e d s w i m m i n g test by a m e c h a ni sm wh i ch s e e m s to involve p r e s y n a p t i c 5-HTIA rec e p t o r s in t h e r a p h e a r e a and a permissive role o f bra i n d o p a m i n e D z r e c e p t o r s .

Acknowledgements This work was partially supported by Consiglio Nazionale delle Ricerche, Rome, Italy, Convenzione Psicofarmacologia. We thank Dr. Angelo Forgione from Ravizza Farmaceutici S.p.a. for the generous gift of ( :i:)-sulpiride.

References Armario, A., A. Galvad~ and O. Marfi, 1988, Forced swimming test in rats: effect of desipramine administration and the period of exposure to the test on struggling behavior, swimming, immobility and defecation rate, Eur. J. Pharmacol. 158, 207. Benavides, J., D. Quarteronet, F. Imbault, C. Malgouris, A. Uzan, C. Renault, M.C. Dubroeucq, C. Gueremy and G. Le Fur, 1983, Labelling of 'peripheral-type' benzodiazepine binding sites in the rat brain by using [3H]PK 11195, an isoquinoline carboxamide derivative: kinetic studies and autoradiographic localization, J. Neurochem. 41, 1744. Bianchetti, A., T. Croci and L. Manara, 1990, The new serotoninergic compound SR 57746A promotes fecal excretion in rats by stimulation of central 5-HTxA receptors, Pharmacol. Res. 22 (Suppl. 2), 53. BiUard, W., V. Ruperto, G. Crosby, L.C. Iorio and A. Barnett, 1984, Characterization of the binding of 3H-SCH 23390, a selective D-1 receptor antagonist ligand, in rat striatum, Life Sci. 35, 1885. Blurton, P.A. and M.D. Wood, 1986, Identification of multiple binding sites for [3H]5-hydroxytryptamine in the rat CNS, J. Neurochem. 46, 1392. Borsini, F., E. Nowakowska, L. Pulvirenti and R. Samanin, 1984, Repeated treatment with amitriptyline reduces immobility in the behavioral 'despair' test by activating dol~aminergic and/3-adrenergic mechanisms. J. Pharm. Pharmacol. 37, 137. Borsini, F., L. Pulvirenti and R. Samanin, 1985, Evidence of dopamine involvement in the effect of repeated treatment with various antidepressants in the behavioral 'despair' test in rats, Eur. J. Pharmacol. 110, 253. Borsini, F., A. Lecci, A. Mancinelli, V. D'Aranno and A. Meli, 1988, Stimulation of dopamine D-2 but not D-1 receptors reduces immobility time of rats in the forced swimming test: implication for antidepressant activity, Eur. J. Pharmacol 148, 301.

Bylund, D.B. and S.H. Snyder, 1976 Beta adrenergic receptor binding in membrane preparations from mammalian brain, Mol. Pharmacol. 12, 568. Cervo, L. and R. Samanin, 1987a, Potential antidepressant properties of 8-hydroxy-2-(di-n-propylamino)tetralin, a selective serotonintA receptor agonist, Eur. J. Pharmacol. 144, 223. Cervo, L. and R. Samanin, 1987b, Evidence that dopamine mechanisms in the nucleus accumbens are selectively involved in the effect of desipramine in the forced swimming test, Neuropharmacology 26, 1469. Cervo, L. and R. Samanin, 1988, Repeated treatment with imipramine and amitriptyline reduced the immobility of rats in the swimming test by enhancing dopamine mechanisms in the nucleus accumbens, J. Pharm. Pharmacol. 40, 155. Cervo, L. and R. Samanin, 1991, Effect of chronic treatment with 8-OH-DPAT in the forced swimming test requires the integrity of presynaptic serotonergic mechanisms, Psychopharmacology 103, 524. Cervo, L., G. Grignaschi and R. Samanin, 1988, 8-Hydroxy-2-(di-npropylamino)tetralin, a selectice serotoninlA receptor agonist, reduces the immobility of rats in the forced swimming test by acting on the nucleus raphe dorsalis, Eur. J. Pharmacol. 158, 53. Cervo, L., G. Grignaschi and R. Samanin, 1990, The role of the mesolimbic dopaminergic system in the desipramine effect in the forced swimming test, Eur. J. Pharmacol. 178, 129. Creese, I., T. Prosser and S.H. Snyder, 1978, Dopamine receptor binding: specificity, localization and regulation by ions and guanyl nucleotides, Life Sci. 23, 495. De Lean, A., P.J. Munson and D. Rodbard, 1978, Simultaneous analysis of families of sigmoidal curves: application to bioassay, radioligand assay, and physiological dose-response curves, Am. J. Physiol. 235, E97. De Vivo, M. and S. Maayani, 1986, Characterization of the 5-hydroxytryptaminelA receptor-mediated inhibition of forskolinstimulated adenylate cyclase activity in guinea pig and rat hippocampal membranes, J. Pharmacol. Exp. Ther. 238, 248. Ehlert, F.J., W.R. Roeske and H.I. Yamamura, 1981, Multiple benzodiazepine receptors and their regulation by y-aminobutyric acid, Life Sci. 29, 235. Fink, H. and W. Oelssner, 1981, LSD, mescaline and serotonin injected into medial raphe nucleus potentiate apomorphine hypermotility, Eur. J. Pharmacol. 75, 289. Garattini, S. and R. Samanin, 1988, Biochemical hypotheses on antidepressant drugs: a guide for clinician or a toy for pharmacologists?, Psychol. Med. 18, 287. Giral, P., P. Martin, P. Soubri6 and P. Simon, 1988, Reversal of helpless behavior in rats by putative 5-HTIA agonists, Biol. Psychiatry 23, 237. Glowinski, J. and L.L. Iversen, 1966, Regional studies of catecholamines in the rat brain. 1. The disposition of [3H]norepinephrine, [3H]dopamine and [3H]DOPA in various regions of the brain, J. Neurochem. 13, 655. Greenberg, D.A., D.C. U'Prichard and S.H. Snyder, 1976, Alphanoradrenergic receptor binding in mammalian brain: differential labeling of agonist and antagonist states, Life Sci. 19, 69. Greengrass, P. and R. Bremner, 1979, Binding characteristics of 3H-prazosin to rat brain t~-adrenergic receptors, Eur. J. Pharmacol. 55, 323. Habert, E., D. Graham, L. Tahraoui, Y. Claustre and S.Z. Langer, 1985, Characterization of [3H]paroxetine binding to rat cortical membranes, Eur. J. Pharmacol. 118, 107. Hall, M.D., S. El Mestikawy, M.B. Emerit, L. Pichat, M. Hamon and H. Gozlan, 1985, [3H]8-Hydroxy-2-(di-n-propylamino)tetralin binding to pre and postsynaptic 5-hydroxytryptamine sites in various regions of the rat brain, J. Neurochem. 44, 1685. Herschel, M. and R.J. Baldessarini, 1979, Evidence for two types of binding of 3H-GABA and 3H-museimol in rat cerebral cortex and cerebellum, Life Sci. 24, 1849.

L. Cervo et al. / European Journal of Pharmacology 253 (1994) 139-147 Hoyer, D., 1988, Functional correlates of serotonin 5-HT I recognition sites, J. Recept. Res. 8, 59. Jenkins, S.W., D.S. Robinson, L.F. Fabre, Jr., J.J. Andary, M.E. Messina and L.A. Reich, 1990, Gepirone in the treatment of major depression, J. Clin. Psychopharmacol. 10 (Suppl.), 77s. Jenner, P. and C.D. Marsden, 1981, Substituted benzamide drugs as selective neuroleptic agents, Neuropharmacology 20, 1285. Invernizzi, R., C. Fracasso, S. Caccia, A. Di Clemente, S. Garattini and R. Samanin, 1989, Effect of L-cysteine on the long-term depletion of brain indoles caused by p-chloroamphetamine and d-fenfluramine in rats. Relation to brain drug concentrations, Eur. J. Pharmacol. 163, 77. Invernizzi, R., M. Carli, A Di Clemente and R. Samanin, 1991, Administration of 8-hydroxy-2-(di-n-propylamino)tetralin in raphe nuclei dorsalis and medianus reduces serotonin synthesis in the rat brain: differences in potency and regional sensitivity, J. Neurochem. 56, 243. Leysen, J.E., C.J.E. Niemegeers, J.M. Van Nueten and P.M. Laduron, 1982, [3H]Ketanserin (R 41 468), a selective 3H-ligand for serotonin 2 receptor binding sites. Binding properties, brain distribution, and functional role, Mol. Pharmacol. 21,301. Ljungberg, T. and U. Ungerstedt, 1978, Classification of neuroleptic drugs according to their ability to inhibit apomorphine-induced locomotion and gnawing: evidence for two different mechanisms of action, Psychopharmacology 56, 239. Lowry, O.H., N.J. Rosebrough, A.L. Farr and R.J. Randall, 1951, Protein measurement with the Folin phenol reagent, J. Biol. Chem. 193, 265. McDevitt, J.T. and P.E. Setler, 1981, Differential effects of dopamine agonists in mature and immature rats, Eur. J. Pharmacol. 72, 69. Middlemiss, D.N. and J.R. Fozard, 1983, 8-Hydroxy-2-(di-n-propylamino)-tetralin discriminates between subtypes of the 5-HT I recognition site, Eur. J. Pharmacol. 90, 151. Middlemiss, D.N., L. Blakeborouge and S.R. Leather, 1977, Direct evidence for an interaction of /3-adrenergic blockers with the 5-HT receptor, Nature 267, 289. Nahorski, S.R. and A.L. Willcocks, 1983, Interactions of/3-adreno-

147

ceptor antagonists with 5-hydroxytryptamine receptor subtypes in rat cerebral cortex, Br. J. Pharmacol. 78, 107P. Neate, R.F., S.L. Fallon, W.C. Boyar, J.W.F. Wasley, L.L. Martin, G.A. Stone, B.S. Glaeser, C.M. Sinton and M. Williams, 1987, Biochemical and pharmacological characterization of CGS 12066B, a selective serotonin-lB agonist, Eur. J. Pharmacol. 136, 1. Nelson, D.R. and D.R. Thomas, 1989, [3H]-BRL 43694 (Granisetron), a specific ligand for 5-HT 3 binding sites in rat brain cortical membranes, Biochem. Pharmacol. 38, 1693. Pazos, A., D. Hoyer and J.M. Palacios, 1984, The binding of serotonergic ligands to the porcine choroid plexus: characterization of a new type of serotonin recognition site, Eur. J. Pharmacol. 106, 539. Porsolt, R.D., G. Anton, M. Deniel, N. Blavet and M. Jalfre, 1978, Behavioural despair in rats: a new model sensitive to antidepressant treatments, Eur. J. Pharmacol. 47, 379. Robinson, D.S., K. Rickels, J. Feighner, L.F. Fabre, Jr., R.E. Gammans, R.C. Shrotriya, D.R. Alms, J.J. Andary and M.E. Messina, 1990, Clinical effects of the 5-HTIA partial agonists in depression: a composite analysis of buspirone in the treatment of depression, J. Clin. Psychopharmacol. 10, 67s. Romandini, S. and R. Samanin, 1984, Methysergide and metergoline reduce morphine analgesia with no effect on the development of tolerance in rats, Psychopharmacology 82, 140. Salomon, Y., C. Londos and M. Rodbell, 1974, A highly sensitive adenylate cyclase assay, Anal. Biochem. 58, 541. Sprouse, J.S. and G.K. Aghajanian, 1986, (-)-Propranolol blocks the inhibition of serotonergic dorsal raphe cell firing by 5-HT1A selective agonists, Eur. J. Pharmacol., 128, 295. Tan Tran, V., R.S.L. Chang and S.H. Snyder, 1978, Histamine H 1 receptors identified in mammalian brain membranes with [3H]mepyramine, Proc. Natl. Acad. Sci. USA 75, 6290. Van Wijngaarden, I., M.Th.M. Tulp and W. Soudijn, 1990, The concept of selectivity in 5-HT receptor research, Eur. J. Pharmacol. 188, 301.