1, Forbes et al., 1993). However, SB A is also equipotent

Transcription

1 Br. J. Pharmacol. (1994), 111, '." Macmillan Press Ltd, 1994 In vivo properties of SB 2646A, a 5-H2C/2B receptor antagonist 'G.A. Kennett, M.D. Wood, A. Glen, S. Grewal, *1. Forbes, *A. Gadre &.P. Blackburn Department of Psychiatry and *Department of Medicinal Chemistry, SmithKline Beecham, Coldharbour Road, he Pinnacles, Harlow, Essex CM 19 5AD 1 SB 2646A, N-(1-methyl-5-indolyl)-N'-(3-pyridyl) urea hydrochloride, the first reported selective 5-H2C,2B over 5-H2A receptor antagonist, (pk, rat 5-H2c receptor 6.9, pa2 rat 5-H2B receptor 7.5, pk, rat 5-H2A receptor 5.2) dose-dependently blocked a putative rat model of 5-H2c receptor activation; 1-(3-chlorophenyl)piperazine (, 5 mg kg- ', i.p. 2 min pretest)-induced hypolocomotion (estimated ID5 19.2mg kg-', p.o.). 2 SB 2646A also blocked another putative in vivo model of 5-H2c receptor function; (5 mg kg-', i.p. 2 min pretest)-induced hypophagia in 23 h food-deprived rats (estimated ID5o 18.3 mg kg-', p.o.). 3 SB 2646A did not antagonize 1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane (DOI)-induced head shakes in rats at doses up to 2mgkg'1, p.o., an effect thought to be mediated by 5-H2A receptors for which SB 2646A has its next highest affinity (5 fold less) after the 5-H2c and 5-H2B sites. 4 SB2646A (2, 4mgkg'1, p.o., 1 h pretest) also reversed (.5mgkg'1, i.p., 3min pretest)-induced anxiety in the social interaction test, under low light familiar conditions. 5 When given alone, under high light unfamiliar conditions, SB2646A (2-4mgkg-', p.o.) increased active social interaction without affecting locomotor activity in the rat social interaction test. his is consistent with an anxiolytic action of SB 2646A. 6 hese results indicate that SB 2646A has in vivo efficacy and that 5-H2C or 5-H2B receptors are indeed likely to mediate -induced hypolocomotion, hypophagia and anxiogenesis. hey also suggest that 5-H2C,2B receptor blockade induces anxiolysis. Keywords: 5-H2c receptors; 5-H2A receptors; 5-H2B receptors; 5-H; anxiety; SB2646A Introduction he 5-Hc receptor was first characterized by Pazos et al. (1984). However, it is now recognised as belonging to the 5-H2 receptor family on the basis of marked amino acid sequence homology (Pritchett et al., 1988; Saltzman et al., 1991), and has therefore been renamed as the 5-H2c receptor site (Humphrey et al., 1993). he receptor is reported to act through the phosphoinositide hydrolysis secondary messenger system (Conn et al., 1986) and is widely distributed in both rat (Pazos & Palacios, 1985) and human (Pazos et al., 1987) brain, with particularly high density in the choroid plexus (Pazos & Palacios 1985; Pazos et al., 1987). In rats, several in vivo responses to the putative 5-H2c receptor agonist, 1-(3-chlorophenyl) piperazine () have been suggested to be mediated by 5-H2c receptor activation. hese include -induced hypolocomotion (Kennett & Curzon, 1988a), hypophagia (Kennett & Curzon, 1988b) and anxiety (Kennett et al., 1989). -induced anxiety is of particular interest as it has been widely reported in healthy volunteers and to a greater extent in patients with either panic disorders or obsessive compulsive disorder (OCD) (see Kennett, 1993). Unfortunately is not a selective 5- H2c receptor agonist and the pharmacological characterization of its effects in vivo result from the use of non-selective 5-H2c receptor antagonists. In the present study we have investigated the effects of the in vivo administration of SB 2646A (N-(1-methyl-5-indolyl)-N'-(3-pyridyl) urea hydrochloride), the first reported antagonist with selectivity for the 5-H2C over the 5-H2A receptor (Forbes et al., 1993; Wood et al., 1993). his compound had 5 fold selectivity for the rat 5-H2C over the 5-H2A receptor, and 8 fold or greater selectivity over other neurotransmitter receptors tested (able Author for correspondence. 1, Forbes et al., 1993). However, SB 2646A is also equipotent at the 5-H2B (rat stomach fundus) receptor with which the 5-H2c receptor shares a high degree of sequence homology (Foquet et al., 1992; Kursar et al., 1992) and at which has agonist properties (Clineschmidt et al., 1985). Methods Animals Male Sprague-Dawley (CD) rats g were housed in groups of six under a 12 h light/dark cycle (lights on 7 h min) with free access to food (CRMX, Special Diet Services) and water. -induced hypolocomotion Rats were placed in a room adjacent to the experimental room on the day of the procedure. hey were dosed orally 1 h pretest with SB 2646A or vehicle, and injected i.p. 2 min pretest with or saline, in groups of four. Rats were returned to their home cages after dosing. At h they were each placed in automated locomotor activity cages (57 x 16.6 x 25.3 cm) made of black perspex with a clear perspex lid and sawdust-covered floor, under red light for 1 min. During this time locomotion was recorded by means of alternately breaking two photocell beams traversing opposite ends of the box 3.9 cm above floor level. -induced hypophagia Rats were individually housed on day 1 and deprived of food. wenty three hours later they were dosed orally with

2 798 G.A. KENNE et al. able 1 Receptor binding profile of SB 2646A H N H N Receptor Affinity (pk,) 5-H2C 5-H2B 5-H2A 5-HA 5-HID 5-H3 BDZ GABAA GABA/C1- channel (BPS) * 5.2 N MAe Receptor Affinity (pk,) Adrenoceptorm, Adrenoceptor2A AdrenoceptOra2B Adrenoceptorp, AdrenoceptorR2 Dopamine D, Dopamine D2 Histamine HI *pa2 value in the rat stomach fundus preparation. For experimental details see Forbes et al. (1993) except affinities for benzodiazepine (BDZ) receptors ([3H]-flunitrazepam in rat cortex), histamine HI (ph]-mepyramine in rat cortex) and ter-butylbicyclophosphorothionate (BPS) ([3H]-BPS) which were carried out as in Sanger & Nelson (1989) and those for GABAA ([3H]-muscimol in rat cortex as described by Peters et al. (1988)). <4. <4. <4.5 antagonist or vehicle and returned to their home cages. Forty minutes later they were given or saline i.p. and again returned to their home cages. After a further 2 min, weighed aliquots of their normal food pellets were placed in their food hoppers and the amount remaining after 2 h was measured. Social interaction Rats were housed singly in a room adjacent to the testing room on day 1. On day 5 they were dosed orally with SB2646A or vehicle in treatment- and weight-matched ( ± 5 g) pairs, which had not previously been housed together, and returned to their home cage. Rats were then placed in a white perspex test box (54 x 37 x 26 cm) for 15 min under bright white light (15 lux) in an adjacent darkened room containing a fan to generate white noise. Active social interaction (sniffing, following, grooming, biting, boxing and crawling over or under) was scored by a 'blind' observer by remote video monitoring and a computerised score pad. At the end of each test the box was thoroughly wiped with moistened tissue paper. Alternatively rats were individually pre-exposed to test boxes of black perspex under red light for 1 min on days 3 and 4 and tested under the same conditions on day 5. DOI-induced head shakes Rats were dosed orally with SB 2646A 3 min before being placed in individual clear perspex observation cages (25.4 x 25.4 x 22.2 (height) cm). hirty minutes later 1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane (DOI) was given i.p. and the number of head shakes produced over the next 3 min were counted using a computerised score pad. Drugs SB 2646A (SB) and mianserin (Organon, Newhouse, Scotland) were ground into 1% methyl cellulose in.9% NaCl with a drop of BRIJ 35 (Sigma Chemical Co., Poole, Dorset) dispersant using a mortar and pestle and given orally as a suspension in a 2 ml kg-' injection volume 1 h pretest. SB 2646A was neutralised to ph 4.5. SB 2646A controls were given 1% methyl cellulose in.9% NaCl and a drop of BRIJ 35 acidified to ph 4.5 with HCL. DOI (Research Biochemcials Inc, Natick, Massachusetts, U.S.A.) and (Sigma Chemical Co., Poole, Dorset) were dissolved in saline and given i.p. in ml kg-' injection volume 1 and 2 min pretest respectively. Statistics he effect of SB 2646A on -induced hypolocomotion was determined by one-way ANOVA and Newman-Keuls test, and on hypophagia and anxiogenesis by two-way ANOVA and Newman-Keuls test. All other data were subjected to one-way ANOVA and Dunnett's test. Estimates of the dose producing 5% inhibition (ID~v) of -induced hypolocomotion or hypophagia were made using the iterative curve-fitting programme 'Allfit' (DeLean et al., 1978). All data are cited as means ± s.e.mean. Results Effect of SB 2646A on -induced hypolocomotion SB 2646A alone at 4 mg kg-', p.o., had no effect on locomotor activity, neither did lower doses in previous experiments (data not shown) or mianserin at 4 mg kg-', p.o. (able 2). (5 mg kg-', i.p.) markedly reduced the number of transits over 1 min and this effect was significantly reversed by SB 2646A (2 and 4 mg kg-', p.o.) and mianserin, used here as a positive control. he iterative curve-fitting programme 'Allfit', estimated the ID.o value of SB 2646A against -induced hypolocomotion as 19.2 ±.2mg kg-', p.o. he ANOVA test result for this study was 1(8,178) = 25., P<.1). Effect of SB 2646A on -induced hypophagia SB 2646A had no significant effect on the food intake of 23 h food-deprived rats over 2 h (F(4,81) = 1.63, NS), although food intake was somewhat reduced after 4 and 6mg kg-', p.o. (able 3). (5 mg kg', i.p.) significantly reduced food intake (E(1,81) = 22.3, P<.1) and this effect was significantly, and dose-dependently, reversed by SB 2646A (1-6 mg kg-', p.o.) (F(4,81) = 1.7, P<.1) (able 3). he estimated ID" for this action was 18.3 ± 1.3 mg kg', p.o.

3 EFFECS OF A 5-H2Cl2B RECEPOR ANAGONIS 799 Effect of SB 2646A on DOI-induced head shakes DOI (3 mg kg-', i.p.) induced 27.9 ± 3. head shakes over 3 min. SB 264A, at doses up to 2 mg kg- ', p.o. 1 h pretest, did not alter this response (F(4,58) =.9, NS) (Figure 1) and did not cause head shake behaviour when given alone at 1 or 2mg kg-', p.o. (vehicle-treated 2.3 ± 1.2, SB 2646A 1 mg kg-', p.o. 2.8 ±.8; 2 mg kg-', p.o., n = 4 per group). Effect of SB 2646A on 'anxiogenic-like' responses to (.5 mg kg-', i.p.) significantly reduced total interaction scores (N 1,49) = 11.5, P<.1), but had no effect on locomotor activity (F(1,49) =.86, NS). SB 2646A had no effect on total interaction scores when given alone, but significantly reversed the effect of (1(2,49) = 3.36, P <.5). SB 26464A also caused a significant effect on locomotion (f1r2,49) = 6.7, P<.1), but this did not result in a significant effect between vehicle- and SB 2646Atreated groups, whether given or not, when analysed by the Newman-Keuls test (Figure 2). Effect of SB 2646A on rat social interaction able 2 Effect of SB 2646A on 1-(3-chlorophenyl) piperazine ()-induced hypolocomotion Pretreatment reatment ransits/1o min SB 2646A 4 mg kg- ' Mianserin 4 mg kg- I SB 2646A 1 mg kg- ' SB 2646A 2 mg kg- ' SB 2646A 4 mg kg- ' Mianserin 4 mg kg'-i 16.6 ± ± ± ±.7** 2.5 ± ± 1.5t 11.4 ± 2.3tt 13.3 ± 2.5tt ID5 = 19.2 ±.2 mg kg-, p.o. by 'Allfit' (5 mg kg-', i.p.) given 2 min pretest, SB 2646A and mianserin given p.o. 1 h pretest. All points means ± s.e.mean, n = Significantly different from vehicle + saline-treated group: **P<.1; from vehicle + -treated group. tp<.5; ttp<.1 by Newman-Keuls test following 1-way ANOVA. able 3 Effect of SB 2646A on 1-(3-chlorophenyl) piperazine ()-induced hypophagia Pretreatment SB2646A 1mgkg-' SB 2646A 2 mg kg- ' SB 2646A 4 mg kg'-i SB 2646A 6 mg kg- ' SB 2646A 1 mg kg-' SB 2646A 2 mg kg- ' SB 2646A 4 mg kg- ' SB 2646A 6 mg kg- ' reatment 2 h Food intake (g) 4.3 ± ± ± ± ±.3 1. ±.7** 2.5 ±.3t 2.4 ±.5t 3.6 ±.6tt 3.5±.5tt Mm =l18.3±l1.3 mgkg-', p.o. by 'Allfit' (5 mg kg-', i.p.) given 3 min pretest, SB 2646A given p.o. 1 h pretest. All points means ± s.e.mean, n = 9. Significantly different from vehicle + saline-treated group: **P<.1; from vehicle + -treated group: tp<.5; ttp<.1 by Newman-Keuls test following significant 2-way ANOVA.._ of I' 51 SB 2646A dose-dependently increased time spent in active social interaction over 15 min (R(6,67) = 28.2, P <.1) (Fig- 4- w_ w ~~~.,.,'..'.., ,... *:--: :.:.:.::.: :...:.:.:.::.. :--.:.:-.:.:-. :-. :-.--:: 2- - ~~~~..... : _.-.-' ' SB 2646 (mg kg-', p.o.) Figure 1 Effect of SB 2646A, given p.o. 1 h pretest, on 1-(2,5- dimethoxy-4iodophenyl)-2-aminopropane (DOI, 3 mg kg'-, i.p. 1 min pretest)-induced head shakes over 3 min. All columns means ± s.e.mean. n = per group. No significant differences t C L 1 o c E - _- _. 7r Veh +Sal Veh f f tt I //// I ///1 / Veh SB2 SB4 Veh SB2 SB4 +Sal +Sal +Sal Figure 2 Effect of SB 2646A (2-4 mg kg-', p.o. 1 h pretest) on 1-(3-chlorophenyl)piperazine (,.5 mg kg-', i.p., 3 min pretest)-induced anxiety in the rat social interaction test. Veh = vehicle, SB = SB 2646A. All columns means ± s.e.mean, n = 8-12 per group. Significantly different from vehicle + saline (Sal)-treated group: **P<.1; from vehicle + -treated group: ttp<.1 by Newman-Keuls test following significant 2-way ANOVA.

4 8 G.A. KENNE et al. a en L-.) 16 :e 1) CO) CO) U) c/ E C. -J ' Veh SB 2 SB 5 SB 1 SB 2 SB 4 CDP reatment (mg kg-', p.o.) Veh SB 2 SB 5 SB 1 SB 2 SB 4 CDP reatment (mg kg-1, p.o.) Figure 3 Effect of SB 2646A (2-4mg kg-'), chlordiazepoxide 5 mg kg-' (CDP) and vehicle (Veh) (all given p.o., 1 h pretest) in the rat 15 min social interaction test under high light unfamiliar conditions. All columns means ± s.e.mean. n = 8-12 per group. Significantly different from vehicle-treated group: *P<.5; **P<.1 by Dunnett's test following significant 1-way ANOVA. ure 3). he drug also caused a small increase in locomotion at 2 mg kg-', p.o. but at no other dose (F(6,67) = 2.4, P <.5) (Figure 3). he magnitude of the increase in total interaction induced by SB 2464A was equal to that seen after chlordiazepoxide (CDP) at a dose of 5 mg kg-', p.o. Discussion SB 2646A is the first reported selective 5-H2C,2B receptor antagonist and has 5 fold selectivity for the rat 5-H2C,2B receptors over the 5-H2A and at least 8 fold selectivity over all other neurotransmitter receptors tested (able 1, Forbes et al., 1993; Wood et al., 1993). he ability of the compound to reverse -induced hypolocomotion and hypophagia, putative 5-H2c receptor-mediated behaviours (Kennett & Curzon, 1988a,b; 1991) demonstrates in vivo efficacy. Since SB 2646A has similar potency in both models it is likely that a similar mechanism of action is indeed involved and this is likely to be via blockade of the 5-H2C site, as previously proposed, or of the recently characterized 5-H2B site. However, given the relatively low potency of the compound in these tests it might be suggested that SB 2646A is blocking some other site, despite its 5 fold selectivity for 5-H2C/2B receptors. he inability of SB 2646A, at much higher doses (2 mg kg-', p.o.), to antagonize 5-H2A-mediated DOI-induced head shakes (Pranzatelli, 199; Kennett & Curzon, 1991) argues against this, as after the 5-H2C/2B site, its next highest affinity is for the 5-H2A receptor (able 1). Although both -induced hypophagia and hypolocomotion appear to be mediated by the same receptor they are independent behaviours. hus, hypophagia caused by is blocked by cyanopindolol and (-)-propranolol (Kennett & Curzon, 1988b) unlike the hypolocomotor response (Kennett & Curzon, 1988a) while intrahypothalamic trifluoromethyl phenylpiperazine (FMPP), a closely related 5-H2c agonist, causes only hypophagia (Hutson et al., 1988). hat SB 2646A fully reversed the hypophagic effect of but only partially reversed hypolocomotion (ables 2 and 3) is, however, probably due to not having administered high enough doses, although it may conceivably indicate the presence of an additional mechanism. SB 2646A also antagonized the anxiogenic-like effect of in the rat social interaction model at similar doses to those which were effective against -induced hypolocomotion and hypophagia. hus, the anxiogenic response is also likely to be mediated by 5-H2C/2B receptors. his is consistent with previous evidence that non-selective 5-H2c, 2B, but not selective 5-H2A or 5-HIA and 5-HlB receptor antagonists blocked the effect of in this paradigm (Kennett et al., 1989). It is also consistent with antagonism of -induced anxiety in man by both the non-selective 5-H, and 5-H2 receptor blockers metergoline (Kalus et al., 199; Pigott et al., 1991) and methysergide (Kalus et al., 199) and, in particular, by the relatively selective (see Kennett, 1993) 5-H2C,2B,2A receptor antagonist, ritanserin (Seibyl et al., 1991). Since activation of 5-H2C (or 5-H2B) receptors seems to cause anxiety, their blockade might be anxiolytic if the system is activated during stress. Indeed, immobilisation stress (Shimzu et al., 1992), tail pinch and handling (Kalen et al., 1989) and placement on the open arms of the elevated X-maze (Wright et al., 1992) have all been shown to enhance extraneuronal 5-H in either the hypothalamus or the hippocampus. infusion into this latter region in the rat, which contains high levels of 5-H2c receptor mrna (Hoffman & Mezey, 1989; Molineaux et al., 1989) and receptors (Mengod et al., 199), has been reported to induce anxiogenic-like responses (Whitton & Curzon, 199). has also been reported to potentiate periaqueductal grey-induced anxiety (Beckett et al., 1992). he anxiolytic-like response to SB 2646A in the social interaction test may therefore be caused by antagonism of these responses and is in agreement with previous findings of the anxiolytic effects of non-selective 5-H2C,2B, but not of 5-H2A or other receptor antagonists, in both the rat social interaction and Geller-Seifter models of anxiety (Kennett, 1992; Kennett et al., 1993). he lack of an anxiolytic-like response in the social interaction test when SB 2646A was given alone under low light unfamiliar conditions (Figure 2) is probably due to low intrinsic levels of anxiety under these conditions (File, 1985). As SB 2646A has low affinity for the benzodiazepine, GABAA and BPS binding sites (able 1) the GABA/C1- complex is unlikely to be the primary site of its anxiolytic action while the presence of 5-H2B receptors in rat brain, let alone the hippocampus is as yet unknown. In conclusion, experiments with SB 2646A, the first selective 5-H2C/2B receptor antagonist, indicate that the compound has in vivo activity and that 5-H2C/2B receptors are likely to mediate the hypolocomotion, hypophagia and anxiety induced by. Furthermore, the results suggest that selective antagonists of 5-H2C,2B receptors possess anxiolyticlike properties. hese may be of clinical relevance given reports of the clinical anxiolytic efficacy of the non-selective 5-H2C,2B receptor antagonists, mianserin (Murphy, 1978; Russell et al., 1978; Conti & Pinder, 1979; Khan et al., 1983) and ritanserin (Ceulemans et al., 1985; Bressa et al., 1987; Pangalila-Ratu et al., 1988). However, although there is as yet no pharmacological evidence to differentiate between

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