'Lakhbir Singh, Mark J. Field, John Hughes, Richard Menzies, Ryszard J. Oles, Caroline A. ... receptor antagonist (Hughes et al., 1990; Horwell et al., 1991).
Br. J. Pharmacol. (1991), 104, 239-245
(D Macmillan Press Ltd, 1991
The behavioural properties of CI-988, a selective cholecystokininB receptor antagonist 'Lakhbir Singh, Mark J. Field, John Hughes, Richard Menzies, Ryszard J. Oles, Caroline A. Vass & Geoffrey N. Woodruff Parke-Davis Neuroscience Research Centre, Addenbrookes Hospital Site, Hills Road, Cambridge CB2 2QB 1 The behavioural effects of a selective cholecystokininB (CCKB) receptor antagonist CI-988 were investigated in rodents. 2 In three rodent tests of anxiety (rat elevated X-maze, rat social interaction test and mouse light/dark box) CI-988 over the dose range 0.001-I0.Omgkg 1, (i.p.) produced an anxiolytic-like action. The magnitude of this effect was similar to that of chlordiazepoxide (CDP). In contrast, the selective CCKA receptor antagonist, devazepide, was inactive. CI-988 also showed anxiolytic-like action in the rat conflict test but the magnitude of this effect was about 2.5 fold less than that of CDP. 3 Central but not peripheral administration of the selective CCKB receptor agonist, pentagastrin, like FG 7142, produced an anxiogenic-like action. 4 The pentagastrin-induced anxiety was dose-dependently antagonized by CI-988, whereas devazepide was inactive. However, ten times higher doses of CI-988 were required to block a similar action of FG 7142. 5 In contrast to CDP, CI-988 up to 3000 fold higher doses than those inducing anxiolysis was inactive in tests measuring sedation and ataxia. It also failed to antagonize pentylenetetrazol-induced tonic seizures. Furthermore, CI-988 did not interact with alcohol or barbiturates. Thus, CI-988 appears to be an anxioselective compound. 6 The anxiolytic-like action of CDP in the rat elevated X-maze was dose-dependently antagonized by flumazenil. In contrast, the benzodiazepine receptor antagonist failed to block a similar effect of CI-988. 7 Thus, CI-988 shows anxiolytic-like activity in several animal models of anxiety. The anxiolytic-like effect of CI-988 involves a novel mechanism of action, that is likely to be mediated by selective antagonism of the brain CCKB receptor. It is suggested that CI-988 should have a better side-effect profile in man than the benzodiazepines. Keywords: CCKA receptor; CCKB receptor; agonist; antagonist; anxiolytic; anxiogenic; sedation
Introduction The peptide cholecystokinin (CCK) discovered originally in the gastrointestinal tract (Ivy & Oldberg, 1928), is also present in high concentrations in certain regions of the brain (Vanderhaegen et al., 1975), where it exists mainly as the octapeptide in the sulphated and desulphated forms (Dockray, 1976; Rehfeld, 1978). The receptors for CCK have been divided into two types (Innis & Snyder, 1980): the CCKA receptor that is blocked selectively by devazepide (formerly MK-329, L-364,718; Chang & Lotti, 1986) and is found in discrete brain regions as well as some peripheral tissues (Hill et al., 1987a,b), and the CCKB receptor that is found throughout the brain (Hill et al., 1987a) and is characterized by the high affinity for the novel antagonists CI-988 (formerly PD 134308) and L-365,260 (Lotti & Chang 1989; Hughes et al., 1990). It has been reported that CCK may control various physiological events (Zetler, 1985) including hypothermia (Katsuura et al., 1981), analgesia (Zetler, 1980; Kubota et al., 1985), sedation (Zetler, 1981) and satiety (Gibbs et al., 1973). Recent studies have also indicated a role of CCK in anxiety. Thus, animal studies have shown that activation of central CCKB receptors induces an anxiogenic-like action, whilst antagonism of this receptor leads to anxiolytic-like effects (Hughes et al., 1990; Singh et al., 1991a). Consistent with these data, it has been shown that intravenous administration of CCK-4 in man induces panic-like attacks (De Montigny, 1989; Bradwejn et al., 1990). The dipeptoid CI-988 is a highly potent and selective CCKB receptor antagonist (Hughes et al., 1990; Horwell et al., 1991). Recently it has been reported that CI-988 produces anxiolytic'Author for correspondence.
like actions in several experimental models of anxiety (Hughes et al., 1990; Singh et al., 1991a). In the present study, evidence is presented that supports and extends our previous findings that the anxiolytic-like action of CI-988 may involve a specific interaction with the CCKB receptor. The present data also indicate that unlike benzodiazepines, CI-988 does not possess either anticonvulsant or sedative properties. Furthermore, it does not interact with CNS depressants. Preliminary accounts of some of these data have already been presented (Field et al., 1991a; Singh et al., 1991b).
Methods Animals Male Hooded Lister rats (200-250 g) were obtained from Olac (Bicester, U.K.) and male TO mice (20-25 g) were obtained from Bantin and Kingman (Hull, U.K.). Animals were housed in groups of 6-10 under a 12 h light/dark cycle (lights on at 07 h 00 min) and unless stated otherwise, with food and water ad libitum. All behavioural tests were carried out between lOh OOmin and 17hOOmin.
Drug administration Drugs were administered either i.p. or orally (p.o.) before test in a volume of lmlkg- for rats and lOmlkg'- for mice. Pentagastrin was administered intracerebroventricularly (i.c.v. in SpI of the vehicle).
Intracerebroventricular injections The i.c.v. injections in mice and rats were carried out as described previously (Singh et al., 1990; 1991a). Briefly, rats
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were anaesthetized and a 22 gauge stainless steel guide cannula was implanted unilaterally by use of a Kopf stereotaxic frame (anterior posterior = 8.2mm anterior of interaural line; lateral = + 1.4 mm; dorso-ventral = 7.6 mm dorsal to the interaural line according to the atlas of Paxinos & Watson, 1976). Test compounds were administered through an injection cannula extending 1 mm ventral to the guide cannula tip. At the end of the experiment animals were selected randomly and injected with a dye to check the placement of the guide cannula. Mice were briefly anaesthetized with halothane and i.c.v. injections were made at lambda with a 27 gauge needle (3.5mm long) attached to a 50Oul Hamilton syringe.
Mouse light/dark box The apparatus was an open-topped box, 45 cm long, 27 cm wide and 27cm high, divided into a small (2/5) and a large (3/5) area by a partition that extended 20cm above the walls (Costall et al., 1989a). There was a 7.5 x 7.5 cm opening in the centre of the partition at floor level. The small compartment was painted black and the large compartment white. The white compartment was illuminated by a 60 W tungsten bulb. The laboratory was illuminated by red light. Each mouse was tested by placing it in the centre of the white area and allowing it to explore the novel environment for 5min. The time spent in the illuminated side was measured (Kilfoil et al., 1989).
Rat social interaction test The apparatus used was an open-topped box (51 x 51 x 20cm high) with 17 x 17cm areas marked on the floor. Two naive rats from separate housing cages were placed into the brightly illuminated box. Their behaviour was scored on a television monitor in an adjacent room, by use of a keyboard linked to an IBM compatible PC over a 5min period. The arena was cleaned with a solution of Dettol in water (18 ml -') between each observation to remove odours. The following behaviours were scored for 5 min as active social interaction (Guy & Gardner, 1985): sniffing, following, grooming, licking, mounting, genital investigation and aggression (which consists of biting, boxing, aggressive grooming, kicking away and full submissive posture).
Rat elevated X-maze A standard elevated X-maze (Handley & Mithani, 1984; Pellow et al., 1985) was automated as previously described (Field et al., 1991b). The animals were placed on the centre of the X-maze facing one of the open arms. For anxiolytic effects the entries and time spent on the end half sections of the open arms was measured during the 5 min test period (Costall et al., 1989b; Singh et al., 1991a). However, to measure anxiogeniclike actions the total entries and time spent on the open arms was measured (Singh et al., 1991a).
Rat conflict test Male Hooded Lister rats (food deprived to 75-80% of their free feeding body weight) were trained to press levers for 45mg food pellets in standard operant chambers (Coulbourn Instruments). The schedule consisted of alternations of four 4min variable interval (VI) 30s unpunished periods signalled by chamber lights on and three 3 min fixed ratio (FR) 5 punished periods signalled by chamber lights off. During the punished periods, a 100 ms footshock was given concomitant to food delivery. To study anxiolytic effects, the degree of footshock (100300upA) was adjusted for each rat to obtain approximately 8-(90% suppression of responding in compariFR5 responding. To measure son with unpunished anxiogenic-like effects the footshock punishment (50-100 pA) was adjusted to obtain approximately 40% suppression. Upon stable baseline responding, six rats were implanted with a intracerebroventricular (i.c.v.) guide cannula. Test sess-
ions were run once a week and on control days, animals were injected with the appropriate vehicle. Drug effect was expressed as the percentage increase or decrease of the leverpressing rates during the punished periods and during the unpunished periods on the test day compared with mean performances obtained the two previous days. Rats were only tested if their 2-day control values remained within the following ranges: (1) daily VI30 responses were within 20% of the 2-day mean; (2) daily FR5 responses were within 20% of the 2-day mean; (3) the number of shocks on each control day was at least 2.
Anticonvulsant studies Pentylenetetrazol (PTZ; 15mgmlP , 0.224mlmin-1) was infused i.v. via a tail vein of restrained mice. The latency to tonic seizure was noted. This convulsion represented the endpoint of the test and mice were killed immediately after it had occurred. The threshold dose of the convulsant required to elicit this seizure was calculated for each mouse.
Sedation and motor coordination Mouse rotarod Untrained mice were placed on the accelerating rotarod (accelerating to 15revmin-' in 120s) and the time spent on the rotating rotarod was determined. Rat locomotor activity cages Rats were placed individually in photocell activity cages (45 x 24 x 20cm). The boxes were fitted with a pair of infrared photocells (in the centre of the long side walls). The locomotor cages were interfaced to an IBM compatible PC. Activity was measured as the number of photocell breaks during a 60 min test period.
Interaction with alcohol and barbiturates The ability of CI-988 or CDP to potentiate the sedative action of ethanol (0.75 g kg- 1, a threshold sedative dose administered p.o.30 min before test) was examined using the mouse rotarod apparatus. An interaction with sodium pentobarbitone (60.0 mgkg- 1, i.p.) was investigated by measuring changes in the sleeping time.
Drugs The following drugs were used: chlordiazepoxide (CDP, Sigma Chemical Co.), pentagastrin and FG 7142 (N-methyl-#carboline-3-carboxamide, Research Biochemicals Inc.). Flumazenil (Roche), sodium pentobarbitone (Sagatal, May and Baker). CI-988 (4-{412-[[3-(1H-indol-3-yl)-2-methyl-1-oxo-2[[(tricyclo - [3.3.1.1.3 ] - dec - 2 - yloxy) - carbonyl] - amino] propyl]-amino]- 1 -phenylethyl]-amino} -4-oxo-[R-(R*,R*)]-
butanoate-N-methyl-D-glucamine), L-365,260 [(3R)-( + )-N-
(2,3-dihydro- 1-methyl-2-oxo-5-phenyl- 1 H- 1,4-benzodiazepin3-yl)-N1-(3-methylphenyl)-urea] and devazepide were synthesized at Parke-Davis Neuroscience Research Centre. Chlordiazepoxide, flumazenil and FG 7142 were suspended in 1.0% w/v carboxymethylcellulose (CMC) with aid of ultrasonification. CI-988 was dissolved in 0.9% w/v NaCl. Devazepide and L-365,260 were dissolved in 100% ethanol and serially diluted in alcohol and the required concentration was achieved by addition of CMC, so that all solutions contained 10% alcohol. Pentagastrin was dissolved in sodium bicarbonate (50 mM).
Results
Effects of CI-988, L-365,260, CDP and devazepide in the mouse light/dark box The i.p. administration of CI-988 (0.01-10.Omgkg-1), L-365, 260 (0.01-10.Omgkg-1) or CDP (2.5-20.Omgkg-1) 40min
BEHAVIOURAL PROPERTIES OF CI-988 Table 1 The effect of cholecystokinin (CCK) receptor antagonists in the mouse light/dark box and the rat social interaction test Compound CDP
CI-988
L-365,260
Devazepide
Dose (mg kg- 1)
Veh 0.3 1.0 3.0 2.5 5.0 10.0 20.0 Veh 0.001 0.01 0.1 1.0 3.0 10.0
Veh 0.01 0.1 0.3 1.0 3.0 6.0 10.0 Veh 0.5 5.0 10.0 20.0 40.0
Light/dark box
Social interaction
TIL (s) 79.4 + 9.7
SI (s) 35.7 + 1.9 42.7 + 3.3 61.3 + 6.4* 68.6 + 5.6*
122.4 + 15.6 158.3 + 15.4* 155.4 + 10.4* 157.6 + 20.5* 80.9 + 7.4
63.2 + 43* 51.3 + 4.1*
114.6 + 14.9 132.5 + 15.1* 130.3 + 10.2*
35.7 + 40.4 + 51.9 + 57.1 + 61.5 + 60.8 +
165.9 + 80.0 + 90.2 + 101.3 +
45.9 + 1.4
21.9* 10.0 18.3 14.3
139.3 + 16.8* 134.6 + 9.7* 87.8 + 18.3 94.2 + 12.0 77.5 + 11.1 59.5 + 9.1 103.4 + 23.8
1.9 3.7 2.9* 2.3* 1.9* 7.7*
+ 3.1 + 3.7
46.2 54.1 59.1 54.6 45.2 45.9
+ 2.9 + 4.3 + 1.4
37.2 54.9 34.8 35.8
+ 2.9 + 7.4 + 1.8 + 3.3
+ 2.0*
Compounds were administered i.p. 40 min before the 5 min test periods. The time spent by mice in the light side (TIL) of the light/dark box and the social interaction (SI) between pairs of rats is shown in seconds. Results are shown as the means (+ s.e.mean) of 8-10 animals per group. * Significantly different from vehicle-treated controls, P < 0.05 (ANOVA followed by Dunnett's t test).
before test, increased dose-dependently the time spent on the illuminated side of the box, with minimum effective doses (MED) of 0.1, 1.0 and 5.0 mgkg-1 respectively (Table 1). In contrast, devazepide was inactive up to 20 mg kg- 1 (Table 1).
Effects of CI-988, CDP, L-365,260 and devazepide in the rat conflict test The i.p. administration of CI-988 (0.001-3.Omgkg-1), CDP (0.3-5.0 mg kg- 1) or L-365,260 (0.3-3.0 mg kg- 1) 40 min before
test dose-dependently increased the active interaction between pairs of rats with MED of 0.01, 1.0 and 3.Omgkg-' respectively (Table 1). As in the light/dark box, the activity of CI-988 was maintained over a wide (1000 fold) dose-range (Table 1). However, at higher doses of L-365,260 and CDP a fall off in the effect was observed (Table 1). As in the light/dark box, devazepide was without effect up to 40mg kg- l (Table 1). CDP (1.0-30.0mgkg-1, i.p.) administered 40min before test, produced a pronounced increase in punished responding at 3 and 10mg kg-1 (Table 2). However, at 30mg kg- 1, CDP caused a large decrease in punished and unpunished responding (Table 2). CI-988 (0.001-1.Omgkg-1, i.p.) induced a small but consistent increase in punished responding at all doses, with a significant effect occurring at 0.01 mg kg-1 (Table 2). In contrast to CDP, it had no effect on unpunished responding at any of the doses tested (Table 2). The i.c.v. administration of pentagastrin (16 pg/rat) 5min before test, produced a large decrease in both punished and unpunished
responding (Table 2).
Effects of FG 7142 and pentagastrin in the rat elevated X-maze and the mouse light/dark box The administration of FG 7142 (10.0-30.Omgkg 1, i.p.) 30min before test decreased dose-dependently the entries and time spent by rats on the open arms of the X-maze without affecting the total entries (Figure 1). The MED was 10.Omgkg-' (Figure 1). Similar administration in the mouse also dose-dependently reduced the time spent in the illuminated side of the light/dark box with a MED of 3.0mgkg-1 (Figure 1). The i.c.v. administration of pentagastrin (0.088.0pg per animal) 15min before test produced similar effects to FG 7142 in the two tests (Figure 1), the MED in both tests being 0.8 pg per animal (Figure 1). However, when administered i.p. 30min before test, pentagastrin (0.1-1.Omgkg-') failed to affect the time spent by mice in the illuminated side of the light/dark box. The mean time (s) spent by control animals in the light side was 117.7 + 16.2 (± s.e.mean, n = 10).
The oral activity of CI-988 in the mouse light/dark box and the rat elevated X-maze The oral administration of CI-988 (0.001-10.Omgkg-1) 40min before test, increased the percentage time spent and entries made by rats on to the end sections of the X-maze in a dose-related manner (Figure 2). However, it did not alter the total number of entries suggesting a lack of affect on spontaneous locomotor activity. Similarly, CI-988 also increased
Table 2 The effect of CI-988, CDP and pentagastrin in the rat conflict test
Unpunished Treatments
CDP
CI-988
Pentagastrin Pentagastrin +
CI-988
Dose (mg kg-')
1.0 3.0 10.0 30.0 0.001 0.01 0.1 1.0 16 pg per rat 16 pg per rat
Punished % Increase or decrease
9.4 + 4.2* 7.8 + 5.6 -14.5 + 16.1 -62.1 + 20.9** -1.9 + 3.9 -2.6 + 2.8 -0.3 + 5.6 -0.3 + 2.1 -48.3 + 12.8*
88.6 + 41.6 84.8 + 15.8** 137.5 + 50.7** 3.5 + 55.0 12.1 + 11.7 25.8 + 11.8* 14.3 + 9.0 7.7 + 11.8 -53.0 + 13.5**
- 10.6 + 10.3*
-3.6 + 13.8*
+
0.1 mgkg-', i.p.
241
Results are expressed as the mean percentage increase or decrease of lever-pressing rates ( + s.e.mean of at least 9 rats per group) on the test day compared with mean performances obtained the two previous days following vehicle administration. Test compounds were administered i.p. 40 min before test. For pentagastrin n = 6. Significantly different from previous control days *P
