- Email: [email protected]
Inhibition of pentagastrin-induced pressor response in conscious rats by the CCK-B receptor antagonist CI-988 and chlordiazepoxide
REGULATORY
ELSEVIER
PEPTIDES Regulatory Peptides 61 (1996) 71-76
Inhibition of pentagastrin-induced pressor response in conscious rats by the CCK-B receptor antagonist CI-988 and chlordiazepoxide J.K. Webb, N.M.J. Rupniak *, S. Boyce Merck, Sharp and Dohme Research Laboratories, Neuroscience Research Centre, Terlings Park, Eastwick Road, Harlow, Essex, CM20 2QR, UK Received 8 May 1995; revised 11 September 1995; accepted 11 September 1995
Abstract
Elevation of blood pressure is a characteristic feature of spontaneous and pharmacologically induced panic attacks in man. We examined whether this symptom could be reproduced in conscious rats and, if so, whether the cardiovascular changes induced by anxiogenic agents could be employed as a functional in vivo screen for CCK-B receptor antagonists which might be predictive of anxiolytic activity. Bolus i.v. administration of pentagastrin (0.1-100 /.~g/kg) or the /3-carboline FG-7142 (0.001-3 mg/kg) caused transient (< 5 min) dose-dependent increases in arterial blood pressure of up to 41 mmHg. The benzodiazepine receptor agonist chlordiazepoxide (10 mg/kg i.v., 15 min previously) attenuated the pressor response induced by either pentagastrin (1 ~g/kg i.v.) or FG-7142 (0.3 mg/kg i.v.). ]tn contrast, the CCK-B receptor antagonist CI-988 (3 mg/kg i.v., 15 min previously) inhibited the pressor response induced by pentagastrin, but not FG-7142. Antagonism of the pressor response elicited by pentagastrin in animals provides a simple method to establish the active dose range for CCK-B receptor antagonists in vivo. Since cardiovascular parameters can be readily monitored in man, this approach may assist in guiding clinical dose ranging studies to establish therapeutically beneficial effects of these compounds in panic disorder. Keywords: Panic attack; Blood pressure; Animal model; Anxiety
1. Introduction
Panic attacks are a severe form of anxiety disorder in which sudden feelings of intense fear, such as of impending death, are accompanied by alarming autonomic symptoms (hyperventilation, palpitations and sweating). The induction of similar symptoms following bolus intravenous injection of CCK-4 [6] or pentagastrin [1] suggests that CCK-B receptor antagonists may be efficacious in the treatment of panic attacks, and possibly other anxiety disorders. Preliminary studies using normal volunteers have provided encouraging evidence in support of this proposal. The prototypical CCK-B receptor antagonists L-365,260 [13] and CI-988 [11] were able to attenuate or prevent the anxiogenic symptoms associated with injection of CCK-4 or lactic acid [24]. Unfortunately, clinical development of
* Corresponding author. 0167-0115/96/$15.00 © 1996 Elsevier Science B.V. All rights reserved SSDI 0 1 6 7 - 0 1 1 5 ( 9 5 ) 0 0 1 4 0 - 9
these compounds has been hindered by their poor bioavailability (CI-988) and aqueous solubility (L-365,260). Preclinical screening for improved CCK-B receptor antagonists requires reliable assays with which to define the active dose range in vivo. Although CCK-B receptor antagonists exhibit activity in a range of behavioural paradigms which may be predictive of anxiolytic activity in man [4,18,25], the effective dose range for these compounds varies widely between different studies, both within and across paradigms. For example, in the mouse light/dark box test, there was a 100-fold discrepancy in the minimum effective dose of CI-988 reported by Singh et al. [26] (0.1 mg/kg) and by Costall et al. [4] (1 /xg/kg). Moreover, CI-988 was found to be effective in the rat elevated plus maze over a wide dose range (0.01-3 mg/kg), but in the rat conflict test only one dose (0.01 m g / k g ) caused significant effects [26]. Finally, the anxiolytic-like effects of both compounds in these assays have not been observed by other investigators [5]. Hence there is no consensus regarding which, if any, of these assays may
72
J.K. Webb et al. / Regulatory Peptides 61 (1996) 71-76
be employed as a functional in vivo screen for CCK-B receptor antagonists. In man, cardiovascular symptoms (tachycardia and increased blood pressure) accompany feelings of anxiety following administration of sodium lactate [12], pentagastrin [2], or the /3-carboline analogue FG-7142 [8]. Similar cardiovascular effects have been reported in animals following administration of CCK-4 [16] or the /3-carboline benzodiazepine receptor inverse agonists /3-CCE and FG7142 [19]. We now examine the effect of pentagastrin and FG-7142 on blood pressure in conscious rats, and compare the ability of CI-988 and chlordiazepoxide to prevent the pressor response induced by these agents. Since cardiovascular parameters can be readily monitored in man, this approach may assist in guiding clinical dose ranging studies to establish therapeutically beneficial effects of CCK-B receptor antagonists in panic disorder.
mal pressor response were chosen for subsequent investigations using chlordiazepoxide and CI-988.
2.3. Effect of CI-988 and chlordiazepoxide on the pressor response to pentagastrin and FG-7142 Rats received an i.v. bolus of either pentagastrin (1 /zg/kg) or FG-7142 (0.3 mg/kg) in order to establish the pressor response to these agents. When blood pressure had returned to pretreatment levels (15 min later) a further, single i.v. injection of either CI-988 (1 or 3 mg/kg) or chlordiazepoxide (3 or 10 mg/kg), or water was administered. This was followed 15 min later by a second challenge dose of either pentagastrin (1 /zg/kg) or FG-7142 (0.3 mg/kg). Animals were then humanely killed using an overdose of pentobarbitone (Euthatal, Rhone-Poulenc Rorer, UK).
2.4. Drug preparation 2. Materials and methods
2.1. Subjects and surgical preparation The subjects were male Sprague-Dawley rats (240-260 g; Bantin and Kingman, UK). Under isoflurane anaesthesia, two incisions were made in the tail to permit cannulation of the caudal artery and vein with polythene tubing (Portex Ltd; o.d. 0.96 ram). The surgical area was sprayed with a local anaesthetic (xylocaine; Astra, UK) and dressed with propax gauze and surgical tape and the animals were allowed to recover from anaesthesia. Animals were placed in individual restrainers (16 cm long, 6 cm diameter) and allowed to habituate for at least 1 h to permit stabilisation of blood pressure prior to administration of test drugs. Blood pressure was recorded on a Graph-Tech Linearcorder via the arterial cannula. The difference between the pretreatment baseline and the peak systolic and diastolic blood pressure was determined by taking readings 1 min before and 1 min after drug administration. Experiments were performed in an environmentally controlled, quiet room to minimise spontaneous perturbations in blood pressure recordings.
Pentagastrin was obtained in the form of ampoules (Peptavlon, 500 /zg in 2 ml; ICI) and diluted to the required concentration with sterile 0.9% saline. N-methyl/3-carboline-3-carboxamide (FG-7142; RBI, UK) complexed with 2-hydroxypropyl-fl-cyclodextrin (8.8 mg FG7142 in 1 g) was dissolved in distilled water to the required concentration. Chlordiazepoxide hydrochloride (Sigma, UK) and CI-988 (synthesised by the Department of Medicinal Chemistry, Merck, Sharp and Dohme, Harlow, UK) were dissolved in distilled water. All doses are expressed as the free base equivalent.
2.5. Statistical analysis For construction of dose-response curves to pentagastrin and FG-7142, systolic and diastolic blood pressure values were subjected to one-way analysis of variance (ANOVA) with repeated measures using the statistical software package BMDP 2V (BBN Software Products Corporation). For examination of the effect of CI-988 or
PentagMtrn i 40 ¸
2.2. Pressor effects of pentagastrin and FG-7142
30j
Systolic
The effect of pentagastrin or FG-7142 on blood pressure was determined using sequential, rising doses administered via the intravenous cannula. A period of 15 min elapsed between the delivery of each dose in order to allow blood pressure to return to the pretreatment baseline level. Doses of pentagastrin ( 0 , 1 - 1 0 0 / ~ g / k g ) were based on those reported to elicit panic-like symptoms in man [2]. The dose range for FG,-7142 (0.001-3 mg/kg) was based" on that reported to elevate blood pressure in rats [19]. Doses of pentagastrin and FG-7142 producing a submaxi-
20
Diastolic
10
0.01
0.1
1
10
100
Dose (~g/kg i.v.)
Fig. 1. Effect of pentagastrin ( 1 - 1 0 0 / ~ g / k g i.v.) on mean arterial blood pressure in conscious rats. Values are the mean + S.E.M. for 8 animals. Each animal received every dose of pentagastrin administered in rising doses at 15-min intervals.
J.K. Webb et al. / Regulatory Peptides 61 (1996) 71-76 Pentagastrin
Chlordiazepoxide
1
f
f
t
t
t
0.1
0.3
1
3
10
30
t 100
f
37.3
mmHgL_ FG 7142
=mmma ~ 3
~ 10
¢ 30
* 100
10
C1-988 "!
t 300
~ 1000
3000
Fig. 2. Typical blood pressure responses to rising doses of pentagastrin and FG-7142, showing the duration of the pressor response and recovery of blood pressure to baseline levels. Numbers under the arrows indicate the dose administered in /zg/kg.
chlordiazepoxide on the pressor response to pentagastrin or FG-7142, the mean arteri~d pressure was calculated using the equation: (Systolic - diastolic)
t
3
1 min
t 1
73
'
'-r,
A ........
0.5
1
3
Fig. 4. Blood pressure responses to C1-988 and chlordiazepoxide. Animals received a single dose of either CI-988 (0.5-3 m g / k g i.v.) or chlordiazepoxide (3 or 10 m g / k g i.v.).
blood pressure). A dose of 1 /xg/kg (which approximated the EDs0 value) was chosen for further investigations using CI-988 and chlordiazepoxide.
+ diastolic
3
Mean arterial pressure values were subjected to one-way A N O V A using BMDP 71) statistical software. Data are expressed as the percentage inhibition of the pressor response by CI-988 or chlordiazepoxide.
3.2. Pressor response to FG-7142
Like pentagastrin, bolus intravenous injection of FG7142 (0.001-3 mg/kg) produced a dose-dependent in-
Pentagastrin-induced pressor response
3. Results
Cl-O~g 120
3.1. Pressor response to pentagastrin
100
Bolus intravenous injection of pentagastrin (0.1-100 /zg/kg) caused a dose-dependent increase in both systolic and diastolic blood pressure compared with vehicle treatment (F7,42 = 19.61, P < I).05 for systolic; F7.42 = 37.99, P < 0,05 for diastolic; Fig. 1). The effect of pentagastrin on blood pressure was transient, lasting less than 3 min (Fig. 2). The maximum response elicited by pentagastrin was observed following administration of the highest dose examined - 100 /xg/kg i.v. (increase of 28.7 5-3.72 mmHg for systolic, and 22.0 + 2.23 mmHg for diastolic FG 7142
80 60
==
*
40
2o ®
er
0
c~
Vehicle 0.5 1.0 Dose of CI-988 (mg/kg i.v.)
U)
0..
~6
Chlordlazepoxide 120
=.z: t-
100
o~
80
Z-,
60
Systolic
"~- "I"
40 20
o,asto, ~m
10 o
0 3 10 Doseof Chlordiazepoxide (mg/kg i.v.)
Vehicle -
0.001
-
0.01
0.1
1
10
Dose (mg/kg i.v.)
Fig. 3. Effect of FG-7142 (0.001-3 m g / k g i.v.) on arterial blood pressure in conscious rats. Values are the mean + S.E.M. for 9 animals. Each animal received every dose of FG-7142 administered in rising aoses at 15-min intervals.
Fig. 5. Effect o f CI-988 (0.5 and ] m g / k g i.v.) or chlordiazepoxide (3 and 10 m g / k g i.v.) on the pentagastrin (1 p , g / k g i.v.) induced pressor
response. Values are the m e a n + l S.E.M. for 3 - 5 animals. Animals received an intravenous (i.v.) dose of pentagastrin (1 g g / k g ) followed 15 min later by a single i.v. dose of either CI-988 or chlordiazepoxide. This was followed 15 rain later by a challenge dose of pentagastrin (1 / z g / k g i.v.). * P < 0.05 compared to vehicle-treated animals (Dunnett's test).
J.K. Webb et al. /Regulatory Peptides 61 (1996) 71-76
74
crease in both systolic and diastolic blood pressure compared with vehicle treatment (systolic, Fie,80 = 17.18, P < 0.05; diastolic, /'10,80 = 11.88, P < 0.05, Fig. 3). The effect of FG-7142 on blood pressure was again transient (Fig. 2), lasting less than 3 min, although at higher doses this became more prolonged, and the trace more erratic, but always returning to the pretreatment baseline level within 5 min. The peak increase in arterial blood pressure recorded was 41.25 + 1.77 mmHg for systolic and 20.0 + 1.77 mmHg for diastolic blood pressure at the highest dose of 3 m g / k g i.v. A dose of 0.3 m g / k g i.v. of FG-7142 was employed for examination of the effects of CI-988 and chlordiazepoxide since this elicited a presser response of similar magnitude to that observed using 1 /.~g/kg i.v. of pentagastrin. 3.3. Effect of CI-988 and chlordiazepoxide on blood pressure Administration of CI-988 (0.5 or 1 m g / k g i.v.) alone caused a transient increase in blood pressure which lasted approximately 1 min and returned to baseline levels within 2 min. In contrast, chlordiazepoxide (3 or 10 m g / k g i.v.)
FG 7142-1nduced pressor response Cl-988
120 1O0 -
[
caused a transient decrease in blood pressure which recovered within 5 min (Fig. 4). 3.4. Effect of CI-988 and chlordiazepoxide on the presser response to pentagastrin and FG-7142 Antagonism of the presser response to pentagastrin and FG-7142 was examined following a 15 min pretreatment with CI-988 or chlordiazepoxide in order to allow recovery from the potentially confounding direct effects of these agents on blood pressure (see above). Intravenous administration of pentagastrin (1 /zg/kg) caused an increase in mean arterial pressure of 26.0 + 1.5 mmHg which was not altered by a 15 min pretreatment with vehicle. This presser response was reduced by approximately 50% following a 15 min pretreatment with 1 m g / k g i.v. of CI-988, but not by a lower dose (0.3 mg/kg; F3,12 = 4.78, P < 0.05; Fig. 5). Similarly, administration of 10, but not 3, m g / k g i.v. of chlordiazepoxide caused a 37% inhibition of the pentagastrin-induced presser response (F2,15 = 4.26, P < 0.05; Fig. 5). The challenge dose of FG-7142 (0.3 m g / k g i.v.) increased mean arterial pressure by 16.9 ___0.9 mmHg. This response was not attenuated by doses of up to 3 m g / k g i.v. of CI-988 (F2,13 = 0.77, P = 0.48; Fig. 6). Higher doses of CI-988 could not be employed owing to limitations of solubility. Unlike CI-988, chlordiazepoxide (10 m g / k g i.v.) inhibited the FG-7142-induced presser response by approximately 65% (F2,15 = 7.59, P < 0.05; Fig. 6).
80-
4. Discussion
6019
40-
o
20
rr
0
==
Vehicle
¢)
1
3
Dose of CI-988 (mg/kg i.v.)
0_
"6
Chlordiazepoxide 120 100
o~
III
80 60 40
20 0
10 Vehicle 3 Dose of Chlordiazepoxide (mg/kg i.v.) Fig. 6. Effect of CI-988 (1 and 3 m g / k g i.v.) or chlordiazepoxide (3 and 10 m g / k g i.v.) on the FG-7142 (0.3 m g / k g i.v.) induced presser response. Values are the m e a n + 1 S.E.M. for 3 - 5 animals. Animals received an intravenous (i.v.) dose of FG-7142 (0.3 m g / k g ) followed 15 min later by a single i.v. dose of either CI-988 or chlordiazepoxide. This was followed 15 rain later by a challenge dose of FG-7142 (0.3 m g / k g i.v.). * P < 0.05 compared to vehicle-treated animals (Dunnett's test).
Elevation of blood pressure is a characteristic feature of panic attacks provoked in man by pentagastrin [2] or FG-7142 [8]. The purpose of the present study was to examine whether this symptom could be reproduced in conscious rats and if so, whether the cardiovascular changes induced by anxiogenic agents could be employed as an in vivo assay to establish the active dose range for CCK-B receptor antagonists. Both pentagastrin and FG-7142 given intravenously produced dose-related elevations in blood pressure in rats which could be reversed by the benzodiazepine receptor agonist chlordiazepoxide (10 m g / k g i.v.). The CCK-B receptor antagonist CI-988 (1 m g / k g i.v.) reversed the presser response induced by pentagastrin, but not that induced by FG-7142. The inhibition of these presser responses by CI-988 and chlordiazepoxide was observed at a time when blood pressure had normalised following their administration, and was therefore not attributable to a direct effect of these agents on blood pressure. The EDs0 for the presser response induced by pentagastrin (approximately 1 /zg/kg i.v.) in conscious rats is consistent with the dose of 0.6 /zg/kg i.v. reported to provoke panic and cardiovascular symptoms in man [1,27].
J.K. Webb et al. / Regulatory Peptides 61 (1996) 71-76
The extremely short duration of the peak pressor response in rats ( < 1 min) may explain the failure to detect cardiovascular effects of pentagastrin in a previous study using rhesus monkeys [23]. It is not clear whether the pressor effect of intravenous injection of CCK-B receptor agonists are mediated centrally or peripherally. Whilst the extremely rapid response and /~g/kg potency suggest that pentagastrin is most likely to elicit the pressor response via a direct activation of CCK-B receptors in the periphery, a central mechanism of action is also possible [15]. However, the ability of CI-988 to block the pentagastrin-induced pressor response at a dose of 1 m g / k g i.v. strongly suggests a peripheral, rather than a central, action at CCK-B receptors. Thus, whilst CI-988 was able to potently displace [125I]Bolton Hunter-CCK-8S binding to whole mouse brain ex vivo following direct intracerebral infusion (IDs0 = 43 /.tg/kg), this compound caused no significant displacement using intravenous doses below 30 m g / k g [17]. Other evidence also indicates that CI-988 penetrates the CNS poorly. Thus, using electrophysiological assays able to detect modifications in spontaneous firing of midbrain dopamine neurones following systemic administration of the brain penetrant CCK-B receptor antagonists LY-262691 [21] and L-365,260 [20], intravenous injection of CI-988 (up to 4 mg/kg) was reported to have little effect [14]. The ability of chlordiazepoxide (10 m g / k g i.v.) to inhibit the pentagastrin-induced pressor response is consistent with evidence that benzodiazepine receptor agonists can antagonise the effects of CCK- both in the periphery and the CNS, and can prevent CCK-4-induced panic attacks in man (see [6]). Similarly, a preliminary report indicated that CCK-4 induced behavioural agitation and cardiovascular effects in vervet monkeys were inhibited by benzodiazepines [9]. Like pentagastrin, intravenous administration of FG7142 at doses up to 3 m g / k g increased blood pressure in conscious rats ([19]; present study). Due to the proconvulsant activity [22] and limited solubility of FG-7142, intravenous administration has not been investigated fully in man [7]. After oral administration to normal volunteers, FG-7142 (200-400 mg) caused recurring waves of intense anxiety which were accompanied by prominent cardiovascular symptoms [8]. In the present study, the failure of CI-988 (up to 3 m g / k g i.v.) to inhibit the FG-7142-induced pressor response i~L rats might reflect poor CNS penetration, or the different pharmacological actions of these compounds. This issue might be addressed by examining the ability of central infusion of CI-988, or systemic administration of a brain penetrant CCK-B receptor antagonist, to block the FG-7142 induced pressor response. Conversely, the ability of chlordiazepoxide (10 m g / k g i.v.) to prevent the effect of FG-7142 on blood pressure is consistent with the benzodiiazepine receptor pharmacology of these compounds and their ability to penetrate the CNS (see [3,22]). Similarly in man, FG-7142-induced anxiety
75
was rapidly reversed by the benzodiazepine receptor agonist lormetazepam [8]. Significant uncertainties remain to be resolved concerning the potential clinical use of CCK-B receptor antagonists for panic disorder. It is not known whether such compounds would need to gain access to the CNS in order to exert therapeutic effects. Although it is often assumed that this would be a requirement [17], it is also possible that the pyschological symptoms of panic may occur in response to somatic symptoms, such as increased heart rate [10]. More detailed information about the ability of CI-988 to control the somatic and psychological symptoms of panic in man might help to clarify this issue.
References [1] Abelson, J.L. and Nesse, R.M., Cholecystokinin-4 and panic. Arch. Gen. Psychiat., 47 (1990) 395. [2] Abelson, J.L. and Nesse, R.M., Pentagastrin infusions in patients with panic disorder. I. Symptoms and cardiovascular responses. Biol. Psychiat., 36 (1994) 73-83. [3] Braestrup, C. and Neilsen, M., Neurotransmitters and CNS disease: anxiety. Lancet, ii (1982) 1030-1034. [4] Costall, B., Domeney, A.M., Hughes, J., Kelly, M.E., Naylor, R.J. and Woodruff, G.N., Anxiolytic effects of CCKB antagonists. Neuropeptides, 19 (1991) 65-73. [5] Dawson, G.R., Rupniak, N.M.J., Tye, S.J., Curnow, R., Iversen, S.D. and Tricldebank, M.D., Lack of effect of CCK B antagonists in animal screens for anxiolytic drugs. Psychopharmacology, 121 (1995) 109-117. [6] De Montigny, C., Cholecystokinin tetrapeptide induces panic-like attacks in healthy volunteers. Arch. Gen. Psychiat., 46 (1989) 511517. [7] Dorow, R., FG7142 and its anxiety-inducing effects in humans. Br. J. Clin. Pharmacol., 23 (1987) 781-782. [8] Dorow, R., Horowski, R., Paschelle, G., Amin, M. and Braestrup, C., Severe anxiety induced by FG7142, a fl-carboline ligand for benzodiazepine receptors. Lancet, ii (1983) 98-99. [9] Ervin, F.R., Palmour, R.M. and Bradwejn, J., A new primate model for panic disorders. Biol. Psychiat., 29 (1991) 430S. [10] Hibbert, G.A., Ideational components of anxiety: their origin and content. Br. J. Psychiat., 144 (1984) 618-624. [11] Hughes, J., Boden, P., Costail, B., Domeney, A., Kelly, E., Horwell, D., Hunter, J.C., Pinnock, R.D. and Woodruff, G.N., Development of a class of selective cholecystokinin type B receptor antagonists having potent anxiolytic activity. Proc. Natl. Acad. Sci USA, 87 (1990) 6728-6732. [12] Liebowitz, M.R., Gorman, J.M., Fyer, A.J., Levitt, M., Dillon, D., Levy, G., Appleby, I.L., Anderson, S., Palij, M., Davies, S.O. and Klein, D.F., Lactate provocation of panic attacks: II Biochemical and physiological findings. Arch. Gen. Psychiat., 42 (1985) 709-719. [13] Lotti, V.J. and Chang, R.S.L., A new and selective non-peptide gastrin antagonist and brain cholecystokinin receptor (CCK-B) ligand: L-365,260. Eur. J. Pharmacol., 162 (1989) 273-278. [14] Meltzer, L., Christoffersen, C.L., Serpa, K.A. and Razmpour, A., Comparison of the effects of the cholecystokinin-B receptor antagonist, PD134308, and the cholecystokinin-A receptor antagonist, L364,718, on dopamine neuronal activity in the substantia nigra and ventral tegmental area. Synapse, 13 (1992) 117-122. [15] Pagani, F.D., Taveira da Silva, A.M., Hamosh, P., Garvey, T.Q. and Gilles, R.A., Respiratory and cardiovascular effects of intraventricular cholecystokinin. Eur. J. Pharmacol., 78 (1982) 128-132.
76
J.K. Webb et aL /Regulatory Peptides 61 (1996) 71-76
[16] Palmour, R.M., Ervin, F.R., Bradwejn, J. and Haubert, J.J., Anxiogenic and cardiovascular effects of CCK-4 in monkeys are blocked by the CCK-B antagonist LY262691. Soc. Neurosci. Abstr., 17 (1991) 637. [17] Patel, S., Chapman, K.L., Heald, A., Smith, A.J. and Freedman, S.B., Measurement of central nervous system activity of systemically administered CCK B receptor antagonists by ex uivo binding. Eur. J. Pharmacol., 253 (1994) 237-244. [18] Powell, K.R. and Barrett, J.E., Evaluation of the effects of PD134308 (CI-988), a CCK a antagonist, on the punished responding of squirrel monkeys. Neuropeptides, 19 (1991) 75-78. [19] Pratt, J.A., Laurie, D.J. and McCulloch, J., The effects of FG7142 upon local cerebral glucose utilisation suggest overlap between limbic structures important in anxiety and convulsions. Brain Res., 475 (1988) 218-231. [20] Rasmussen, K., Czachura, J.F., Stockton, M.E. and Howbert, J.J., Electrophysiological effects of diphenylpyrazolidinone cholecystokinin-B and cholecystokinin-A antagonists on midbrain dopamine neurons. J. Pharmacol. Exp. Ther., 264 (1992) 480-488. [21] Rasmussen, K., Stockton, M.E., Czachura, J.F. and Howbert, J.J.,
[22]
[23]
[24] [25]
[26]
[27]
Cholecystokinin (CCK) and schizophrenia: the selective CCK B antagonist LY262691 decreases midbrain dopamine unit activity. Eur. J. Pharmacol., 209 (1991) 135-138. Rossier, J., Dodd, R., Felblum, S., Valin, A., Prado De Carvalho, L., Potier, P. and Naquet, R., Methylamide fl-carboline (FG7142) an anxiogenic benzodiazepine antagonist, is also a proconvulsant. Lancet, I (1983) 77-78. Rupniak, N.M.J., Schaffer, L., Siegl, P. and Iversen, S.D., Failure of intravenous pentagastrin challenge to induce panic-like effects in rhesus monkeys. Neuropeptides, 25 (1993) 115-119. SCRIP, Progress with peptide antagonists. SCRIP (1949) 20. Singh, L., Lewis, A.S., Field, M.J., Hughes, J. and Woodruff, G.N., Evidence for an involvement of the brain cholecystokinin B receptor in anxiety. Proc. Natl. Acad. Sci. USA, 88 (1991) 1130-1133. Singh, L., Field, M.J., Hughes, J., Menzies, R., Oles, R.J., Van C.A. and Woodruff, G.N., The behavioural properties of CI-988, a selective cholecystokinin B receptor antagonist. Br. J. Pharmacol., 104 (1991) 239-245. Traub, M., Lines, C. and Ambrose, J., CCK and anxiety in normal volunteers. Br. J. Clin. Pharmacol., 36 (1993) 504P.
ELSEVIER
PEPTIDES Regulatory Peptides 61 (1996) 71-76
Inhibition of pentagastrin-induced pressor response in conscious rats by the CCK-B receptor antagonist CI-988 and chlordiazepoxide J.K. Webb, N.M.J. Rupniak *, S. Boyce Merck, Sharp and Dohme Research Laboratories, Neuroscience Research Centre, Terlings Park, Eastwick Road, Harlow, Essex, CM20 2QR, UK Received 8 May 1995; revised 11 September 1995; accepted 11 September 1995
Abstract
Elevation of blood pressure is a characteristic feature of spontaneous and pharmacologically induced panic attacks in man. We examined whether this symptom could be reproduced in conscious rats and, if so, whether the cardiovascular changes induced by anxiogenic agents could be employed as a functional in vivo screen for CCK-B receptor antagonists which might be predictive of anxiolytic activity. Bolus i.v. administration of pentagastrin (0.1-100 /.~g/kg) or the /3-carboline FG-7142 (0.001-3 mg/kg) caused transient (< 5 min) dose-dependent increases in arterial blood pressure of up to 41 mmHg. The benzodiazepine receptor agonist chlordiazepoxide (10 mg/kg i.v., 15 min previously) attenuated the pressor response induced by either pentagastrin (1 ~g/kg i.v.) or FG-7142 (0.3 mg/kg i.v.). ]tn contrast, the CCK-B receptor antagonist CI-988 (3 mg/kg i.v., 15 min previously) inhibited the pressor response induced by pentagastrin, but not FG-7142. Antagonism of the pressor response elicited by pentagastrin in animals provides a simple method to establish the active dose range for CCK-B receptor antagonists in vivo. Since cardiovascular parameters can be readily monitored in man, this approach may assist in guiding clinical dose ranging studies to establish therapeutically beneficial effects of these compounds in panic disorder. Keywords: Panic attack; Blood pressure; Animal model; Anxiety
1. Introduction
Panic attacks are a severe form of anxiety disorder in which sudden feelings of intense fear, such as of impending death, are accompanied by alarming autonomic symptoms (hyperventilation, palpitations and sweating). The induction of similar symptoms following bolus intravenous injection of CCK-4 [6] or pentagastrin [1] suggests that CCK-B receptor antagonists may be efficacious in the treatment of panic attacks, and possibly other anxiety disorders. Preliminary studies using normal volunteers have provided encouraging evidence in support of this proposal. The prototypical CCK-B receptor antagonists L-365,260 [13] and CI-988 [11] were able to attenuate or prevent the anxiogenic symptoms associated with injection of CCK-4 or lactic acid [24]. Unfortunately, clinical development of
* Corresponding author. 0167-0115/96/$15.00 © 1996 Elsevier Science B.V. All rights reserved SSDI 0 1 6 7 - 0 1 1 5 ( 9 5 ) 0 0 1 4 0 - 9
these compounds has been hindered by their poor bioavailability (CI-988) and aqueous solubility (L-365,260). Preclinical screening for improved CCK-B receptor antagonists requires reliable assays with which to define the active dose range in vivo. Although CCK-B receptor antagonists exhibit activity in a range of behavioural paradigms which may be predictive of anxiolytic activity in man [4,18,25], the effective dose range for these compounds varies widely between different studies, both within and across paradigms. For example, in the mouse light/dark box test, there was a 100-fold discrepancy in the minimum effective dose of CI-988 reported by Singh et al. [26] (0.1 mg/kg) and by Costall et al. [4] (1 /xg/kg). Moreover, CI-988 was found to be effective in the rat elevated plus maze over a wide dose range (0.01-3 mg/kg), but in the rat conflict test only one dose (0.01 m g / k g ) caused significant effects [26]. Finally, the anxiolytic-like effects of both compounds in these assays have not been observed by other investigators [5]. Hence there is no consensus regarding which, if any, of these assays may
72
J.K. Webb et al. / Regulatory Peptides 61 (1996) 71-76
be employed as a functional in vivo screen for CCK-B receptor antagonists. In man, cardiovascular symptoms (tachycardia and increased blood pressure) accompany feelings of anxiety following administration of sodium lactate [12], pentagastrin [2], or the /3-carboline analogue FG-7142 [8]. Similar cardiovascular effects have been reported in animals following administration of CCK-4 [16] or the /3-carboline benzodiazepine receptor inverse agonists /3-CCE and FG7142 [19]. We now examine the effect of pentagastrin and FG-7142 on blood pressure in conscious rats, and compare the ability of CI-988 and chlordiazepoxide to prevent the pressor response induced by these agents. Since cardiovascular parameters can be readily monitored in man, this approach may assist in guiding clinical dose ranging studies to establish therapeutically beneficial effects of CCK-B receptor antagonists in panic disorder.
mal pressor response were chosen for subsequent investigations using chlordiazepoxide and CI-988.
2.3. Effect of CI-988 and chlordiazepoxide on the pressor response to pentagastrin and FG-7142 Rats received an i.v. bolus of either pentagastrin (1 /zg/kg) or FG-7142 (0.3 mg/kg) in order to establish the pressor response to these agents. When blood pressure had returned to pretreatment levels (15 min later) a further, single i.v. injection of either CI-988 (1 or 3 mg/kg) or chlordiazepoxide (3 or 10 mg/kg), or water was administered. This was followed 15 min later by a second challenge dose of either pentagastrin (1 /zg/kg) or FG-7142 (0.3 mg/kg). Animals were then humanely killed using an overdose of pentobarbitone (Euthatal, Rhone-Poulenc Rorer, UK).
2.4. Drug preparation 2. Materials and methods
2.1. Subjects and surgical preparation The subjects were male Sprague-Dawley rats (240-260 g; Bantin and Kingman, UK). Under isoflurane anaesthesia, two incisions were made in the tail to permit cannulation of the caudal artery and vein with polythene tubing (Portex Ltd; o.d. 0.96 ram). The surgical area was sprayed with a local anaesthetic (xylocaine; Astra, UK) and dressed with propax gauze and surgical tape and the animals were allowed to recover from anaesthesia. Animals were placed in individual restrainers (16 cm long, 6 cm diameter) and allowed to habituate for at least 1 h to permit stabilisation of blood pressure prior to administration of test drugs. Blood pressure was recorded on a Graph-Tech Linearcorder via the arterial cannula. The difference between the pretreatment baseline and the peak systolic and diastolic blood pressure was determined by taking readings 1 min before and 1 min after drug administration. Experiments were performed in an environmentally controlled, quiet room to minimise spontaneous perturbations in blood pressure recordings.
Pentagastrin was obtained in the form of ampoules (Peptavlon, 500 /zg in 2 ml; ICI) and diluted to the required concentration with sterile 0.9% saline. N-methyl/3-carboline-3-carboxamide (FG-7142; RBI, UK) complexed with 2-hydroxypropyl-fl-cyclodextrin (8.8 mg FG7142 in 1 g) was dissolved in distilled water to the required concentration. Chlordiazepoxide hydrochloride (Sigma, UK) and CI-988 (synthesised by the Department of Medicinal Chemistry, Merck, Sharp and Dohme, Harlow, UK) were dissolved in distilled water. All doses are expressed as the free base equivalent.
2.5. Statistical analysis For construction of dose-response curves to pentagastrin and FG-7142, systolic and diastolic blood pressure values were subjected to one-way analysis of variance (ANOVA) with repeated measures using the statistical software package BMDP 2V (BBN Software Products Corporation). For examination of the effect of CI-988 or
PentagMtrn i 40 ¸
2.2. Pressor effects of pentagastrin and FG-7142
30j
Systolic
The effect of pentagastrin or FG-7142 on blood pressure was determined using sequential, rising doses administered via the intravenous cannula. A period of 15 min elapsed between the delivery of each dose in order to allow blood pressure to return to the pretreatment baseline level. Doses of pentagastrin ( 0 , 1 - 1 0 0 / ~ g / k g ) were based on those reported to elicit panic-like symptoms in man [2]. The dose range for FG,-7142 (0.001-3 mg/kg) was based" on that reported to elevate blood pressure in rats [19]. Doses of pentagastrin and FG-7142 producing a submaxi-
20
Diastolic
10
0.01
0.1
1
10
100
Dose (~g/kg i.v.)
Fig. 1. Effect of pentagastrin ( 1 - 1 0 0 / ~ g / k g i.v.) on mean arterial blood pressure in conscious rats. Values are the mean + S.E.M. for 8 animals. Each animal received every dose of pentagastrin administered in rising doses at 15-min intervals.
J.K. Webb et al. / Regulatory Peptides 61 (1996) 71-76 Pentagastrin
Chlordiazepoxide
1
f
f
t
t
t
0.1
0.3
1
3
10
30
t 100
f
37.3
mmHgL_ FG 7142
=mmma ~ 3
~ 10
¢ 30
* 100
10
C1-988 "!
t 300
~ 1000
3000
Fig. 2. Typical blood pressure responses to rising doses of pentagastrin and FG-7142, showing the duration of the pressor response and recovery of blood pressure to baseline levels. Numbers under the arrows indicate the dose administered in /zg/kg.
chlordiazepoxide on the pressor response to pentagastrin or FG-7142, the mean arteri~d pressure was calculated using the equation: (Systolic - diastolic)
t
3
1 min
t 1
73
'
'-r,
A ........
0.5
1
3
Fig. 4. Blood pressure responses to C1-988 and chlordiazepoxide. Animals received a single dose of either CI-988 (0.5-3 m g / k g i.v.) or chlordiazepoxide (3 or 10 m g / k g i.v.).
blood pressure). A dose of 1 /xg/kg (which approximated the EDs0 value) was chosen for further investigations using CI-988 and chlordiazepoxide.
+ diastolic
3
Mean arterial pressure values were subjected to one-way A N O V A using BMDP 71) statistical software. Data are expressed as the percentage inhibition of the pressor response by CI-988 or chlordiazepoxide.
3.2. Pressor response to FG-7142
Like pentagastrin, bolus intravenous injection of FG7142 (0.001-3 mg/kg) produced a dose-dependent in-
Pentagastrin-induced pressor response
3. Results
Cl-O~g 120
3.1. Pressor response to pentagastrin
100
Bolus intravenous injection of pentagastrin (0.1-100 /zg/kg) caused a dose-dependent increase in both systolic and diastolic blood pressure compared with vehicle treatment (F7,42 = 19.61, P < I).05 for systolic; F7.42 = 37.99, P < 0,05 for diastolic; Fig. 1). The effect of pentagastrin on blood pressure was transient, lasting less than 3 min (Fig. 2). The maximum response elicited by pentagastrin was observed following administration of the highest dose examined - 100 /xg/kg i.v. (increase of 28.7 5-3.72 mmHg for systolic, and 22.0 + 2.23 mmHg for diastolic FG 7142
80 60
==
*
40
2o ®
er
0
c~
Vehicle 0.5 1.0 Dose of CI-988 (mg/kg i.v.)
U)
0..
~6
Chlordlazepoxide 120
=.z: t-
100
o~
80
Z-,
60
Systolic
"~- "I"
40 20
o,asto, ~m
10 o
0 3 10 Doseof Chlordiazepoxide (mg/kg i.v.)
Vehicle -
0.001
-
0.01
0.1
1
10
Dose (mg/kg i.v.)
Fig. 3. Effect of FG-7142 (0.001-3 m g / k g i.v.) on arterial blood pressure in conscious rats. Values are the mean + S.E.M. for 9 animals. Each animal received every dose of FG-7142 administered in rising aoses at 15-min intervals.
Fig. 5. Effect o f CI-988 (0.5 and ] m g / k g i.v.) or chlordiazepoxide (3 and 10 m g / k g i.v.) on the pentagastrin (1 p , g / k g i.v.) induced pressor
response. Values are the m e a n + l S.E.M. for 3 - 5 animals. Animals received an intravenous (i.v.) dose of pentagastrin (1 g g / k g ) followed 15 min later by a single i.v. dose of either CI-988 or chlordiazepoxide. This was followed 15 rain later by a challenge dose of pentagastrin (1 / z g / k g i.v.). * P < 0.05 compared to vehicle-treated animals (Dunnett's test).
J.K. Webb et al. /Regulatory Peptides 61 (1996) 71-76
74
crease in both systolic and diastolic blood pressure compared with vehicle treatment (systolic, Fie,80 = 17.18, P < 0.05; diastolic, /'10,80 = 11.88, P < 0.05, Fig. 3). The effect of FG-7142 on blood pressure was again transient (Fig. 2), lasting less than 3 min, although at higher doses this became more prolonged, and the trace more erratic, but always returning to the pretreatment baseline level within 5 min. The peak increase in arterial blood pressure recorded was 41.25 + 1.77 mmHg for systolic and 20.0 + 1.77 mmHg for diastolic blood pressure at the highest dose of 3 m g / k g i.v. A dose of 0.3 m g / k g i.v. of FG-7142 was employed for examination of the effects of CI-988 and chlordiazepoxide since this elicited a presser response of similar magnitude to that observed using 1 /.~g/kg i.v. of pentagastrin. 3.3. Effect of CI-988 and chlordiazepoxide on blood pressure Administration of CI-988 (0.5 or 1 m g / k g i.v.) alone caused a transient increase in blood pressure which lasted approximately 1 min and returned to baseline levels within 2 min. In contrast, chlordiazepoxide (3 or 10 m g / k g i.v.)
FG 7142-1nduced pressor response Cl-988
120 1O0 -
[
caused a transient decrease in blood pressure which recovered within 5 min (Fig. 4). 3.4. Effect of CI-988 and chlordiazepoxide on the presser response to pentagastrin and FG-7142 Antagonism of the presser response to pentagastrin and FG-7142 was examined following a 15 min pretreatment with CI-988 or chlordiazepoxide in order to allow recovery from the potentially confounding direct effects of these agents on blood pressure (see above). Intravenous administration of pentagastrin (1 /zg/kg) caused an increase in mean arterial pressure of 26.0 + 1.5 mmHg which was not altered by a 15 min pretreatment with vehicle. This presser response was reduced by approximately 50% following a 15 min pretreatment with 1 m g / k g i.v. of CI-988, but not by a lower dose (0.3 mg/kg; F3,12 = 4.78, P < 0.05; Fig. 5). Similarly, administration of 10, but not 3, m g / k g i.v. of chlordiazepoxide caused a 37% inhibition of the pentagastrin-induced presser response (F2,15 = 4.26, P < 0.05; Fig. 5). The challenge dose of FG-7142 (0.3 m g / k g i.v.) increased mean arterial pressure by 16.9 ___0.9 mmHg. This response was not attenuated by doses of up to 3 m g / k g i.v. of CI-988 (F2,13 = 0.77, P = 0.48; Fig. 6). Higher doses of CI-988 could not be employed owing to limitations of solubility. Unlike CI-988, chlordiazepoxide (10 m g / k g i.v.) inhibited the FG-7142-induced presser response by approximately 65% (F2,15 = 7.59, P < 0.05; Fig. 6).
80-
4. Discussion
6019
40-
o
20
rr
0
==
Vehicle
¢)
1
3
Dose of CI-988 (mg/kg i.v.)
0_
"6
Chlordiazepoxide 120 100
o~
III
80 60 40
20 0
10 Vehicle 3 Dose of Chlordiazepoxide (mg/kg i.v.) Fig. 6. Effect of CI-988 (1 and 3 m g / k g i.v.) or chlordiazepoxide (3 and 10 m g / k g i.v.) on the FG-7142 (0.3 m g / k g i.v.) induced presser response. Values are the m e a n + 1 S.E.M. for 3 - 5 animals. Animals received an intravenous (i.v.) dose of FG-7142 (0.3 m g / k g ) followed 15 min later by a single i.v. dose of either CI-988 or chlordiazepoxide. This was followed 15 rain later by a challenge dose of FG-7142 (0.3 m g / k g i.v.). * P < 0.05 compared to vehicle-treated animals (Dunnett's test).
Elevation of blood pressure is a characteristic feature of panic attacks provoked in man by pentagastrin [2] or FG-7142 [8]. The purpose of the present study was to examine whether this symptom could be reproduced in conscious rats and if so, whether the cardiovascular changes induced by anxiogenic agents could be employed as an in vivo assay to establish the active dose range for CCK-B receptor antagonists. Both pentagastrin and FG-7142 given intravenously produced dose-related elevations in blood pressure in rats which could be reversed by the benzodiazepine receptor agonist chlordiazepoxide (10 m g / k g i.v.). The CCK-B receptor antagonist CI-988 (1 m g / k g i.v.) reversed the presser response induced by pentagastrin, but not that induced by FG-7142. The inhibition of these presser responses by CI-988 and chlordiazepoxide was observed at a time when blood pressure had normalised following their administration, and was therefore not attributable to a direct effect of these agents on blood pressure. The EDs0 for the presser response induced by pentagastrin (approximately 1 /zg/kg i.v.) in conscious rats is consistent with the dose of 0.6 /zg/kg i.v. reported to provoke panic and cardiovascular symptoms in man [1,27].
J.K. Webb et al. / Regulatory Peptides 61 (1996) 71-76
The extremely short duration of the peak pressor response in rats ( < 1 min) may explain the failure to detect cardiovascular effects of pentagastrin in a previous study using rhesus monkeys [23]. It is not clear whether the pressor effect of intravenous injection of CCK-B receptor agonists are mediated centrally or peripherally. Whilst the extremely rapid response and /~g/kg potency suggest that pentagastrin is most likely to elicit the pressor response via a direct activation of CCK-B receptors in the periphery, a central mechanism of action is also possible [15]. However, the ability of CI-988 to block the pentagastrin-induced pressor response at a dose of 1 m g / k g i.v. strongly suggests a peripheral, rather than a central, action at CCK-B receptors. Thus, whilst CI-988 was able to potently displace [125I]Bolton Hunter-CCK-8S binding to whole mouse brain ex vivo following direct intracerebral infusion (IDs0 = 43 /.tg/kg), this compound caused no significant displacement using intravenous doses below 30 m g / k g [17]. Other evidence also indicates that CI-988 penetrates the CNS poorly. Thus, using electrophysiological assays able to detect modifications in spontaneous firing of midbrain dopamine neurones following systemic administration of the brain penetrant CCK-B receptor antagonists LY-262691 [21] and L-365,260 [20], intravenous injection of CI-988 (up to 4 mg/kg) was reported to have little effect [14]. The ability of chlordiazepoxide (10 m g / k g i.v.) to inhibit the pentagastrin-induced pressor response is consistent with evidence that benzodiazepine receptor agonists can antagonise the effects of CCK- both in the periphery and the CNS, and can prevent CCK-4-induced panic attacks in man (see [6]). Similarly, a preliminary report indicated that CCK-4 induced behavioural agitation and cardiovascular effects in vervet monkeys were inhibited by benzodiazepines [9]. Like pentagastrin, intravenous administration of FG7142 at doses up to 3 m g / k g increased blood pressure in conscious rats ([19]; present study). Due to the proconvulsant activity [22] and limited solubility of FG-7142, intravenous administration has not been investigated fully in man [7]. After oral administration to normal volunteers, FG-7142 (200-400 mg) caused recurring waves of intense anxiety which were accompanied by prominent cardiovascular symptoms [8]. In the present study, the failure of CI-988 (up to 3 m g / k g i.v.) to inhibit the FG-7142-induced pressor response i~L rats might reflect poor CNS penetration, or the different pharmacological actions of these compounds. This issue might be addressed by examining the ability of central infusion of CI-988, or systemic administration of a brain penetrant CCK-B receptor antagonist, to block the FG-7142 induced pressor response. Conversely, the ability of chlordiazepoxide (10 m g / k g i.v.) to prevent the effect of FG-7142 on blood pressure is consistent with the benzodiiazepine receptor pharmacology of these compounds and their ability to penetrate the CNS (see [3,22]). Similarly in man, FG-7142-induced anxiety
75
was rapidly reversed by the benzodiazepine receptor agonist lormetazepam [8]. Significant uncertainties remain to be resolved concerning the potential clinical use of CCK-B receptor antagonists for panic disorder. It is not known whether such compounds would need to gain access to the CNS in order to exert therapeutic effects. Although it is often assumed that this would be a requirement [17], it is also possible that the pyschological symptoms of panic may occur in response to somatic symptoms, such as increased heart rate [10]. More detailed information about the ability of CI-988 to control the somatic and psychological symptoms of panic in man might help to clarify this issue.
References [1] Abelson, J.L. and Nesse, R.M., Cholecystokinin-4 and panic. Arch. Gen. Psychiat., 47 (1990) 395. [2] Abelson, J.L. and Nesse, R.M., Pentagastrin infusions in patients with panic disorder. I. Symptoms and cardiovascular responses. Biol. Psychiat., 36 (1994) 73-83. [3] Braestrup, C. and Neilsen, M., Neurotransmitters and CNS disease: anxiety. Lancet, ii (1982) 1030-1034. [4] Costall, B., Domeney, A.M., Hughes, J., Kelly, M.E., Naylor, R.J. and Woodruff, G.N., Anxiolytic effects of CCKB antagonists. Neuropeptides, 19 (1991) 65-73. [5] Dawson, G.R., Rupniak, N.M.J., Tye, S.J., Curnow, R., Iversen, S.D. and Tricldebank, M.D., Lack of effect of CCK B antagonists in animal screens for anxiolytic drugs. Psychopharmacology, 121 (1995) 109-117. [6] De Montigny, C., Cholecystokinin tetrapeptide induces panic-like attacks in healthy volunteers. Arch. Gen. Psychiat., 46 (1989) 511517. [7] Dorow, R., FG7142 and its anxiety-inducing effects in humans. Br. J. Clin. Pharmacol., 23 (1987) 781-782. [8] Dorow, R., Horowski, R., Paschelle, G., Amin, M. and Braestrup, C., Severe anxiety induced by FG7142, a fl-carboline ligand for benzodiazepine receptors. Lancet, ii (1983) 98-99. [9] Ervin, F.R., Palmour, R.M. and Bradwejn, J., A new primate model for panic disorders. Biol. Psychiat., 29 (1991) 430S. [10] Hibbert, G.A., Ideational components of anxiety: their origin and content. Br. J. Psychiat., 144 (1984) 618-624. [11] Hughes, J., Boden, P., Costail, B., Domeney, A., Kelly, E., Horwell, D., Hunter, J.C., Pinnock, R.D. and Woodruff, G.N., Development of a class of selective cholecystokinin type B receptor antagonists having potent anxiolytic activity. Proc. Natl. Acad. Sci USA, 87 (1990) 6728-6732. [12] Liebowitz, M.R., Gorman, J.M., Fyer, A.J., Levitt, M., Dillon, D., Levy, G., Appleby, I.L., Anderson, S., Palij, M., Davies, S.O. and Klein, D.F., Lactate provocation of panic attacks: II Biochemical and physiological findings. Arch. Gen. Psychiat., 42 (1985) 709-719. [13] Lotti, V.J. and Chang, R.S.L., A new and selective non-peptide gastrin antagonist and brain cholecystokinin receptor (CCK-B) ligand: L-365,260. Eur. J. Pharmacol., 162 (1989) 273-278. [14] Meltzer, L., Christoffersen, C.L., Serpa, K.A. and Razmpour, A., Comparison of the effects of the cholecystokinin-B receptor antagonist, PD134308, and the cholecystokinin-A receptor antagonist, L364,718, on dopamine neuronal activity in the substantia nigra and ventral tegmental area. Synapse, 13 (1992) 117-122. [15] Pagani, F.D., Taveira da Silva, A.M., Hamosh, P., Garvey, T.Q. and Gilles, R.A., Respiratory and cardiovascular effects of intraventricular cholecystokinin. Eur. J. Pharmacol., 78 (1982) 128-132.
76
J.K. Webb et aL /Regulatory Peptides 61 (1996) 71-76
[16] Palmour, R.M., Ervin, F.R., Bradwejn, J. and Haubert, J.J., Anxiogenic and cardiovascular effects of CCK-4 in monkeys are blocked by the CCK-B antagonist LY262691. Soc. Neurosci. Abstr., 17 (1991) 637. [17] Patel, S., Chapman, K.L., Heald, A., Smith, A.J. and Freedman, S.B., Measurement of central nervous system activity of systemically administered CCK B receptor antagonists by ex uivo binding. Eur. J. Pharmacol., 253 (1994) 237-244. [18] Powell, K.R. and Barrett, J.E., Evaluation of the effects of PD134308 (CI-988), a CCK a antagonist, on the punished responding of squirrel monkeys. Neuropeptides, 19 (1991) 75-78. [19] Pratt, J.A., Laurie, D.J. and McCulloch, J., The effects of FG7142 upon local cerebral glucose utilisation suggest overlap between limbic structures important in anxiety and convulsions. Brain Res., 475 (1988) 218-231. [20] Rasmussen, K., Czachura, J.F., Stockton, M.E. and Howbert, J.J., Electrophysiological effects of diphenylpyrazolidinone cholecystokinin-B and cholecystokinin-A antagonists on midbrain dopamine neurons. J. Pharmacol. Exp. Ther., 264 (1992) 480-488. [21] Rasmussen, K., Stockton, M.E., Czachura, J.F. and Howbert, J.J.,
[22]
[23]
[24] [25]
[26]
[27]
Cholecystokinin (CCK) and schizophrenia: the selective CCK B antagonist LY262691 decreases midbrain dopamine unit activity. Eur. J. Pharmacol., 209 (1991) 135-138. Rossier, J., Dodd, R., Felblum, S., Valin, A., Prado De Carvalho, L., Potier, P. and Naquet, R., Methylamide fl-carboline (FG7142) an anxiogenic benzodiazepine antagonist, is also a proconvulsant. Lancet, I (1983) 77-78. Rupniak, N.M.J., Schaffer, L., Siegl, P. and Iversen, S.D., Failure of intravenous pentagastrin challenge to induce panic-like effects in rhesus monkeys. Neuropeptides, 25 (1993) 115-119. SCRIP, Progress with peptide antagonists. SCRIP (1949) 20. Singh, L., Lewis, A.S., Field, M.J., Hughes, J. and Woodruff, G.N., Evidence for an involvement of the brain cholecystokinin B receptor in anxiety. Proc. Natl. Acad. Sci. USA, 88 (1991) 1130-1133. Singh, L., Field, M.J., Hughes, J., Menzies, R., Oles, R.J., Van C.A. and Woodruff, G.N., The behavioural properties of CI-988, a selective cholecystokinin B receptor antagonist. Br. J. Pharmacol., 104 (1991) 239-245. Traub, M., Lines, C. and Ambrose, J., CCK and anxiety in normal volunteers. Br. J. Clin. Pharmacol., 36 (1993) 504P.