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European Journal of Pharmacology262 (1994) 233-245

Pharmacological properties of ureido-acetamides, new potent and selective non-peptide CCKJgastrin receptor antagonists Philippe Bertrand a G. Andrees B6hme a,, Christiane Durieux d Claude Guyon b Marc Capet b, Bernadette Jeantaud a, Philippe Boudeau a, Bertrand Ducos d, Charles E. Pendley c, Gregory E. Martin c, Anne Floch a, Adam Doble a Departments of a Biology and b Chemistry, Rh~ne-Poulenc Rorer S.A., Centre de Recherches de Vhry-Alfortville, Vitry-Sur-Seine, France c Department of General Pharmacology, Rh3ne-Poulenc Rorer Inc., Collegeville Research Center, Collegeville, PA, USA d Laboratoire de Pharmacoehimie Mol~culaire, CNRS URA 1500/INSERM U-266, Facult~ de Pharmacie, Paris, France Received 17 March 1994; revised MS received 16 June 1994; accepted 21 June 1994

Abstract

We present here the pharmacological properties of 3 ureido-acetamide members of a novel family of non-peptide cholecystokinin-B (CCK B) receptor antagonists. RP 69758 (3-{3-[N-(N-methyl N-phenyl-carbamoylmethyl) N-phenylcarbamoylmethyl] ureido} phenylacetic acid), RP 71483 ((E)-2-[3-(3-hydroxyiminomethyl phenyl) ureido] N-(8-quinolyl) N[(1,2,3,4-tetrahydro 1-quinolyl) carbonylmethyl] acetamide) and RP 72540 ((RS)-2-{3-{3-[N-(3-methoxy phenyl) N-(N-methyl N-phenyl-carbamoylmethyl) carbamoylmethyl] ureido} phenyl} propionic acid) displayed nanomolar affinity for guinea-pig, rat and mouse CCKa receptors labelled with [3H]pCCK-8 or with the selective CCK B receptor ligand [3H]pBC264. RP 69758 and RP 72540 showed selectivity factors in excess of 200 for CCK B versus CCK A receptors. All three compounds had also high affinity for gastrin binding sites in the stomach. The ureido-acetamides behaved as potent antagonists of CCK-8-induced neuronal firing in rat hippocampal slices in vitro, a functional model of brain CCK B receptor mediated responses. RP 69758 is also a potent gastrin receptor antagonist in vivo that dose dependently inhibits gastric acid secretion induced by i.v. injection of pentagastrin in the rat. None of the three ureido-acetamides, at concentrations up to 1/zM, significantly blocked CCK-8-evoked contractions of the guinea-pig ileum in vitro, a CCK A receptor bioassay. In ex vivo binding studies, i.p. administration of RP 69758 and RP 72540 resulted in a dose-dependent inhibition of [3H]pCCK-8 binding in mouse brain homogenate. However, the relative penetration of these ureido-acetamides into the forebrain after peripheral administration was below 0.01%. RP 71483 did not appear to cross the blood-brain barrier in quantities sufficient to prevent [3H]pCCK-8 binding at low doses, a property that makes it suitable for the exploration of the peripheral versus central origin of the behavioural effects observed following systemic administration of CCK. RP 69758, RP 71483 and RP 72540 are highly potent and selective non-peptide CCK B receptor antagonists which are useful tools to explore the physiological functions of CCK B receptors.

Key words: Binding; Brain; Electrophysiology; Gastric acid secretion; Neuropeptide; CCK B receptor

I. Introduction

Cholecystokinin (CCK) and gastrin belong to a family of related peptides whose members play hormonal and neurotransmitter roles in the gastrointestinal tract and the central nervous system (CNS) (Rehfeld, 1981;

* Corresponding author. Rh6ne-Poulenc Rorer S.A., Centre de Recherches de Vitry-Alfortville, 13, quai Jules Guesde, 94403 VitrySur-Seine Cedex, France. Tel. 33 (1) 45 73 81 41, fax 33 (1) 45 73 76

53. 0014-2999/94/$07.00 © 1994 Elsevier Science B.V. All rights reserved SSDI 0 0 1 4 - 2 9 9 9 ( 9 4 ) 0 0 3 7 8 - K

Dockray, 1982). In the digestive system, CCK is released from the intestine mainly as the 33-amino acid peptide CCK-(1-33), and acts upon the gall bladder to initiate biliary discharge and upon the pancreas to stimulate exocrine secretion (Mutt, 1988). It also mediates peripheral satiety signalling by a vagal mechanism (Smith et al., 1981). In the central nervous system, the predominant form of CCK is the sulphated octapeptide CCK-8 (Rehfeld et al., 1985), which is widespread in cortical interneurones and also found in certain species in long-distance transmission pathways such as the mesolimbic and mesocortical systems, where it is colo-

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P. Bertrand et al. / European Journal of Pharmacology 262 (1994) 233-245

calised with dopamine (H6kfelt et al., 1985). Gastrin is a 17-amino acid peptide (G-17) which shares a common pentapeptide C-terminal sequence with CCK-8. It is liberated within the stomach wall, where it stimulates acid secretion from parietal cells (Mulholland and Debas, 1988). At the present time, there is no evidence that gastrin exists within the central nervous system. The actions of CCK/gastrin peptides are mediated by specific receptors, which have been differentiated pharmacologically (Innis and Snyder, 1980). In the gut, CCK interacts with the C C K A receptor, while in the CNS, the principal target receptor for CCK is the CCK B receptor (Moran et al., 1986). The latter receptors are indistinguishable from gastrointestinal gastrin receptors on the basis of their binding spectrum for a range of CCK/gastrin peptides, as well as amino acid sequence comparisons deduced from cloned cDNAs encoding both receptors in rodent and human tissue (Pisegna et al., 1992; Wank et al., 1992; Lee et al., 1993). CCKB/gastrin receptors are encoded by a single gene, localized on chromosome 11, for which alternative splicing has been recently demonstrated (Song et al., 1993). While the digestive functions of CCK are well established, its function in the brain remains largely unknown. CCK has been suggested to be involved in a variety of physiopathological processes in the CNS such as schizophrenia (Crawley, 1991), panic attacks (Bradwejn et al., 1992), nociception (Baber et al., 1989) and memory formation (Itoh and Lal, 1990; Lemaire et al., 1992, 1994). Understanding the role of the different CCK receptors in the physiology of this peptide requires the availability of potent and selective antagonists. This paper reports the pharmacological properties of selective CCK B receptor antagonists. Asperlicin, the first, albeit weak, non-peptide CCK receptor antagonist was discovered in 1985 as a natural fermentation product from an Aspergillus (Chang et al., 1985). Optimization of this structure led to the benzodiazepine L-364,718 (Evans et al., 1986), a highly potent CCK A receptor antagonist also known as MK-329 or devazepide. Further structural modifications yielded the CCKB/gastrin receptor selective benzodiazepine L365,260 (Bock et al., 1989). A second generation of potent and selective CCKB/gastrin receptor antagonists was derived by rational design from the C-terminal tetrapeptide CCK-4 (Horwell et al., 1990). These peptoids comprise PD-134,308 (also called CI-988) and PD-135,158 (CAM 1028), of which the latter displays full agonist activity a t C C K A receptors (H6cker et al., 1993). More recently, several diphenyl-pyrazolidinones were described as a third family of compounds whose members have either C C K A o r CCKB/gastrin receptor antagonist properties (Yu et al., 1992; Rasmussen et al., 1993). Here we present three members of the

A' NA..j. N O

o.y;

H

o

R /N~R2 NR1Rz

Ar

X

RP 69758

~N/OH

RP 71483

Oj

RP 72540

Fig. 1. Chemical structure of the ureido-acetamides.

ureido-acetamides, a new family of potent and selective non-peptide CCKB/gastrin receptor antagonists.

2. Materials and methods

2.1. Chemicals RP 69758 (3-{3-[N-(N-methyl N-phenyl-carbamoylmethyl) N-phenyl-carbamoylmethyl] ureido} phenylacetic acid), RP 72540 ((RS)-2-{3-{3-[N-(3-methoxy phenyl) N-(N-methyl N-phenyl-carbamoylmethyl) carbamoylmethyl] ureido} phenyl} propionic acid), RP 71483 ((E)-2-[3-(3-hydroxyiminomethyl phenyl) ureido]N-(8-quinolyl) N-[(1,2,3,4-tetrahydro 1-quinolyl) carbonylmethyl] acetamide), L-365,260, devazepide and CI-988 were synthesized in the Medicinal Chemistry Departments of Rh6ne-Poulenc Rorer Central Research (Vitry, France; Collegeville, PA, USA). The structures of the ureido-acetamides described in this article are represented in Fig. 1. CCK-8, gastrin and pentagastrin were obtained from Cambridge Research Biochemicals (UK), Research Plus (USA) and Sigma Chemical Co. (USA), respectively. [3H]Propionylated CCK-8 ([3H]pCCK-8, 75 Ci/mmol) was purchased from Amersham International (UK), [[LeulS]125I]gastrin-17-I from D u p o n t / New England Nuclear (USA). BC264 and [3H]propionylated BC264 ([3H]pBC264, 98-100 Ci/mmol) were prepared in the laboratory of Prof. B.P. Roques as previously described (Charpentier et al., 1988; Durieux et al., 1989). All other reagents were obtained from Sigma Chimie (France).


2. 2. In vitro binding studies 2.2.1. [3H]pCCK-8 receptor binding assay CCK A and CCK B receptor binding assays were performed by using guinea-pig or rat pancreas and guineapig cerebral cortex, respectively. 2.2.1.1. Membrane preparation. Membranes from male guinea-pig (Dunkin-Hartley, 250-350 g) brain tissues were prepared according to a modification of the method described by Saito et al. (1981). Cerebral cortices were homogenized in sucrose (0.25 M)/imidazole (3 mM) buffer (pH 7.4), using an Ultraturrax homogenizer, and centrifuged at 700 × g (10 min, 4°C). The supernatant was then centrifuged at 90000 × g (65 min, 4°C). The pellet was resuspended in the same buffer, and the resulting membranes (8-10 mg protein/ml) were stored at - 80°C until use. For pancreas, the homogenate was prepared in Pipes-HCl buffer (10 mM, pH 6.5) containing 1 mM EGTA, 30 mM MgCI2, 0.02% bacitracin, 0.02% soybean trypsin inhibitor and 0.3 M sucrose, and was passed through a gauze filter to remove fatty particles. The homogenate was then centrifuged at 80000 × g (10 min, 4°C), and the resulting pellet was homogenized in a large excess of ice-cold buffer and centrifuged again under the same conditions. The final pellet was resuspended in Pipes-HC1 buffer, and the membranes (5-10 mg protein/ml) were stored at -80°C. Protein concentration was determined by the BCA protein assay (Smith et al., 1985). Rat (Sprague-Dawley, male, 140-180 g) pancreatic membranes were obtained as described by Charpentier et al. (1988). 2.2.1.2. Incubation conditions. The binding assays were performed in Pipes-HC1 buffer (10 mM, pH 6.5) con-

235

taining 200 mM NaCI, 10 mM MgC12 and 1 mM EGTA for membranes from guinea-pig cerebral cortex, and in the same buffer with 30 mM MgC12 for guineapig pancreatic membranes. Before incubation, cortical and pancreatic membranes were diluted into the appropriate Pipes-HC1 buffer supplemented with either bacitracin (0.025%) or a mixture of bacitracin (0.02%) and trypsin inhibitor (0.02%). Peptide solutions were prepared in Pipes-HCl buffer supplemented with either 0.6% bovine serum albumin (cortical membranes) or 0.02% bacitracin (pancreatic membranes). Membranes (0.1-0.2 mg protein/ml) were incubated with the tritiated ligand at 25°C for 120 min (when equilibrium had been reached) in a final volume of 1.2 ml. The non-specific binding was determined in the presence of 100 nM CCK-8. Incubations were terminated by filtration under vacuum through Wathman G F / B filters, which were rinsed twice with 2.5 ml of ice-cold Pipes-HC1 buffer. The radioactivity retained on the filters was counted by liquid scintillation spectrometry in 4 ml of scintillant (Ready Solv HP, Beckman) per filter. For competition experiments, cortical and pancreatic membranes were incubated with 0.4 nM and 0.03 nM [3H]pCCK-8, respectively. The inhibition of CCK binding by ureido-acetamides was assessed in saturation isotherm experiments by using concentrations of [3H]pCCK-8 ranging from 0.01 to 5 nM. Binding assays with rat pancreatic membranes were carried out at pH 7.4 as described by Charpentier et al. (1988).

2.21.3. Data analysis. In competition experiments, binding data were used to determine the half-maximal inhibitory concentration (ICs0) of each compound with an equilibrium binding data analysis program (G.A. McPherson's EBDA program, Elsevier-Biosoft, Cambridge). The K i value was calculated from the Cheng-

Table 1 Summary of receptor types other than CCK receptor subtypes screened for ureido-acetamide binding a 1-Adrenoceptors Bradykinin Calcitonin gene-related peptide (CGRP) Dopamine D 2 Histamine H 1 Muscarinic Neuropeptide Y Neurotensin Opioid 5-HT 2 Serotonin uptake sites Haloperidol-sensitive tr sites Somatostatin Tachykinin NK x Vasoactive intestinal peptide (VIP) Ca 2+ channels Na 2+ channels

Labelled ligand

Non-specific binding

References

[3H]Prazosin (0.1 nM) ~ [3HlBradykinin (0.9 nM) [x25I]CGRP (0.01 nM) [3H]Spiperone (0.04 nM) [3H]Pyrilamine (0.7 nM) [3H]QNB (0.04 nM) [3HINPY (0.15 nM) [3H]Neurotensin (0.4 nM) [3H]Ethylketocyclazocine (0.35 nM) [3H]Ketanserin (0.4 nM) [3H]Paroxetine (0.2 nM) [3H]3-PPP (3.5 nM) [t25I]Somatostatin (0.02 nM) [3HlSubstance P (0.3 nM) [125I]VIP (20 pM) [3H]Desmethoxyverapamil (5 nM) [3H]Batrachotoxinin A20 (5 nM)

Phentolamine (3 p,M) b Bradykinin (1/xM) CGRP (100 nM) Sulpiride (1/zM) Mepyramine (10/zM) Atropine (1 /LM) NPY (3 tzM) Neurotensin (1/zM) Tifluadon (10/zM) Methysergide (10/.~M) Indalpine (10 ~M) Haloperidol (2/xM) Somatostatin (100 nM) Substance P (1 ~M) VIP (100 nM) Verapamil (10/zM) Veratridine (600/zM)

Greengrass and Bremner, 1979 Manning et al., 1986 Yoshizaki et al., 1987 Urwyler and Coward, 1987 Laduron et al., 1982 Yamamura and Snyder, 1974 Unden et al., 1984 Schotte et al., 1986 Kosterlitz et al., 1981 Leysen et al., 1982 Habert et al., 1985 Largent et al., 1986 Tran et al., 1985 Lee et al., 1983 Robberecht et al., 1978 Galizzi et al., 1984 Pauwels et al., 1986

a Concentration of radioligand used for competition, b Concentration of displacer used for determination of non-specific binding.

236

P. Bertrand et al. / European Journal of Pharmacolo,~' 262 (1994) 233-245

Prusoff equation, K i = IC50/(1 + L / K D ) . The dissociation constant ( K o) and the maximal number of binding sites (Bmax) for [3H]pCCK-8 were obtained from Scatchard analysis of saturation isotherms.

2.2.2. [ 3H]pBC264 receptor binding assay Binding experiments were performed as previously described (Durieux et al., 1992), using membranes prepared from rat cerebral cortex and whole mouse brain. Assay conditions and analysis of binding data were as reported (Durieux et al., 1992). 2.2.3. Gastrin receptor binding assay The assay for gastrin receptors was carried out as recently described by Pendley et al. (1993), using guinea-pig gastric glands prepared according to a modification of the method of Praissman et al. (1983). 2. 2. 4. Other binding assays The ureido-acetamides were tested in a series of assays for other neurotransmitter or hormone receptors carried out with frozen crude membrane preparations from the rat brain (or the guinea-pig brain, for histamine H 1, neurotensin and opiate receptors). Details on the receptor types, radioligands and substances used to define non-specific binding are summarized in Table 1. 2.3. Ex c,ic,o binding studies An ex vivo binding assay was performed in order to evaluate the brain penetration of the ureido-acetamides following intraperitoneal (i.p.) or oral (p.o.) administration. CD 1 mice (20-25 g) (Charles River, France) were treated with the compound (prepared as a suspension or a solution in 0.25% polysorbate) at doses ranging from 1 to 80 mg/kg. The animals were killed by decapitation 30 and 60 min after i.p. and p.o. administration, respectively, and their brains were quickly removed. Forebrain tissue (brain without cerebellum and brainstem) was dispersed (1:4, w/v, to avoid excessive dilution of the administered compound) in Pipes-HC1 buffer (10 mM, pH 6.5) containing 200 mM NaC1, 10 mM MgC12, 1 mM E G T A and 0.025% bacitracin with a glass-Teflon pestle homogenizer. Aliquots (100/xl) of the brain homogenate were incubated with 0.1 ml of [3H]pCCK-8 (final concentration 0.4 nM) and 0.1 ml of either CCK-8 (final concentration 10 txM) or buffer at 25°C for 30 min (at which time steady state conditions were already reached). The samples were then diluted with 4 ml of ice-cold buffer and immediately filtered under vacuum through Whatman G F / B filters, which were rinsed twice with 4 ml of buffer. The radioactivity was counted for each filter in 10 ml of scintillant. Specific binding was defined as that displaceable by 10 /xM CCK-8. Each determination was performed in

triplicate. The inhibition of [3H]pCCK-8 binding was measured for each dose of compound, and the halfmaximal inhibitory dose (IDs0) was determined from binding data by linear regression analysis of the relationship between the logarithm of the administered dose and the percentage inhibition observed. In order to evaluate the apparent forebrain levels and relative forebrain penetration of the compounds, standard curves were constructed by incubating crude brain homogenate from untreated mice with various concentrations of each inhibitor under the same conditions as above.

2.4. Electrophysiological studies Extracellular recordings from rat hippocampal neurones were obtained by following classical methods as previously described (B6hme et al., 1988). Briefly, male Sprague-Dawley rats (150-200 g) were killed by decapitation and the brains were rapidly removed. The hippocampus from one hemisphere was dissected out and mounted on the stage of a McIlwain tissue chopper. The tissue was cooled with gassed (95% 0 2 / 5 % CO2), chilled artificial cerebrospinal fluid (aCSF) of the following composition: NaCl 124 mM, KC1 5 raM, MgSO 4 2 raM, CaC12 2 mM, NaHCO 3 26 mM, Kt-I2PO 4 1.25 mM, and glucose 10 mM. Hippocampal slices, approximately 500 /xm thick, were cut transversally to the longest axis of the structure and mounted in a submersion-type recording chamber. The chamber (volume approximately 5 ml) was continuously gravity-perfused with warm (32°C) aCSF at a flow rate of 2.5-3 ml/min. A metal microelectrode was placed into the CA! area of the slices. The spontaneous spike discharge frequency of single neurones was monitored on a chart recorder by using conventional extracellular AC recording, signal detection and integration. CCK-8, made up in aCSF, was applied to the slices for periods of 4-5 min at intervals of not less than 20 min. Nontachyphylactic CCK-sensitive neurones were selected by repeated application of a maximally active concentration of CCK-8 (0.5 p~M). Antagonists were added to the perfusate for 10 rain before being co-perfused with CCK-8. Their effect was evaluated by measuring the extent to which the integrated neuronal responses were reduced with respect to the last control response before antagonist application. The ICs0 values with 95% confidence limits (95% C.L.) were estimated by linear regression analysis from the central part of the concentration-response plot.

2.5. Bioassay on isolated ileum preparations The experiments were performed according to a modification of the method of Chang and Lotti (1986). Segments of ileum (1 cm) obtained from male Hartley

P. Bertrand et aL / European Journal of Pharmacology 262 (1994) 233-245

guinea-pigs (400-500 g) were suspended in organ baths containing 20 ml of Krebs solution bubbled with a mixture of 95% 0 2 and 5% CO 2. One end of each segment was hooked to a tissue holder and the opposite end to a fine stainless steel rod, so that the segment could be connected to a force displacement

237

transducer and the contractions could be recorded on a polygraph which was fitted with appropriate amplifiers. The reactivity of each preparation was tested with 200 nM acetylcholine. After washout, and once their basal tone had been restored, the preparations were challenged with 3 nM CCK-8. To determine the inhibition of responses to CCK-8, each compound was added to the bath 15 min before the addition of CCK-8 (3 nM). 2.6. In vivo gastrin antagonist activity

120

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60

0 "~

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Log [compound] (M)

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Pentagastrin-stimulated acid secretion was evaluated in the in situ perfused rat stomach model according to a modification of the method of Ghosh and Schild (1958). Briefly, male Wistar rats (180-220 g) were anaesthetized with urethane (1.5 g/ kg i.p.) and the trachea and one or both jugular veins were cannulated. The stomach was perfused with warmed physiological saline via Perspex cannulae in the oesophagus and pyloric antrum. Samples of the luminal effluent were collected every 15 min, and acid secretion was determined by titration with NaOH. Pentagastrin was infused intravenously (i.v.) at a 30 / x g / k g / h dose (1 m l / h ) for 3 h. After an hour of pentagastrin infusion (determined in preliminary experiments to be the time required for a maximal response), the test compounds were administered as an i.v. bolus in a water/polyethylene glycol 200 (50:50) vehicle. For treated animals, the inhibition of pentagastrin activity was measured as the maximum decrease in acid secretion (usually seen at 105 min after pentagastrin infusion).

0 3. Results

0 ,...J -I

3.1. Binding to CCK B receptors in cerebral cortex t

i

t

t

-10

-9

-8

-7

The three ureido-acetamides RP 69758, RP 71483 and RP 72540 potently displaced the binding of [3H]pCCK-8 to membrane preparations of guinea-pig cerebral cortex with IC50 values of 19, 1.6 and 4.8 nM, respectively (Fig. 2A). In this respect they showed

Log [compound] (M) 200

C 160

x

120

tl_

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4o

o

20

4o

eo

B (fmol/mg)

eo

loo

Fig. 2. Inhibition of [3H]pCCK-8 binding to guinea-pig cerebral cortex membranes. (A) Competition curves were determined for CCK-8 (e), RP 69758 (©), RP 71483 ( v ) , RP 72540 ( l ) , L-365,260 ( A ) and CI-988 (13) as described in Materials and methods. Each point corresponds to the mean ( + S.E.M.) of at least three independent experiments, each performed in triplicate. (B) Hill transformation of the same data; n H values were not different from unity. (C) Scatchard analysis of specific [3H]pCCK-8 binding in the absence (e) and the presence of 12.5 nM RP 69758 ( o ) or 3.3 nM RP 72540 ( l ) . The data shown were obtained from a single representative experiment with each point measured in triplicate. The K D and Bmax values were 0.45 nM and 76 f m o l / m g protein (control), 2.0 nM and 93 f m o l / mg protein (RP 69758), and 1.4 nM and 84 f m o l / m g protein (RP 72540).

238


similar potency to that of the benzodiazepine CCKa receptor antagonist, L-365,260 (ICs0 = 21 nM), and the dipeptoid CCK B receptor antagonist, CI-988 (ICs0 = 12 nM). The Hill coefficients of the displacement curves were greater than 0.95 (Fig. 2B), which is compatible with a simple bimolecular interaction with CCK B receptor binding sites. Further support for a competitive interaction with [3H]pCCK-8 at the CCK B receptor was provided by saturation experiments performed in the presence of RP 69758 or RP 72540, in which the K D of [3H]pCCK-8 was increased significantly without modification of the Bma x (Fig. 2C). Moreover, the three ureido-acetamides also possess a similar high affinity for CCKB receptor binding sites in rat cerebral cortex and mouse brain (Table 2), since they potently displaced the binding of [3H]pBC264, a selective CCK B receptor agonist. 3.2. Binding to C C K A receptors in guinea-pig and rat pancreas

for gastrin binding sites were similar to those of L365,260 and CI-988 (Table 2). 3.4. Binding to other neurotransmitter receptors

None of the three ureido-acetamides had significant affinity (IC50 greater than 1/zM) for a variety of other neurotransmitter or hormone receptors. These included a~-adrenoceptors, dopamine (D2), histamine (H~), muscarinic, opioid, 5-HT2, bradykinin, calcitonin gene-related peptide, neurotensin, neuropeptide Y, somatostatin, tachykinin (NK 0 and vasoactive intestinal peptide receptors (Table 1). Furthermore, RP 69758, RP 71483 and RP 72540 did not interact with serotonin uptake sites and verapamil binding sites on voltage-dependent calcium channels, and did not affect [3H]batrachotoxin binding to voltage-dependent sodium channels, or [3H]3-(3-hydroxyphenyl)-N-(1-propyl)piper±dine (3-PPP) binding to or sites (Table 1). 3.5. Ex vivo binding studies in mice

RP 69758 and RP 72540 displayed a very low potency at inhibiting binding to CCK A receptors on guinea-pig and rat pancreatic membranes labelled with [3H]pCCK-8 (Table 2). Similar data were obtained for L-365,260 and CI-988, while the ureido-acetamide RP 71483 had higher affinity (guinea-pig pancreatic membranes, K i = 164 nM) for the CCK A receptor, and was thus less selective for CCK B receptors. 3.3. Gastrin receptor assay

The binding of [[Leu151125I]gastrin-17-I to guinea-pig gastric glands was also inhibited by RP 69758, RP 71483 and RP 72540, with K i values of 3.7, 1.2 and 1.2 nM respectively. The affinities of ureido-acetamides

A series of experiments was performed to characterize CCK B receptor binding sites under our conditions using crude brain homogenates from mice. In all cases, total [3H]pCCK-8 binding was about 1000 c.p.m., of which about 150 c.p.m, represented non-specific binding. Scatchard analysis demonstrated the presence of a single population of high-affinity sites with a K D of 2.4 nM and an apparent Bmax of 4.3 pmol/g tissue. The inhibition of [3H]pCCK-8 binding measured in the same conditions as in the ex vivo studies gave ICs0 values of 17.6, 81, 5.7, 21.6 and 8.7 nM for RP 69758, RP 71483, RP 72540, L-365,260 and CI-988, respectively. A significant dose-dependent inhibition of CCK

Table 2 Inhibition of CCK A, CCK B and gastrin receptor binding by ureido-acetamides and CCK n receptor antagonists, L-365,260 and CI-988, in different species RP 69758 CCK B receptor binding Guinea-pig cerebral cortex Rat cerebral cortex Mouse brain CCKA receptor binding Guinea-pig pancreas Rat pancreas Gastrin receptor binding Guinea-pig gastric glands

9.0 ( ± 1.1) 4.3 ( ± 1.3) 5.0 ( ± 0.8) 1254 ( ± 359) 4 734 ( ± 705) 3.7 ( ± 0.2)

RP 71483 0.76 ( ± 0.09) 1.2 ( ± 0.3) 5.6 ( ± 1.5) 164 ( ± 35) 28 ( ± 10) 1.2

RP 72540

L-365,260

CI-988

2.4 ( ± 0.2) 1.2 ( ± 0.1) 3.8 ( + 0.7)

11 ( + 1.1) 11 ( ± 2.0) 5.2 ( + 0.6)

5.7 ( _+0.4) 6.1 ( ± 0.5) 1.2 ( _+0.2)

2 338 ( ± 519) 2 756 ( ± 533)

883 ( + 107) 1 170 ( + 660)

1 440 ( + 149) 2 436 ( + 723)

4.8 ( _+0.6)

2.0 ( _+0.3)

1.2 ( ± 0.3)

The inhibition of [3H]pCCK-8 binding (guinea-pig cerebral cortex, guinea-pig and rat pancreas), [3H]pBC264 (rat cerebral cortex, mouse brain) and [[LeulSl125Ilgastrin-17-1 binding (guinea-pig gastric glands) was determined as described in Materials and methods. The K i values (riM) correspond to the means ( ± S.E.M.) of at least three independent experiments, each performed in triplicate, except for RP 71483 binding to gastric glands.


Table 3 Relative penetration of ureido-acetamides, L-365,260 and CI-988 in mouse forebrain, 30 min after i.p. administration and 60 min after p.o. administration

binding was observed after i.p. administration of RP 72540, with an IDs0 estimated at 9.8/zmol. k g - t (Fig. 3A). While similar potencies were observed for L365,260 (IDs0 = 12.8 ~ m o l - k g - t ) and CI-988 (IDso = 10.5 /zmol. kg-~), RP 69758 was less potent (IDso = 51.4 / z m o l . k g - t ) , and the inhibitory effect of RP 71483 was very low (IDs0 > 100/zmol • kg-t). None of the three ureido-acetamides administered orally gave more than 50% inhibition of [ 3 H ] p C C K - 8 binding at doses up to 100 /zmol. kg -~ (Fig. 3B). From calculations using standard curves and an average weight of 0.33 g for mouse forebrain, it was estimated that the apparent forebrain levels of ureido-acetamides did not exceed 0.01% of the dose 30 min after i.p. injection or 1 h after p.o. administration (Table 3). In this study, the CCK B receptor antagonist L-365,260 was identified as having the highest relative forebrain penetration, achieving ratios of about 0.03% (i.p.) and 0.017% (p.o.).

RP 69758 RP 71483 RP 72540 L-365,260 CI-988

The study was conducted on a total of 39 CCK-sensitive neurones located in the CA1 pyramidal cell-body area. As originally described by Dodd and Kelly (1981), bath application of CCK-8 caused a concentration-dependent excitation of neurones in the CA1 region of the hippocampal slice (Fig. 4A). The threshold concentration of the neuropeptide that increased the spontaneous firing rate was 3 nM, giving a maximal excitation of 5.5 + 1.7 spikes/s (mean + S.E.M., n = 3). Maximal activation was observed with micromolar concentrations of CCK-8, yielding a half-maximal effective concentration (ECso) of 0.033 /~M for this neuropeptide (95% C.L.: 0.019-0.039 /zM) (Fig. 4B). These concen-

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trations are similar to those at which a variety of other neuropeptides such as vasopressin (Miihlethaler et al., 1982), substance P (Dreifuss and Raggenbass, 1986) or corticotropin-releasing factor (Aldenhoff et al., 1983) activate hippocampal neurones in brain slices in vitro. The three ureido-acetamides, as well as the CCK B receptor antagonists L-365,260 and CI-988, concentration dependently inhibited the stimulation of neuronal

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For each compound, the ID50 values correspond to an average forebrain level which was estimated from calibration curves made in two independent experiments. These apparent brain levels were 100, 520, 35, 103 and 64 p g / m g of tissue with RP 69758, RP 71483, RP 72540, L-365,260 and CI-988, respectively. Relative CNS penetration was thus estimated by calculating the ratio between the average forebrain level (expressed in ng equivalents per forebrain) and the corresponding ID50 value (expressed in /zg of compound per mouse).

3.6. Antagonism of CCK-8-evoked neuronal firing

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240

f i r i n g i n d u c e d by C C K - 8 (Fig. 5). T h e IC50 ( a n d 9 5 % C . L . ) v a l u e s w e r e 0.18 ( 0 . 1 4 - 0 . 3 6 ) / z M f o r R P 69758, 0.34 ( 0 . 0 6 - 0 . 3 6 ) t x M f o r R P 71483, 0.018 ( 0 . 0 0 0 8 - 0 . 0 3 4 ) / x M for R P 72540, 0.93 ( 0 . 9 2 - 0 . 9 6 ) / x M f o r C I - 9 8 8 a n d 2.1 ( 1 . 8 - 2 . 3 ) / x M for L-365,260, r e s p e c t i v e l y .

3. 7. Antagonism o f CCK-8-induced contraction o f isolated ileum C o n t r a c t i o n o f t h e g u i n e a - p i g i l e u m in v i t r o p r o v i d e s an assay o f C C K A r e c e p t o r f u n c t i o n . T h e s e l e c t i v e benzodiazepine CCK A receptor antagonist, devazepide, potently inhibited CCK-8-induced contract i o n s at n a n o m o l a r c o n c e n t r a t i o n s (Fig. 6). T h e c o n t r a c t i l e activity o f C C K - 8 was n o t a n t a g o n i z e d signifi-

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Fig. 4. Excitatory effect of CCK-8 on hippocampal neurones in vitro. (A) Representative rate-meter recording of a hippocampal neurone challenged with increasing concentrations of CCK-8 during the time shown by the bars. Consecutive tracings are shown vertically. Calibration bars: Vertical = 20 spikes/s. Horizontal = 5 min. (B) Mean (+ S.E.M.) activation of spontaneous action potential discharge frequency at the maximal effect of CCK-8 for n = 3 neurones per concentration.

3.8. Antagonism of pentagastrin-stimulated acid secretion in uiuo in rats T h e i n h i b i t i o n by u r e i d o - a c e t a m i d e s o f p e n t a g a s t r i n - s t i m u l a t e d a c i d s e c r e t i o n in t h e rat s t o m a c h p e r f u s e d in situ is i l l u s t r a t e d in Fig. 7. I n t r a v e n o u s a d m i n i s t r a t i o n o f R P 69758 d o s e d e p e n d e n t l y a n t a g o n i z e d


241

4. Discussion

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pentagastrin-induced secretion; its half-maximal inhibitory effect corresponded to a dose of 1.14 /zmolkg -1. In this comparative study, RP 69758 was as potent as CI-988, while L-365,260 was found to be approximately 5- to 7-fold less potent.

c

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Dose (pmol kg -1) Fig. 7. In vivo inhibition of pentagastrin-stimulated gastric acid secretion by RP 69758 (©), L-365,260 ( • ) and CI-988 ([]). The data are expressed as the percentage of the peak secretory response to pentagastrin (observed at 45-60 min after pentagastrin infusion), and correspond to the means (_+S.E.M.) of at least three independent experiments. The EDs0 values (with 95% confidence limits) obtained by linear regression analysis were 1.14 (0.72-3.12), 5.27 (2.51-7.78) and 0.75 (0.39-7.81)/zmol .kg -1 for RP 69758, L-365,260 and CI-988 respectively.

This report describes the properties of three members of a new class of CCKB/gastrin receptor antagonists called the ureido-acetamides. We have shown that these compounds have nanomolar affinity for CCK B receptors in guinea-pig cerebral cortex, but only moderate to low affinity for CCK A receptors from guineapig pancreas. RP 69758, RP 71483 and RP 72540 are endowed with selectivity ratios in favour of CCK B receptors of 139, 216 and 974, respectively. Furthermore, the ureido-acetamides do not interact with a wide array of other neurotransmitter or hormone receptors. Their binding properties thus compare favourably with those of previously described CCK B receptor antagonists of lower or similar selectivity in our hands (Table 2), such as the benzodiazepine derivative L-365,260 (Lotti et Chang, 1989) or the peptoid CI-988 (Hughes et al., 1990). The selectivity of the ureido-acetamides for CCK B over CCK A receptors is borne out by the results obtained in the CCK A receptor bioassay in guinea-pig ileum. As expected from their binding profile, RP 69758 and RP 72540 produced no inhibition of CCKinduced contractile activity at concentrations 100 times above the half-maximal effective concentration of devazepide. Interestingly, RP 71483, at a concentration of 1 /.~M, did not significantly block CCK-8-evoked contractions in the guinea-pig ileum either, despite its affinity for peripheral CCK A binding sites. This may reflect a low penetration of this compound into the ileal tissue. Comparison of the results from binding studies with different species reveals that the ureido-acetamides show no marked differences in affinity for CCK B receptors from guinea-pig, rat and mouse brain. Chimeric constructs between clones encoding canine and human CCKB/gastrin receptor indicate that a single amino acid sequence divergence can markedly influence the C C K A / C C K B receptor selectivity profile of benzodiazepine-derived antagonists such as L-365,260 and devazepide (Beinborn et al., 1993a), but not that of the dipeptoid antagonists (Beinborn et al., 1993b). Whether this holds true for the ureido-acetamides remains to be established. The agonist or antagonist nature of the intrinsic activity of the ureido-acetamides was determined with hippocampal slices in vitro. This model provides a functional response to CCK-8 that is selectively mediated by CCK B receptors, as shown by the agonist-induced response profile (Boden and Hill, 1988; B6hme et al., 1989; Daug6 et al., 1990). Similarly, CI-988 was previously shown to antagonize the excitatory effects of CCK-8 in the ventromedial nucleus in slices from the rat hypothalamus (Hughes et al., 1990). The inhibitory activities of the tested compounds yielded the following

242


potency rank order: RP 72540 > RP 69758 > RP 71483 > CI-988 > L-365,260. ICs0 values correlate quite well with the binding affinities of the compounds in rat tissue, except for RP 71483. Again, this may reflect difficulties in tissue penetration of this derivative. The correlation shows that the ICso values in the functional test are 1-2 orders of magnitude higher than in the binding test. This would be expected from competitive antagonists given the high concentration of CCK-8 used (0.5 /.~M), although it cannot be excluded that a low affinity state of the C C K J g a s t r i n receptor mediates these functional responses. In addition to their antagonist activity at CCK B receptors, the ureido-acetamides also have high affinity for gastrin receptors in guinea-pig gastric glands. Functionally, the ureido-acetamide that we have tested (RP 69758) behaves as an antagonist with a potency comparable to that of CI-988, and appears slightly more potent than L-365,260. A similar rank order of potency among these compounds was observed at the level of central CCK B receptors in the hippocampal slices. That CCK B receptor antagonists are also gastrin receptor antagonists confirms previous reports (Hayward et al., 1991) and provides further evidence for the pharmacological identity between these two receptors. Consequently, CCK B receptor antagonists may act as gastric anti-secretagogues and may therefore have potential for preventing gastric damage in ulcerative disease. This hypothesis is supported by recent observations of Pendley et al. (1993) showing that i.v. administration of L-365,260 in the rat reduces the number of ulcerous lesions in acid-dependent gastrointestinal damage models. This protective effect was well correlated with the inhibition of basal acid secretion by L-365,260, although the doses required were higher than those necessary for blocking pentagastrin-stimulated acid secretion. The development of selective CCKB/gastrin receptor antagonists has allowed extensive investigation of the physiological role of CCK in the central nervous system. CCK B receptor antagonists such as CI-988 or L-365,260 have already been evaluated in vivo in several behavioural tests. For example, they have been shown to be active after systemic administration in certain animal models of anxiotytic drug action (Hughes et al., 1990; Rataud et al., 1991; Singh et al., 1991a). L-365,260 and CI-988 are also known to potentiate opiate analgesia (Dourish et al., 1990; Wiesenfeld-Halfin et al., 1990; Xu et al., 1993). More recently, evidence from our laboratories has demonstrated that CCKB receptor antagonists facilitate olfactory memory formation in a social recognition paradigm (Lemaire et al., 1992, 1994). The behavioural effects of CCKB/gastrin receptor antagonists may be related to their capability to penetrate into the brain. The ability of RP 69758, RP 71483

and RP 72540 to cross the blood-brain barrier has thus been examined in an ex vivo binding assay. This technique has previously demonstrated the brain penetration of various drugs including neuroleptics, opioids or, more recently, adenosine receptor antagonists (Baumgold et al., 1992; Marshall et al., 1993). We found a significant and dose-dependent reduction of cerebral CCK binding after i.p. administration of RP 69758 and RP 72540. These results are likely to reflect the ability of these ureido-acetamides to penetrate into the brain (i.e. forebrain in the present study), although a contribution of compound present in the brain vasculature to the detected inhibitory activity cannot be ruled out. L-365,260 and C1-988 were found to be much more potent at inhibiting CCK binding after oral administration than were the ureido-acetamides, probably because of differences in the absorption a n d / o r metabolism of these drugs. However, our comparative study has revealed that ureido-acetamides, benzodiazepine or peptoid CCK B receptor antagonists penetrate rather poorly into the mouse brain. Since behavioural responses in mice are observed at lower doses (about 0.02-2.00 ~ m o l . k g 1) (Singh et al., 1991b; Rataud et al., 1991; Chopin and Briley, 1993), our results may thus indicate that only very low levels of CCKB receptor occupancy are required for generating anxiolytic activity in the mouse. Alternatively, certain behavioural responses may be elicited in areas where the blood-brain barrier is more permeable such as the area postrema, where functional CCK B receptors have been identified (Branchereau et al., 1992). The use of ureido-acetamides such as RP 71483 with high affinity for CCK B receptors and a very low brain penetration ability should help to clarify the peripheral versus central origin of the behavioural effects observed after systemic administration of CCK. For instance, the CCK B receptor agonist CCK-4 induces panic-attack symptoms in humans (De Montigny, 1989; Bradwejn et al., 1990) and in monkeys (Palmour et al., 1992). Whether this anxiogenic activity of CCK-4 is mediated by peripheral or central site(s) remains unclear (Bradwejn et al., 1992). This question could be in part addressed by seeing whether this behaviour could be blocked by a low-penetrating CCK ~ receptor antagonist such as RP 71483. In conclusion, we have discovered in the ureidoacetamides a new family of potent and selective CCKB/gastrin receptor antagonists, agents which should be useful in unravelling the physiological roles of CCK and gastrin.

Acknowledgements Note that the first two authors contributed equally to this article. We are grateful to Jean-Charles Blan-


chard, Claude Cotrel, Marie-Christine Dubroeucq and Bernard P. Roques for their contributions to the CCK-antagonist research programme. We thank Bruce F. Molino and his colleagues for the synthesis of CI-988, Jacques Lavayre and his colleagues for performing the non-CCK binding site screening, and John C.R. Randie for helpful comments and critical reading of the manuscript.

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