The Effect of CCKB/Gastrin Antagonists on Stimulated Gastric Acid Secretion in the Anaesthetized Rat 1N.J

Br. J. Pharmacol. (1991), 104, 973-977 q:) Macmillan Press Ltd, 1991

The effect of CCKB/gastrin antagonists on stimulated gastric acid secretion in the anaesthetized rat 1N.J. Hayward, M. Harding, S.A.C. Lloyd, A.T. McKnight, J. Hughes & G.N. Woodruff

Parke-Davis Neuroscience Research Centre, Addenbrookes Hospital Site, Hills Road, Cambridge CB2 2QB

1 The urethane-anaesthetized, vagotomised rat preparation was used to investigate the effects of the histamine H2-antagonist ranitidine, the proton pump inhibitor omeprazole and the CCKB/gastrin antagonists CI-988, PD 136450 and L-365,260 on pentagastrin-, histamine- and bethanechol-induced gastric acid secretion. 2 The novel CCKB/gastrin antagonists CI-988 and PD 136450, and L-365,260 dose-dependently inhibited pentagastrin-induced secretion. The ED50 value for PD 136450 was 0.05 pmol kg ',the same following intravenous or subcutaneous administration. 3 CI-988 and PD 136450 administered subcutaneously at dose levels highly effective for antagonism of pentagastrin responses had no effect on basal acid secretion. 4 Ranitidine inhibited pentagastrin-, bethanechol-, and histamine-induced acid secretion, whereas the CCKB/gastrin antagonists inhibited only the secretory response to pentagastrin. 5 The selective CCKA antagonist, devazepide, was inactive at up to 300 pmolkg- i.p. against the three stimulants of acid secretion. 6 CI-988 and PD 136450 will be useful research tools with which to investigate the role of CCKB/gastrin receptors in gastric acid secretion and the trophic activities of gastrin and cholecystokinin (CCK) on the gastrointestinal tract. Keywords: CCKA receptor; CCKB receptor; gastrin receptor; vagotomised; gastric acid secretion; cholecystokinin

Introduction for the CCKA class of receptor found predominantly, though not exclusively in peripheral organs, and L-365,260 (Lotti & The primary endogenous mediators of gastric acid secretion Chang, 1989; Bock et al., 1989) which is relatively selective for are acetylcholine, gastrin and histamine. Each of these secre- the CCKB ('brain') receptor and the gastrin receptor, no tagogues acts through its own receptor to trigger unique series antagonist is known that discriminates between the CCKB of biochemical events within the parietal cell but the final step and gastrin receptor. More recently, non-peptide, non- involves activation of the enzyme, H', K+-ATPase, located in benzodiazepine compounds, CI-988 (PD 134308) and PD the membrane of intracellular tubulovesicles. As a result of 136450 have been described that have affinity at the this activation, potassium ions are reabsorbed from the lumen CCKB/gastrin receptor comparable to that seen with L-365, into the cytosol in exchange for the hydrogen ions that are 260, but are more selective for the CCKB receptor over the excreted into the lumen of the canaliculi. CCKA receptor (Hughes et al., 1990; Horwell et al., 1991). The interrelationship of the three physiological gastric While the antisecretory effects of L-365,260 have been secretagogues in the stimulation of gastric acid secretion described in several animal models of acid secretion (Lotti & remains unresolved. The physiological problem is in the Chang, 1989), the activity of the more selective compounds relationship between gastrin and histamine, both being power- has not yet been reported. The importance of such studies ful stimulants of gastric acid secretion and both synthesized in with the CCKB/gastrin antagonists is not only for an exami- the mucous membrane of the stomach. As early as 1938, nation of their potential utility as anti-secretory agents, but MacIntosh had proposed that stimulation of the vagus also as possible adjuncts to therapies with currently available resulted in the release of histamine, and Code (1965), and later agents that carry the attendant risk of hypergastrineamia that Kahlson & Rosengren (1972) extended that idea to gastrin, is a consequence of the achlorhydria produced by long-term making histamine the final common chemostimulant. antisecretory therapy. However, many believe gastrin to be a direct hormone of In the present study, an in situ perfused stomach prep- secretion in its own right; thus, the question of the function aration of the rat has been used for the quantitative investiga- and interrelationship of the three secretagogues in the tion of the effect of the selective CCK/gastrin receptor stomach remains unsettled. The pharmacological problem at antagonists devazepide, L-365,260, CI-988 and PD 136450 on the basis of this has been the lack of antagonists for the gastric acid secretion evoked by pentagastrin, histamine or gastrin receptor that have sufficient affinity and selectivity. bethanechol. A preliminary account of these findings has been Proglumide, a non-selective CCK receptor antagonist, blocks presented to the British Pharmacological Society (Hayward et gastrin receptors (Magous & Bali, 1983), and has been used in al., 1991). the treatment of peptic ulcer disease in Europe and Japan (Rovati, 1976; Weiss, 1979). However, neither the specificity Methods nor the affinity of proglumide has been sufficient to permit its use in vivo to define the role of gastrin in the regulation of Anaesthetized rat perfused stomach preparation gastric acid secretion. In recent years other CCK receptor antagonists from several chemical classes have been devel- The perfused stomach preparation was that described by oped, and although there are now available such benzodiaze- Parsons (1969) as a modification of the Ghosh & Schild (1958) pines as devazepide (MK-329, L-364,718 Chang & Lotti, method. Briefly, male Wistar rats (180-220g) were anaes- 1986; Evans et al., 1986) with high affinity and high selectivity thetized with urethane (1.5gkg-1, i.p.) and the trachea and one or both jugular veins were cannulated. The stomach was 'Author for correspondence. perfused with 5.4% (w/v) glucose in water (370C) at a rate of 974 N.J. HAYWARD et at.

3 ml min-1 via perspex cannulae in the oesophagus and Pentagastrin, histamine dihydrochloride and carbamyl-/i- pyloric antrum; the perfusate issued through a perspex funnel methylcholine chloride (bethanechol) were stored as frozen ali- in the non-secretory lumen and passed over a conventional quots of 0.lmgml-', 10mgmlP', and lmgmlP' in 0.9% pH electrode recording continuously onto a flatbed chart (w/v) NaCl respectively and diluted to the required concentra- recorder. The cannula in the oesophagus was placed below the tion with 0.9% w/v NaCl. CI-988, PD 136450, ranitidine and diaphragm and the oesophagus was cut. atropine were dissolved in 0.9% w/v NaCl with brief sonica- In preliminary experiments to determine doses of the tion. L-365,260 and devazepide were made up fresh in 10% separate agonists that produce comparable, submaximal levels (v/v) ethanol in 90% carboxymethyl cellulose/Tween 80 of acid output, the dose-effect relationship with pentagastrin, (aqueous suspension, 1% w/v with respect to carboxymethyl histamine or bethanechol was determined. Agonists were cellulose, 0.1% w/v with respect to Tween 80). Omeprazole infused intravenously at a constant rate (1ml h-1) and the was made up fresh in distilled water with brief sonication. dose increased cumulatively when the effect of the preceding CI-988, PD 136450, ranitidine, atropine and omeprazole were dose had reached a peak. The response of a given dose was administered intravenously or subcutaneously in a dose measured as hydrogen ion concentration at the peak of the volume of 1 ml kg- ', whilst L-365,260 and devazepide due to response minus basal hydrogen ion concentration. Hydrogen their poor solubility were administered via the intraperitoneal ion concentration at any time of interest was measured as the route in a dose volume of 4 ml kg-1. negative antilogarithm of the pH at that time. For the work with antagonists only one dose of blocker was tested in each preparation. Once basal acid secretion was Results stable submaximal doses of either pentagastrin (1 pugkg 1 min- 1), histamine (30opgkg- ' min- 1) or Stimulation ofgastric acid secretion bethanechol (6 pgkg- min-1) were administered intravenously via the right jugular vein. The stimulation of acid In preliminary experiments the dose-response relationship was secretion produced by the secretagogue was allowed to reach established for the secretagogues pehtagastrin, histamine and a plateau for about 30min before the dose of antagonist was bethanechol. All three secretagogues caused a dose-dependent administered by intravenous injection via the left jugular vein, increase in gastric acid secretion. Doses of histamine or subcutaneously into the right flank. Acid secretion contin- (30pg kg-1 min 1, i.v.) and bethanechol (6,ug kg- min 1, i.v.) ued to be monitored for as long as possible after drug admin- that were on the linear portion of their log dose-response istration. Results are expressed as percentage inhibition of curves, and that produce increases in acid output comparable secretagogue-induced gastric acid secretion: to that obtained with pentagastrin (1,ug kg-1 min , i.v.) were used to determine the specificity of CI-988 and PD 136450 as / reversal-basal %i1nhibition=simlae. bsa!xX 100- 100 antagonists of pentagastrin in vivo (Figure 1). Each secre- ~stimulated-basal tagogue stimulated a maintained plateau of acid output; pentagastrin and histamine 1 h following the start of infusion gave where: acid output levels of 1113.1 + 413.5 and inhibition = hydrogen ion concentration ([He]) at the 1415.4 + 159.0 pmol 1-' H +, and at 3 h gave acid output levels point of maximal inhibition of stimulated acid of 866.4 + 422.1 and 1254.2 + 184.2Pmol I1 H+ respectively. secretion by the compound under test In the case of bethanechol, stable levels of acid output were stimulated = [H+] at the stable level of stimulated acid obtained only after 2 h following the start of infusion, the level secretion being 1572 + 486.6 pmol I' H+; at 4 h the acid output was basal = unstimulated [He] 1767.9 + 290.6pmol 1 H+. In those experiments where the antagonists had little or no Effects of CCK/gastrin antagonists, ranitidine, effect on stimulated acid secretion the preparation was vali- omeprazole and atropine on stimulated gastric acid dated at the end of the day by testing for complete block of stimulated acid secretion by administering the H2-antagonist, secretion ranitidine (1 pmol kg- s.c./i.v.). No preparations were rejected In the anaesthetized rat preparation, CI-988 powerfully inhib- on these grounds. ited pentagastrin-induced gastric acid secretion following ED50 values with 95% confidence limits were calculated for intravenous administration (Figure 2). CI-988, PD 136450, the inhibition of gastric acid secretion. At least four rats were used H2-antagonist ranitidine, the proton pump inhibitor omepra- for each dose group. zole, administered subcutaneously, and the CCKB antagonist L-365,260 following intraperitoneal administration, were all Compounds able to antagonize dose-dependently pentagastrin-induced The following compounds were used: atropine sulphate a (Sigma Chemical Co. Ltd.), carbamyl-fi-methylcholine chlo- 24u b ride (Sigma Chemical Co. Ltd.), histamine dihydrochloride 2400 2400C (Sigma Chemical Co. Ltd.), pentagastrin (Bachem), ranitidine 2000 2000 2000 hydrochloride (Sigma Chemical Co. Ltd), omeprazole (Astra). CI-988 (PD 134308), [R - (R*, R*)]-4-[[2-[[3-(1H-Indol-3-yl)- 1600- 1600- 1600 2-methyl- l-oxo-2-[[(tricyclo[3.3.1.13'7]dec-2-yloxy)carbonyl]- E amino]propyl]amino] - 1 - phenylethyl]amino] - 4- oxobutanoic r 1200 1200. 1200 acid, and PD 136450, [R - (R*, R*)]-4-[[2-[[3-(lH-Indol-3- 0 yl)-2-methyl- I -oxo-2- [[(tricyclo[3.3.1.13 7]dec-2-oxy)carbon- IV 800 800 800 yl]amino]propyl]amino] - 1 - phenylethyl]amino] - 4 - oxo - 2 - 0 butenoic acid, were synthesized in the Department of Medici- w400- 400. 400 nal Chemistry, Parke-Davis Neuroscience Research Centre, 0 0. L-LL-- 0 Cambridge, UK. Devazepide (MK-329, L-364,718 Evans et al., 0.3 1 3 10 30 10 30100 300 1 3 6 10 1986) and L-365,260 (3R( + )-N42,3-dihydro-l-methyl-2-oxo-5- Dose (,ug kg-1 min-, iv.) phenyl- I H- 1,4-benzodiazepin-3-yl)-N'-(3-methyl-phenyl)urea; Figure 1 Histograms illustrating the dose-related stimulant effect of Bock et al., 1989) were synthesized in the Department of pentagastrin (a), histamine (b), and bethanechol (c) on gastric acid Medicinal Chemistry, Parke-Davis Ann Arbor, U.S.A. All secretion. (Each column is the mean of 4-6 observations. Vertical bars compound doses are expressed in terms of free base. represent s.e.mean). CCKB ANTAGONISTS AND GASTRIC ACID SECRETION 975

As shown in Table 1, at doses 100 times their respective Pentagastrin CI-988 ED50 value against pentagastrin, the CCKB/gastrin antago- (1 ptg kg-' min-', i.v.) (0.5 pRmol kg-', i.v.) nists L-365,260, CI-988 and PD 136450 were without effect on gastric acid secretion by either histamine or bethanechol, thus highlighting the selectivity of the former compounds for the CCKB/gastrin receptor. As expected, the proton pump inhibi- 0 + 600- * I 500- tor omeprazole, which inhibits the enzyme H', K+-ATPase o 400- involved in the final step in the secretion of hydrogen ions in a, the stomach, and interestingly, the H2-antagonist ° 300- ranitidine, .2E dose-dependently inhibited gastric acid secretion produced by < 200- either histamine or bethanechol with values for ED50 comparable to those against pentagastrin-induced acid secretion. m The selective CCKA antagonist, devazepide, was found to 10 min be inactive at up to 300 umolkg-1, i.p. against the three Figure 2 The effect of CI-988 (0.5jumol kg-', i.v.) on pe:ntagastrin stimulants of acid secretion (Table 1). (1 pgkg-' min- , i.v.)-stimulated gastric acid secretion in the anaesthetized rat. Discussion secretion (Figure 3). On a molar basis PD 136450 was found Recently, non-peptide agents of the benzodiazepine structure to be 3.8, 9.4, and 32.2 times more active than ranitidine, with high affinity for peripheral CCKA receptors and only low omeprazole and L-365,260 respectively (Table 1). affinity for gastrin and CCKB receptors have been described PD 136450 was as active at inhibiting pentagastrin- (Chang et al., 1985; Chang & Lotti, 1986). The related com- stimulated acid secretion following intravenous administration pound with the reverse profile of affinity, L-365,260, was sub- (ED50 0.052 (0.01-0.26) umol kg- 1), as after subcutaneous sequently described (Bock et al., 1989; Lotti & Chang, 1989). administration; thus indicating good absorption of the com- The present paper describes the effects of the new, non- pound following subcutaneous administration. Ranitidine on benzodiazepine compounds, CI-988 and PD 136450, that the other hand was approximately 3 times more potent when exhibit high affinity for the stomach gastrin and brain CCKB administered intravenously compared to subcutaneously with receptors, relative to the peripheral, pancreatic CCKA receptor an ED50 value of 0.067 (0.02-0.20) ,umol kg- '. (Hughes et al., 1990). CI-988 and PD 136450 are antagonists After subcutaneous administration, at dose levels highly at CCKB receptors in the brain, as highlighted by the observa- effective for antagonism of the pentagastrin response (1- tion that both compounds inhibit responses to agonists at this 100pmol kg-1), neither CI-988 nor PD 136450 had any sig- receptor in the ventromedial nucleus of the hypothalamus of nificant effect upon basal acid secretion in this model. All the rat (Hughes et al., 1990; Boden & Pinnock, personal other antagonists tested had no effect upon basal acid secre- communication). tion. CI-988 and PD 136450 inhibited pentagastrin-induced acid secretion, confirming the view that they are antagonists at the 'gastrin receptor'. The potency of CI-988 was comparable to V that of the H2-antagonist ranitidine, while PD 136450 was 4 n 10 times more potent, with an identical ED50 value when admin- CD istered subcutaneously or intravenously. The intraperitoneal cC: administration of the CCKB/gastrin antagonist L-365,260 was *' 80 +- active in producing a dose-dependent block of pentagastrin- m . T induced acid secretion in the anaesthetized rat, confirming m Q) 6060. observations of Lotti & Chang (1989) in the anaesthetized .2 v mouse and conscious rat following oral or intraduodenal 40 administration. The effect of this CCKB/gastrin antagonist o Q was relatively weak compared to PD 136450, whilst the selec- o 20- y tive CCKA antagonist devazepide was inactive at up to .0 300pumolkg-1 against the three stimulants. This latter obser- S O vation is in keeping with the low affinity of devazepide for ).010 0.100 1.000 10.000 both the gastrin, and brain CCKB receptors (Chang & Lotti, Dose (,umol kg-') 1986; Evans et al., 1986). However, our results with devaze- Figure 3 The effect of CI-988 s.c. (O), PD 136450 s.c. (-), L-365,260 pide are in conflict with those in a similar study by Hirst et al. i.p. (A), ranitidine s.c. (A) and omeprazole s.c. (V) on pentagastrin- (1991). These authors observed that L-365,260 and devazepide induced acid secretion in the anaesthetized rat. (Each point is the are both active at a dose of 10,umol kg ', administered intra- mean of 4-5 observations. Vertical lines represent s.e.mean). venously, in blocking acid secretion by either pentagastrin or

Table 1 The effect of various compounds on pentagastrin, bethanechol, and histamine stimulated gastric acid secretion ED50 (95% confidence limits) pmol kg-' Compound Pentagastrin Histamine Bethanechol CI-988 0.25 (0.06-1.05) >25 >25 PD 136450 0.05 (0.01-0.26) >5 >5 L-365,260 1.61 (0.61-4.21) >160 >160 Devazepide >300 >300 >300 Ranitidine 0.19 (0.11-0.36) 0.25 (0.03-2.30) 0.20 (0.08-0.49) Omeprazole 0.47 (0.17-1.33) 0.23 (0.03-1.96) 0.19 (0.09-0.40) Atropine >0.2 >0.2 0.0021 (0.0003-0.0118) All compounds were administered subcutaneously, except devazepide and L-365,260 which were administered via the intraperitoneal route. (Each value represents the mean with 95% confidence limits in parentheses). 976 N.J. HAYWARD et al. histamine. The model used in the latter study is almost identi- well as H2-antagonists have been shown to possess specific cal, to that in the present study, but in their case with the actions on the rabbit isolated parietal cell against cholinocep- vagus nerve trunks kept intact. It may be that this fact alone tor agonists and histamine, respectively (Soll & Berglindh, is sufficient to reveal a role for the CCKA receptor in affecting 1987). It has also been shown that ranitidine does not inhibit acid secretion induced by either pentagastrin or histamine. It the action of bethanechol at muscarinic receptors in isolated is curious however, that in their hands, even with intravenous guinea-pig ileum even when tested at concentrations greatly in administration, L-365,260 is comparatively weak as a blocker excess of those required to block H2-receptors (Daly et al., of the response to pentagastrin (cf. ED50 value after i.p. 1981). These findings are thus at odds with those described in administration in the present study, was only 1.6,umol kg- 1). this study and with other in vivo observations (Brimblecombe Our studies confirm the findings by Bunce & Parsons et al., 1975; Parsons, 1975) that H2-antagonists block cholin- (1978), in the conscious vagotomized rat, that H2-antagonists ergically stimulated secretion as well as that by histamine inhibit gastric acid secretion in response to pentagastrin, his- itself. Also, gastrin and acetylcholine have been shown to tamine and bethanechol. The selectivity of CI-988 and PD cause release of histamine from rabbit gastric glands 136450 was highlighted by the observation that they inhibited (Bergqvist et al., 1980). Thus, the powerful inhibition by raniti- the response to pentagastrin, without altering the responses to dine on bethanechol-induced acid secretion may imply that histamine and bethanechol, in the vagotomised, anaesthetized part of the stimulant effect of bethanechol on acid secretion is rat at least. due to the release of endogenous histamine; thus suggesting At present there are two hypotheses for the regulation of the pivitol role of histamine in the control of acid secretion. gastric acid secretion; the 'permissive' and 'transmissive' theo- The apparent contradiction may reflect the influences of the ries. The permissive theory states that parietal cells express endogenous stimulants and their interactions (Soll & Ber- acetylcholine, gastrin and histamine receptors on their surface glindh, 1987) as observed in the intact animal. but that gastrin acting at its receptor is only fully effective if Species differences are apparent among the different models histamine has already occupied its own receptor, that is, that have been used to explore the regulation of acid secre- gastrin acts by 'permission' of histamine (Grossman & Kontu- tion. In the dog, the cholinergic pathway seems to be of great rek, 1974; Soll, 1977). Our studies in the anaesthetized rat, importance since atropine is able to block not only the power- confirm this view since ranitidine inhibited equally the acid ful effect of cholinomimetics to stimulate acid secretion, but secretion by either pentagastrin or histamine, whereas CI-988 also the response to histamine and pentagastrin. In the rabbit and PD 136450 selectively inhibited the responses to penta- on the other hand, as in the vagotomized rat, highlighted in gastrin alone. However, the corollary to this hypothesis is that this study, the potent stimulant effects on acid secretion of pentagastrin will be inactive in the absence of histamine secre- pentagastrin and histamine are not inhibited by atropine tion. An interesting finding then, is that pentagastrin is fully (Albinus & Sewing, 1969; Hedges & Parsons, 1977; Muluihill active on the in vitro mouse-stomach preparation under stan- et al., 1985). dardized conditions where there is no pharmacological evi- In conclusion, the present studies demonstrate that the dence that histamine secretion occurs (Black & Shankley, selective CCKB/gastrin antagonists CI-988 and PD 136450 1985). However, the ability of the supplementation by exoge- potently inhibit pentagastrin-stimulated gastric acid secretion nous histamine to restore partially the response to pentagas- without having any efficacy by themselves on acid secretion. trin in mast cell-deficient mice (Stechschulte et al., 1990) sug- The inability of both compounds to antagonize either gests that gastrin requires the presence of histamine to act histamine- or bethanechol-induced acid secretion, compared effectively as a secretagogue in the mouse. The ability of rani- to the H2-antagonist ranitidine, and the proton pump inhibi- tidine to block pentagastrin-induced acid secretion as shown tor omeprazole, supports the view that, for the anaesthetized, in this study also argues for the effect of gastrin on histamine- vagotomized rat at least, pentagastrin stimulates acid secre- containing cells and against a significant direct effect of tion, indirectly via histamine. Additionally however, the data gastrin on parietal cells; the transmissive theory. This theory do not disprove a permissive role for otherwise subthreshold states that blood-borne gastrin and neurally-released acetyl- levels of histamine. Although this model is useful for demon- choline acting on histamine-secreting cells located adjacent to strating antisecretory activity, a conscious animal model the parietal cells release histamine, which then acts on parietal would be more useful in elucidating the physiological control cells; thus histamine 'transmits' the actions of gastrin and the of acid secretion, since maximal acid output in the anaes- vagus (MacIntosh, 1938; Code, 1965; Kahlson & Rosengren, thetized rat is considerably lower than in conscious rats. 1972). Earlier studies have demonstrated the ability of gastrin CI-988 and PD 136450 have provided us with research or pentagastrin to initiate histamine release from gastric tools to investigate the role of CCKB receptors in the control mucosa (Ekblad, 1985) and mast cells (Tharp et al., 1984), thus of anxiety (Hughes et al., 1990); in gastric acid secretion and confirming the view that part of the gastric acid secretion the trophic activities of gastrin and CCK on the gastro- effect of gastrin is through histamine. In the rat, histamine is intestinal tract, with their potential application in the preven- stored in endocrine-like cells (Hakanson et al., 1976) and tion or treatment of gastrointestinal carcinoids. Moreover it gastrin stimulates the formation of histamine by inducing the may be possible to use these agents to block the rebound activity of histidine decarboxylase (Kahlson & Rosengren, effect whereby gastrin levels rise when patients are on long 1968; Hakanson & Leidberg, 1970) and diminishes gastric term anti-secretory therapy and there are hazards in produc- mucosal histamine (Haverback et al., 1964; Bunce & Parsons, ing achlorhydria, such as gastrointestinal carcinoids. 1977; Main & Pearce, 1982). Just as CCKB/gastrin antagonists were seen to be selective The authors are grateful to Dr D.C. Horwell and colleagues at Parke- for the one secretagogue, pentagastrin, atropine was selective Davis Neuroscience Research Centre, Cambridge for the synthesis of in blocking acid secretion induced by the muscarinic agonist CI-988 and PD 136450, and to the Chemistry Department of Parke- bethanechol, confirming earlier studies (Albinus & Sewing, Davis, Ann Arbor, U.S.A. for the synthesis of devazepide and L-365, 1969; Hedges & Parsons, 1977). Anticholinoceptor agents as 260.

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