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Prokinetic benzamides stimulate peristaltic activity in the isolated guinea pig ileum by activation of 5-HT4 receptors
EJP 52167
Karl-Heinz Buchheit and Thomas Buhl Pwchiral
Rcwarch.
Sarrdo: Pharrrra Ltd.. CH--100,’ Easel. Swirxrland
Received 4 February IYYI. revised MS received
Substituted
in various
bcnzamides
contribute
to their
substituted
benzamides
which
such as meloclopramidc.
species. As they are antagonists at 5HT, gastrointestinal
prcssurc-induced
prokinetic
on gut motility pcrislaltic
cisapridc,
zacopride.
accepted II) September IWI
renzapride
or BRL 206617. stimulate
intestinal
motility
and agonists a( S-HT, receptors and as both mechanisms could potentialI!
effect.
the underlying
was investigated
contractions
1 July IW.
mechanism
in the isolated guinea
are measured.
All
benzamides
is unclear.
To clarify
this. the effccl
pig ileum using the Trendclcnburg stimulated
the peristaltic
of some
technique.
in
reflex with the rank
order of potency: renzapridc > cisapridc > BRL 20647 > ( A_ )-zacopride > metoclopramide. ICS 205930. granisctron and 2methyl-5-HT did not change the peristaltic response. 5HT and 5methoxyiryptamine potently mimicked the effect of the benzamides. The effect of S-HT was not blocked by ICS 205-930 (IO-’ ML These results indicate :hat the Trendclenburg preparation
is suitable
for the investigation
results suggest that the intestinal 5-HT,
of intestinal
effect of bcnzamidcs
prokinetic
effects
results from activation
of the substituted of 5-HT,
receptors
benzamides.
Furthermore.
rather than from blockade
the of
receptors. Benzamides (substituted): Small intestinal motility: 5HT 3 receptors: 5-HT4 receptors: Trendelenburg
1. Introduction Substituted benzamides derived from metoclopramide, such as cisapride, renzapride. zacopride. BRL 20627. stimulate gastric and small intestinal motility in man and in various animals (for review see King and Sanger, 1988) and are being introduced into the clinic as so-called prokinetics for the treatment of gastrointestinal motility disorders. The mechanism of the prokinetic action of these compounds is unclear. Recently, effects at 5hydroxytryptamine (5HT) receptors have been implicated in the prokinetic actions of metoclopramide and its analogues. After the discovery that metoclopramide blocks 5-HT, receptors (Fozard and Mobarok Ali, 19781, other prokinetic analogues of metoclopramide such as renzapride, zacopride and BRL 20627 have also been described as 5-HT, receptor antagonists (Sanger. 1987: Smith et al.. 1988; Dunbar et al.. 1986). In addition, the prokinetic benza-
Correspondence to: K.-H. Buchheit. Preclinical Research (3861543). Sand07 Pharma Ltd.. CH-4002 Bawl. Switzerland. * Dedicated to Prof. Ernst Mutschler (Frankfurt/Main) on the occasion of his 60th birthday. Part of these results was presented at the 3151 Spring Meeting of the German Society ior Pharmacology and Toxicology. Mainz. March 13-16. 1990.
preparation
mides have recently been found to be agonists at a 5-HT receptor [hat has tentatively been named 5-HT, and which hss been identified in the brain (Dumuis et al., 1989) and in the enteric nervous system (Craig and Clarke, 1990). Besides having the substitute3 benzamides as agonists. this receptor is also characterized by a high sensitivity to both 5-HT and 5-methoxytryptamine (5-MeOT). In addition, the site shows a low affinity (pK, about 6.2) for the potent 5-HT, receptor antagonist. ICS 205-930 (Dumuis et al., 1988). Regarding the gut nervous system. activation of 5MT, receptors causes contraction of the longitudinal muscle layer of the guinea pig ileum (Eglen et al., 1990). Such an effect is potentially prokinetic as an increase in motility requires increased muscle activity. On the other hand, blockade of 5-HT, receptors could also be considered as a potential prokinetic effect. as 5-HT, receptor antagonists such as ICS 205-930. have a stimulatory effect on gastric motility in man (Akitermans et al., 1988) and in experimental animals (Buchheit et al. 1985a, Gamse, 1989). This raises the question of whether an interaction with 5-HT receptors is involved in the prokinetic action of the substituted benzamides and, in particular. which of the two mechanisms, 5-HT, receptor antagonism or 5-HT, receptor agonism, accounts for the action. TO answer this question, the effects of some substituted
to the serosa! side of the preparation. Previous expcrirems had confirmed that identical conccntration-response curves were obtained on cumu!:ltivc and non-cumulative administration of the test compounds. The maximal concentrations added were either !O~-i M or concentrations which consistently produced tonic contractions of the preparation. If such tonic contractions occurred, they were not considered for the quantitative assessment of drug effects. The tissue was left in contact with each drug concentration for IO min bcforc responses were quantified. Each preparation was used for the investigation of one drug only. Concentration-response curv2s were made by nlotting the mean area under the contraction cu7r2s from -I to I I experiments, expressed as a percentage of the control preparations. The potency of the drugs was characterized by the concentration yielding a half-maxima! effect. expressed as pD, values. These values were determined graphically from the mean concentrationresponse curves. Statistically significant differences from the control were determined for individual points of the concentration-response curves, using the nonparametric one-sample Wilcoxon signed rank test. min intervals
Femafe guinea pigs. 200-&I0 g, kept in colonies under standard conditions and diet were stunned and bled. The irtst 10 cm of the ileum were discarded. Segments of the remaining ileum, 4-5 cm long, were used. The lumen was carefully flushed to remove a!! remnants of food. Extensive distension of the preparation was a\oidcd during this process. The tissue was suspended in an organ bath under a l-g load by connLxtin_r the proximal (oral) end of the preparation to a iever transducer. The distal (anal) end was pulled over a glas t&e and connected to a pressure bttle. filled with Tyrodc solution. The preparation was bathed in Tyrode solution (NaCI 136-S: CaCI, 1.8: KC! 2.7; NaH -PO, 0.Q: MgC!. 1.05: NaHCO, 11.9: glucose 5.6 mM)at pH 7.1 maintained at 37 ’ C and bubbled with 5% CO, in oxygen.
2.4. Correlatiorr analysis The correlation between the lenburg experiment and those neurons was calculated based using a commercial software bridge, MA).
data from the Trendefrom mouse colliculi on a least squares fit, package (BBN, Cam-
2.5. Drugs
Peristaltic contractions were elicited for 30 s by increasing intralumina! pressure from 0 to I cm HZ0 at 6-mii; intervals. Longitudinal muscle responses were recorded using an isotonic force displacement transducer (Hugo Sachs. March-Hugstetten, Germany). A pressure transducer (Statham 23V, O-200 mm Hg) was used to measure changes of intralumina! pressure as an indication of circular muscle activity. Longitudinal and circular muscle activity were quantified by determining the area under the curve of peristaltic contractions, by means of a computerized integration procedure (developed at Sandoz Pinarma Ltd.) on an IBM persona! computer AT 03. Only those preparations were used which gave at least three consistent pressure-induced responses before administration of any test compound and where no signs of spastic, tonic contractions were observed.
5-HT IFluka, Buchs, Switzerland), metoclopramide (RBI, Natick, USA) and 5methoxyttyptamine (5 MeOT, Sigma, Munich, Germany) were purchased. ICS 205-930 ([3cY-tropanyl-I-lH-indole-3-carboxylic acid ester), granisetron, 2-methyl-5-HT (2-Me-5-HT), ( f Irenzapride, and ( f I-zacopride were synthesized at Sandoz Pharma Ltd. (Base!). Cisapride and BRL 20627 [(2cr,6&9a(u)-( f I-4-amino-5-chloro-2-methoxy-N-(octahydro-6-methyl-2H-quinolizin-2-y!)benzamide] were kind gifts of Janssen (Beerse, Belgium) and Beecham (Harlow, UK), respectively. A!! drugs were dissolved in saline, wit11 (bases) or without (salts) the addition of tartaric acid.
3. Results
2.3. Qwntitatire assessment of effects
3. I. Characterization of the peristaltic reflex
Cumulative concentration-response curves were recorded under equilibrium conditions by adding increasing concentrations of the test compounds at 12-
Peristaltic activity in the isolated guinea pig ileum was initiated by increasing the intralumina! pressure by 1 cm Hz0 over that in the organ bath by elevating the
3
TABLE
x or
I
S~imulatoty effecl on longitudinal muscle activity in the Trendelenhurg preparation. compared with the potency to stimulate the formation of CAMP in isolated mouse colliculi neurons by activation of 5.HT, receptors. Compound
Trendelenhurg experiment J PD,
S-HT S-MeOT 2-Me-5 -HT ICS 205-930 Granisetron Metoclopramide Cisapride Renzapride Zacopride BRL 20627
P
8.0 7.7 <5 <7 <7 5.3 7.5 8.6 5.7 6.6
E,, 280 (8) 220 (6) - (9) - (8) - (61 340 (5) 220 (5) 170 (4) 17OflI~ 200 (4)
S%‘“’ I 250 225
Mouse colliculi neurons
1 200.
PD, ’
175
7.0 7.0
150
5.3 7.1 6.9 6.0 5.5
’ pD, = - ,,,log of the concentration which caused a half-maximal effect; E,,, indicates the maximal increase in phasic contractility. expressed as percentage of the control: the numbers in parentheses indicate the number of experiments: h data taken from Dumuis et al. (19891 and Dumuis et al. (1988).
level of the buffer in the pressure bottle which was connected to the lumen of the preparation. This caused radial distension of the muscle and elicited the peristaltic reflex consisting of coordinated phasic contractions and relaxations of the longitudinal and the circular muscle layer. Normally, tonic spasms were not observed under the conditions used. Although there were differences in the amplitude and frequency of the phasic contractions between different preparations under control conditions, the responses expressed as area under the contraction time curves (which integrates contraction amplitude and frequency) were reproducible and remained constant for several hours, without signs of fatigue. 3.2. Ejfects of benzamides on the peristaltic reflex Upon administration to the serosal side of the preparation, metoclopramide, BRL 20627, renzapride, zacopride and cisapride augmented with different potencies the contractile activity elicited in the longitudinal muscle layer by an increase of intraluminal pressure (table 1). All five compounds caused qualitatively identical responses. None elicited isolated tonic or phasic contractions (i.e. contractions which were not induced by increases in intraluminal pressure). Mptoclopramide and zacopride were only weakly active (pD, values: 5.3 and 5.7, respectively). BRL 20627 was slightly more potent (pD, = 6.6). Renzapride and cisapride augmented contractions, with pD2 values of 8.6 and 7.5, respectively. This resulted in the following rank order of potency: renzapride > cisapride > BRL 20627 > (& )-zacopride > metoclopramide (fig. 1).
125
- log II Agonist Fig. 1. Comparison of the stimulatory eff&ts of the benzamides. metoclopramide to), cisapride (0). renzapride (0 1. zacopride f BRL 20627 f A) arid of 5-MeOT (Cl. on peristaltic activity in the Trendelenburg preparation of the isolated guinea pig ileum fn = 41 Il. Effects on longitudinal muscle activity elicited by pressure stimuli of 1 cm of water are shown. All values represent the area under the contraction curve expressed as means (activity of control = 101)C;1. Error bars are omitted for the sake of clarity. All values except those for the lowest concentration of each curve are significantly fP < 0.Z) difierent from the control (one-sample Wilcoxon signed rank test).
Metoclopramide was the most efficacious stimulator of longitudinal muscle activity. causing an increase in contractile activity to 340% of the control (figs. I. 2). The E,,, for all other benzamides fell within the range of 170 and 220% of the control. None of the compounds had a stimulatory effect on the circular muscIe layer but all reduced contractile activity at higher concentrations (shown for metoclopramide in fig. 2). These reductions of circular muscle activity usually parallelled the stimulatory effect on longitudinal muscle and are not specific to the benzamides since they are also observed after diverse stimuli such as carbachol, cholecystokinin or histamine (Buchheit. unpublished). % of Control 4OO350-300.250-200-150-loo-50-o-
8
$
b
s
-LOG M METOCLOPRI\WIOE Fig. 2. Effect of metoclopramide on peristaltic activity in the Trendeation in the isolated guinea pig ileum. Effects on ) and circular (0) muscle activity elicited by a pressure stimulus of I cm of water are shown. All values represent the area under the contraction curve expressed as means? S.D.: n = _S (activity c,f the control = 1OOci).
and ftl’-” M tend muscle Iayers at ftl
Fhc cxmtractions of fwth ’ M. The S-HT, rWcpFCw agonist.
reduced
2-Me-5HT. did not affect
the peristaltic
reflcs iIt
con-
centrations below It1 c, M.
In the ahsenc~ of selecrive L-HT, rcccptw agonists. 5-HT
and 5-nleFh~~~~F~~taminL‘ (5MeOTA
-LOG I4 5-w Fig. 4. Effect of 5-HT on peristaltic activity in the Trendelenhurg preparbm in thr is&&d guinrs pig ileum (n = Xt. Effects on ll~ff~itud~~~it&t ) and circular t ‘1 muscle activity ekited by a pressure s!imulus of I cm of water are shown. All values represent the areit under the amtruction curve expressed as means f S.D. (activity of the control = IOW ).
which boFh
actkate
5-HT4 r&xprors in the isolated guinea pig iicurn tfraig and Clarke. WOf were used as probes. After sen~saf adminisrrrttion. 5HT sensitized the ileum to pressure stimuli and thus augmented the rhythmic sristaltic cr,ntractions elicited by the increase of intraluminaf prwure.
As a wnscquence.
longitudinal
con-
tractkns wre increased (fig. 4). As in ths case of the knramides ~~nFr~ctions of the circufar muscle layer wcrz
decwawd.
fO_-
M S-HT
fhs
maximal
increase
obtained
at
was 20Wi of the control value, and a pD, value of S.0 was determined (table I ). The effect of 5-m
on longitudinal
musck
contractifity
conccntrat~on-res~nse cume for S-HT was not shifted under the influence of KS 205-930. 5-MeOT caused a stimulatory response closely similar to that of S-HT with a pD2 value of 7.7. The maximal effect of longitudinal muscle activity amounted to 2205 of the control (fig. 1, table 11. Concentrations of S-MeOT higher than 10-’ M, however, were found to have an inhibitory effect on longitudinal muscle contractions and thus the concentration-response curve of S-MeOT was bell-shaped (data not shown).
WBS unaf-
fected by KS /1WUO at a c~n~~~~rat~~n uf 10-’ RI. which is sufficient to block S-NT, receptors in the isolated guinea pig ileum (Buchheit et al. 198Sb). The
60
10i
Fig. 3. Effect of KS 2&9_W on peristaltic activity in the Trrndelrnburg preparation in the isolated guinea pig ileum (n = 8). Effects on longitudinal (0 1 and circular ( 2 I muscle activity eliciled by a pressure stimulus of 1 cm of water are shown. All values represent the area under the contraction curve expressed as means + S.D. tactivity of the control = IQW; 1.
Substituted benzamides stimulate small intestinal motility in man and in various species in vivo Wing and Sanger, 1988). The present experiments indicate that such effects are also exerted in the Trendelenburg preparation of the isolated guinea pig ileum, (Trendelenburg, 1917), as all the benzamides investigated increased peristaltic contractions in response to an increase in intraluminaf pressure (fig. 1). The model is therefore suitable for the investigation oi’ the mechanism of action of this group of com~unds. The mechanism of action of the substituted benzamides has remained unclear despite various attempts at its elucidation. Recently, blockade of 5HT, receptors (Buchheit et al., 1985a) and activation of 5-HT, receptors lDumuis et al., f9SY; Tonini et al., 1989; Craig and Clarke, 1990) have been considered as potential prokinetic mechanisms. The availability of compounds which act as 5HT, receptor antagonists without agonist effects at 5HT, receptors, e.g. KS 205930, (Richardson et a!., 19854, and of 5-HT, receptor agonists which are - at feast in vitro - devoid of affinity
for S-HT, receptors. e.g. 5-MeOT, (Fozard. 1985; 1990; Craig and Clarke, 1990; Dumuis et al.. 3988) has enabled us to analyze the mode of action of the henzamides in the Trendelenburg preparation. Excitatory 5-HT, receptors have been identified on neurons of the myenteric plexus in the guinea pig i!cum (Mawe et al., 19861 and thus are good candidates as targets for the modulation of the peristaltic reflex. The results of the current experiments. however, argue against such an assumption. Neither ICS 205-930 (Richardson et al., 1985) nor granisetron (Sanger and Nelson, 1988k both potent antagonists at 5-HT, receptors, had any influence on the peristaltic reflex when investigated in concentrations below lo-’ M. which are specific for 5-HT, receptor blockade. Furthermore. 2-Me-5-HT. a moderately selective agonist at 5-HT, receptors (Richardson et al. 1985). did not cause any change in the peristaltic reflex. Thus there is no evidence for an involvement of 5-HT, receptors in the regulation of the peristaltic reflex in the Trendelenburg preparation, which essentially rules out the possibility that the substitued benzamides stimulate the peristaltic reflex as a consequence of their S-HT, receptor antagonist properties. This is in agreement with previous results obtained for stomach (Gullikson et al.. 19891 and small intestine (Tonini et al.. 1989; Craig and Clarke, 1990). On the other hand, 5-HT and S-MeOT are potent stimulators of i!eal motility. Since both are agonists at 5-HT, receptors (Dumuis et al.. 1988; Craig and Clarke. 1990; Fozard. 1990). the data would be consistent with a role for the 5-HT., receptor subtype in the peristaltic reflex, although neither compound is selective for 5-HT, receptors. The fact that the stimulatory effect of 5-HT is insensitive to ICS 205-930. used at a concentration of lo-’ M which is 10 times the K, concentration of ICS 205-930 for the 5-HT, receptors in this preparation (Buchheit et al., 1985bJ rules out the possibility that the effect of 5-HT is mediated by activation of 5-HT, receptors. 5-MeOT, on the other hand. is devoid of effects at 5-HT, receptors in the isolated guinea pig ileum (Fozard, 1985; 1990). However. in addition to effects at 5-HT., receptors, agonist effects of 5-MeOT have also been reported at 5-HT, and 5-HT, receptors (Hoyer, 1989; Fozard 1990). Recently results from our laboratory, however, indicate that stimulation of SHT,,, 5-HT,,. and 5-HT, receptors does not influence the peristaltic reflex (Buchheit and Buhl, 1990). A major obstacle to the characterization of S-HT, receptors is the lack of a potent and selective antagonist. Although ICS 205-930 has been shown to inhibit activation of %-IT, receptors at micromolar concentrations, we found it impossible to use such high concentrations in the present experiments since peristaltic activity was reduced by ICS 205-930 in an unpredictable way. The reason for this is unknown. Other
groups have succeeded in using ICS 305-930 at micromolar concentrations in a similar setup (Tonini et al.. 1989; Craig and Clarke. 1991). These discrepancies might be due to species differences in sensitivity to the blockade of potassium and sodium channels which has been described for ICS 205-930 (Scholtysik et al.. 1988). A plausible way to characterize a receptor in the absence of selective antagonists is to compare rank orders of potency in the system investigated and in other well defined assays. Quantitative data describing the effects of substituted benzamides at 5-HT, receptors are available from an in vitro assay that measures stimulation of CAMP in mouse colliculi neurons (Dumuis et al. 19891. Comparison of the pD, values of S-HT. 5MeOT and the substituted benzamides used in the present study for the stimulation of the peristaltic reflex with the pD2 values for the stimulation of aafenylate cyclase in mouse colliculi neurons reveals a significant correlation (P = 0.013: correlation coefficient r = 0.8583 between the two data sets (table 1). Although this by no means constitutes definite proof. it does suggest that 5-HT, receptor activation increases peristaltic activity. and that the substituted benzamides stimulate the peristaltic reflex in vitro by activation of 5HT, receptors. In conclusion. the present results suggest that the agonistic effecr. at 5-HT, receptors and not the blockade of S-HT, receptors is responsible for the prokinetic effect of substituted benzamides on the small intestine.
Acknowledgements The
authors
comments
computerized Adrian
to
are indehtrd
on the manuscript integration
Bertholrt.
Dr.
John Fozard
and to Perrr
procedure.
Jean-Louis
for his critical
Maegli for providing the
The skillful technical
nork
Runscr and Peter Wingrove
of
is grate-
fully acknwledged.
References Akkrrmans.
L.M.A..
Horouitz.
A. Vos. A. Hoekstra.
J.M.M.
Roelofs
l9XH. Effect of ICS 205-930 (a specific 5-HT;
antagonist)
on gastric rmpving
and M. receptor
af a solid meal in normal
suh-
jrcts. Gut 2). IXY. Buchheit.
K.H..
B.P.
B. Costall. G. Engel. S.J. Gunning
Richardson.
IYXSa. 5-Hydroqtryptamine
nism hy mrtoclopramide to enhancement hy electrical K.H..
of contractions
field strmulation G. Engel.
isolated K.H.
stimulate
and T.
The second IUPHAR
p. 67.
muscle
and B. Richardson.
strip
Arch. Pharmacol. Buhl.
the peristaltic
of gastric rmpt>ing
37. 664.
effect of 5-hydrowtryptamine
lor%tudinal
Naunyn-Schmiedeh. Buchheit.
of stomach muscle strips induced
and facilitatmn
E. Mutschler
Study of the contractile the
antago-
and ICS 205-930 in the guinea pig leads
in viva. J. Pharm. Pharmacol. Buchheit.
R.J. Naylor and receptor
from
19X5h.
6HT)
guinea-pig
in
ileum.
329. 36.
1990. 5-HT,,-
and S-HT,
reflex in the isolated guinea
satellite meeting on serotonin.
agonists
pig ileum. Basel. l9W
Gullikaon. G.W.. M.A. Virina. R. Loeftler. M. Mortray and R.G. Bianchi. 1989. S-HT, antagonists in gastroparesis: comparison with prokinetic benzamides in a canine model. J. Gastrointest. Mot. 1. 79. t layer. D.. lYS9. 5-Hydrox~tlyptamine receptors and effector coupling mechanisms in peripheral tissues. in: The Peripheral Actions of S-Hydroqtryptamine. ed. J. Fozard (Oxford University Press. Oxford. U.K.) p. 72. King. F.D. and G.J. Sanger. 1988. Gastrointestinal motility enhancing agents. Ann. Rep. Med. Chem. 23. 201. Mawe. G.M.. T.A. Brattchek and M.D. Gershon. 1986. Peripheral neural serotortin receptors: Identification and characterization with specific antagonists and agonists, Proc. Natl. Acad. Sci. U.S.A. X3. 9799. Richardson. B.P.. G. Engel. P. Dottatsch and P.A. Stadler. 1985. Identification of serotonin M-receptor subtypes and their specific blockade hy a new class of drugs. Nature 316. 126. Sangrr. G.J.. 1987. Increased gut cholinergic activity and antagonism of 5-hydroxytryptamine M-receptors by BRL 24924: potential clinical importance of BRL 24924. Br. J. Pharmacol. 91. 77. Sartger. G.J. and D.R. Nelson. 1988. Selective and functional 5-hydroaytryptamine 1 receptor antagonism by BRL 43694 (granisetrott). Eur. J. Phannacol. 159. 113. Scholtpik. G.. Y. Imoto. A. Yatani and A.M. Brown. 1988, S-Hydroxytryptamine antagonist ICS 205-930 blocks cardiac potassium. sodium and calcium currents. J. Pharmacol. Exp. Ther. 245. 173. Smith. W.W.. L.F. Sancilio. J.B. Owera-Atepo. R.J. Naylor and L. Lambert. 1988. Zacopride. a potent 5-HT, antagonist, J. Pharm. Pharmacol. 40.301. Tonini. M.. J.J. Galligan and R.A. North. 1989, Effects of cisapride on cholinergic neurotransmission and propulsive motility in the guinea pig ileum, Gastroenterology 96. 1257. Trendelenburg. P.. 1917. Physiologische und pharmakologische Versuche iiber die Diinndarmperistaltik, Arch. Exp. Pathol. Pharmakol. XI. 55.
Karl-Heinz Buchheit and Thomas Buhl Pwchiral
Rcwarch.
Sarrdo: Pharrrra Ltd.. CH--100,’ Easel. Swirxrland
Received 4 February IYYI. revised MS received
Substituted
in various
bcnzamides
contribute
to their
substituted
benzamides
which
such as meloclopramidc.
species. As they are antagonists at 5HT, gastrointestinal
prcssurc-induced
prokinetic
on gut motility pcrislaltic
cisapridc,
zacopride.
accepted II) September IWI
renzapride
or BRL 206617. stimulate
intestinal
motility
and agonists a( S-HT, receptors and as both mechanisms could potentialI!
effect.
the underlying
was investigated
contractions
1 July IW.
mechanism
in the isolated guinea
are measured.
All
benzamides
is unclear.
To clarify
this. the effccl
pig ileum using the Trendclcnburg stimulated
the peristaltic
of some
technique.
in
reflex with the rank
order of potency: renzapridc > cisapridc > BRL 20647 > ( A_ )-zacopride > metoclopramide. ICS 205930. granisctron and 2methyl-5-HT did not change the peristaltic response. 5HT and 5methoxyiryptamine potently mimicked the effect of the benzamides. The effect of S-HT was not blocked by ICS 205-930 (IO-’ ML These results indicate :hat the Trendclenburg preparation
is suitable
for the investigation
results suggest that the intestinal 5-HT,
of intestinal
effect of bcnzamidcs
prokinetic
effects
results from activation
of the substituted of 5-HT,
receptors
benzamides.
Furthermore.
rather than from blockade
the of
receptors. Benzamides (substituted): Small intestinal motility: 5HT 3 receptors: 5-HT4 receptors: Trendelenburg
1. Introduction Substituted benzamides derived from metoclopramide, such as cisapride, renzapride. zacopride. BRL 20627. stimulate gastric and small intestinal motility in man and in various animals (for review see King and Sanger, 1988) and are being introduced into the clinic as so-called prokinetics for the treatment of gastrointestinal motility disorders. The mechanism of the prokinetic action of these compounds is unclear. Recently, effects at 5hydroxytryptamine (5HT) receptors have been implicated in the prokinetic actions of metoclopramide and its analogues. After the discovery that metoclopramide blocks 5-HT, receptors (Fozard and Mobarok Ali, 19781, other prokinetic analogues of metoclopramide such as renzapride, zacopride and BRL 20627 have also been described as 5-HT, receptor antagonists (Sanger. 1987: Smith et al.. 1988; Dunbar et al.. 1986). In addition, the prokinetic benza-
Correspondence to: K.-H. Buchheit. Preclinical Research (3861543). Sand07 Pharma Ltd.. CH-4002 Bawl. Switzerland. * Dedicated to Prof. Ernst Mutschler (Frankfurt/Main) on the occasion of his 60th birthday. Part of these results was presented at the 3151 Spring Meeting of the German Society ior Pharmacology and Toxicology. Mainz. March 13-16. 1990.
preparation
mides have recently been found to be agonists at a 5-HT receptor [hat has tentatively been named 5-HT, and which hss been identified in the brain (Dumuis et al., 1989) and in the enteric nervous system (Craig and Clarke, 1990). Besides having the substitute3 benzamides as agonists. this receptor is also characterized by a high sensitivity to both 5-HT and 5-methoxytryptamine (5-MeOT). In addition, the site shows a low affinity (pK, about 6.2) for the potent 5-HT, receptor antagonist. ICS 205-930 (Dumuis et al., 1988). Regarding the gut nervous system. activation of 5MT, receptors causes contraction of the longitudinal muscle layer of the guinea pig ileum (Eglen et al., 1990). Such an effect is potentially prokinetic as an increase in motility requires increased muscle activity. On the other hand, blockade of 5-HT, receptors could also be considered as a potential prokinetic effect. as 5-HT, receptor antagonists such as ICS 205-930. have a stimulatory effect on gastric motility in man (Akitermans et al., 1988) and in experimental animals (Buchheit et al. 1985a, Gamse, 1989). This raises the question of whether an interaction with 5-HT receptors is involved in the prokinetic action of the substituted benzamides and, in particular. which of the two mechanisms, 5-HT, receptor antagonism or 5-HT, receptor agonism, accounts for the action. TO answer this question, the effects of some substituted
to the serosa! side of the preparation. Previous expcrirems had confirmed that identical conccntration-response curves were obtained on cumu!:ltivc and non-cumulative administration of the test compounds. The maximal concentrations added were either !O~-i M or concentrations which consistently produced tonic contractions of the preparation. If such tonic contractions occurred, they were not considered for the quantitative assessment of drug effects. The tissue was left in contact with each drug concentration for IO min bcforc responses were quantified. Each preparation was used for the investigation of one drug only. Concentration-response curv2s were made by nlotting the mean area under the contraction cu7r2s from -I to I I experiments, expressed as a percentage of the control preparations. The potency of the drugs was characterized by the concentration yielding a half-maxima! effect. expressed as pD, values. These values were determined graphically from the mean concentrationresponse curves. Statistically significant differences from the control were determined for individual points of the concentration-response curves, using the nonparametric one-sample Wilcoxon signed rank test. min intervals
Femafe guinea pigs. 200-&I0 g, kept in colonies under standard conditions and diet were stunned and bled. The irtst 10 cm of the ileum were discarded. Segments of the remaining ileum, 4-5 cm long, were used. The lumen was carefully flushed to remove a!! remnants of food. Extensive distension of the preparation was a\oidcd during this process. The tissue was suspended in an organ bath under a l-g load by connLxtin_r the proximal (oral) end of the preparation to a iever transducer. The distal (anal) end was pulled over a glas t&e and connected to a pressure bttle. filled with Tyrodc solution. The preparation was bathed in Tyrode solution (NaCI 136-S: CaCI, 1.8: KC! 2.7; NaH -PO, 0.Q: MgC!. 1.05: NaHCO, 11.9: glucose 5.6 mM)at pH 7.1 maintained at 37 ’ C and bubbled with 5% CO, in oxygen.
2.4. Correlatiorr analysis The correlation between the lenburg experiment and those neurons was calculated based using a commercial software bridge, MA).
data from the Trendefrom mouse colliculi on a least squares fit, package (BBN, Cam-
2.5. Drugs
Peristaltic contractions were elicited for 30 s by increasing intralumina! pressure from 0 to I cm HZ0 at 6-mii; intervals. Longitudinal muscle responses were recorded using an isotonic force displacement transducer (Hugo Sachs. March-Hugstetten, Germany). A pressure transducer (Statham 23V, O-200 mm Hg) was used to measure changes of intralumina! pressure as an indication of circular muscle activity. Longitudinal and circular muscle activity were quantified by determining the area under the curve of peristaltic contractions, by means of a computerized integration procedure (developed at Sandoz Pinarma Ltd.) on an IBM persona! computer AT 03. Only those preparations were used which gave at least three consistent pressure-induced responses before administration of any test compound and where no signs of spastic, tonic contractions were observed.
5-HT IFluka, Buchs, Switzerland), metoclopramide (RBI, Natick, USA) and 5methoxyttyptamine (5 MeOT, Sigma, Munich, Germany) were purchased. ICS 205-930 ([3cY-tropanyl-I-lH-indole-3-carboxylic acid ester), granisetron, 2-methyl-5-HT (2-Me-5-HT), ( f Irenzapride, and ( f I-zacopride were synthesized at Sandoz Pharma Ltd. (Base!). Cisapride and BRL 20627 [(2cr,6&9a(u)-( f I-4-amino-5-chloro-2-methoxy-N-(octahydro-6-methyl-2H-quinolizin-2-y!)benzamide] were kind gifts of Janssen (Beerse, Belgium) and Beecham (Harlow, UK), respectively. A!! drugs were dissolved in saline, wit11 (bases) or without (salts) the addition of tartaric acid.
3. Results
2.3. Qwntitatire assessment of effects
3. I. Characterization of the peristaltic reflex
Cumulative concentration-response curves were recorded under equilibrium conditions by adding increasing concentrations of the test compounds at 12-
Peristaltic activity in the isolated guinea pig ileum was initiated by increasing the intralumina! pressure by 1 cm Hz0 over that in the organ bath by elevating the
3
TABLE
x or
I
S~imulatoty effecl on longitudinal muscle activity in the Trendelenhurg preparation. compared with the potency to stimulate the formation of CAMP in isolated mouse colliculi neurons by activation of 5.HT, receptors. Compound
Trendelenhurg experiment J PD,
S-HT S-MeOT 2-Me-5 -HT ICS 205-930 Granisetron Metoclopramide Cisapride Renzapride Zacopride BRL 20627
P
8.0 7.7 <5 <7 <7 5.3 7.5 8.6 5.7 6.6
E,, 280 (8) 220 (6) - (9) - (8) - (61 340 (5) 220 (5) 170 (4) 17OflI~ 200 (4)
S%‘“’ I 250 225
Mouse colliculi neurons
1 200.
PD, ’
175
7.0 7.0
150
5.3 7.1 6.9 6.0 5.5
’ pD, = - ,,,log of the concentration which caused a half-maximal effect; E,,, indicates the maximal increase in phasic contractility. expressed as percentage of the control: the numbers in parentheses indicate the number of experiments: h data taken from Dumuis et al. (19891 and Dumuis et al. (1988).
level of the buffer in the pressure bottle which was connected to the lumen of the preparation. This caused radial distension of the muscle and elicited the peristaltic reflex consisting of coordinated phasic contractions and relaxations of the longitudinal and the circular muscle layer. Normally, tonic spasms were not observed under the conditions used. Although there were differences in the amplitude and frequency of the phasic contractions between different preparations under control conditions, the responses expressed as area under the contraction time curves (which integrates contraction amplitude and frequency) were reproducible and remained constant for several hours, without signs of fatigue. 3.2. Ejfects of benzamides on the peristaltic reflex Upon administration to the serosal side of the preparation, metoclopramide, BRL 20627, renzapride, zacopride and cisapride augmented with different potencies the contractile activity elicited in the longitudinal muscle layer by an increase of intraluminal pressure (table 1). All five compounds caused qualitatively identical responses. None elicited isolated tonic or phasic contractions (i.e. contractions which were not induced by increases in intraluminal pressure). Mptoclopramide and zacopride were only weakly active (pD, values: 5.3 and 5.7, respectively). BRL 20627 was slightly more potent (pD, = 6.6). Renzapride and cisapride augmented contractions, with pD2 values of 8.6 and 7.5, respectively. This resulted in the following rank order of potency: renzapride > cisapride > BRL 20627 > (& )-zacopride > metoclopramide (fig. 1).
125
- log II Agonist Fig. 1. Comparison of the stimulatory eff&ts of the benzamides. metoclopramide to), cisapride (0). renzapride (0 1. zacopride f BRL 20627 f A) arid of 5-MeOT (Cl. on peristaltic activity in the Trendelenburg preparation of the isolated guinea pig ileum fn = 41 Il. Effects on longitudinal muscle activity elicited by pressure stimuli of 1 cm of water are shown. All values represent the area under the contraction curve expressed as means (activity of control = 101)C;1. Error bars are omitted for the sake of clarity. All values except those for the lowest concentration of each curve are significantly fP < 0.Z) difierent from the control (one-sample Wilcoxon signed rank test).
Metoclopramide was the most efficacious stimulator of longitudinal muscle activity. causing an increase in contractile activity to 340% of the control (figs. I. 2). The E,,, for all other benzamides fell within the range of 170 and 220% of the control. None of the compounds had a stimulatory effect on the circular muscIe layer but all reduced contractile activity at higher concentrations (shown for metoclopramide in fig. 2). These reductions of circular muscle activity usually parallelled the stimulatory effect on longitudinal muscle and are not specific to the benzamides since they are also observed after diverse stimuli such as carbachol, cholecystokinin or histamine (Buchheit. unpublished). % of Control 4OO350-300.250-200-150-loo-50-o-
8
$
b
s
-LOG M METOCLOPRI\WIOE Fig. 2. Effect of metoclopramide on peristaltic activity in the Trendeation in the isolated guinea pig ileum. Effects on ) and circular (0) muscle activity elicited by a pressure stimulus of I cm of water are shown. All values represent the area under the contraction curve expressed as means? S.D.: n = _S (activity c,f the control = 1OOci).
and ftl’-” M tend muscle Iayers at ftl
Fhc cxmtractions of fwth ’ M. The S-HT, rWcpFCw agonist.
reduced
2-Me-5HT. did not affect
the peristaltic
reflcs iIt
con-
centrations below It1 c, M.
In the ahsenc~ of selecrive L-HT, rcccptw agonists. 5-HT
and 5-nleFh~~~~F~~taminL‘ (5MeOTA
-LOG I4 5-w Fig. 4. Effect of 5-HT on peristaltic activity in the Trendelenhurg preparbm in thr is&&d guinrs pig ileum (n = Xt. Effects on ll~ff~itud~~~it&t ) and circular t ‘1 muscle activity ekited by a pressure s!imulus of I cm of water are shown. All values represent the areit under the amtruction curve expressed as means f S.D. (activity of the control = IOW ).
which boFh
actkate
5-HT4 r&xprors in the isolated guinea pig iicurn tfraig and Clarke. WOf were used as probes. After sen~saf adminisrrrttion. 5HT sensitized the ileum to pressure stimuli and thus augmented the rhythmic sristaltic cr,ntractions elicited by the increase of intraluminaf prwure.
As a wnscquence.
longitudinal
con-
tractkns wre increased (fig. 4). As in ths case of the knramides ~~nFr~ctions of the circufar muscle layer wcrz
decwawd.
fO_-
M S-HT
fhs
maximal
increase
obtained
at
was 20Wi of the control value, and a pD, value of S.0 was determined (table I ). The effect of 5-m
on longitudinal
musck
contractifity
conccntrat~on-res~nse cume for S-HT was not shifted under the influence of KS 205-930. 5-MeOT caused a stimulatory response closely similar to that of S-HT with a pD2 value of 7.7. The maximal effect of longitudinal muscle activity amounted to 2205 of the control (fig. 1, table 11. Concentrations of S-MeOT higher than 10-’ M, however, were found to have an inhibitory effect on longitudinal muscle contractions and thus the concentration-response curve of S-MeOT was bell-shaped (data not shown).
WBS unaf-
fected by KS /1WUO at a c~n~~~~rat~~n uf 10-’ RI. which is sufficient to block S-NT, receptors in the isolated guinea pig ileum (Buchheit et al. 198Sb). The
60
10i
Fig. 3. Effect of KS 2&9_W on peristaltic activity in the Trrndelrnburg preparation in the isolated guinea pig ileum (n = 8). Effects on longitudinal (0 1 and circular ( 2 I muscle activity eliciled by a pressure stimulus of 1 cm of water are shown. All values represent the area under the contraction curve expressed as means + S.D. tactivity of the control = IQW; 1.
Substituted benzamides stimulate small intestinal motility in man and in various species in vivo Wing and Sanger, 1988). The present experiments indicate that such effects are also exerted in the Trendelenburg preparation of the isolated guinea pig ileum, (Trendelenburg, 1917), as all the benzamides investigated increased peristaltic contractions in response to an increase in intraluminaf pressure (fig. 1). The model is therefore suitable for the investigation oi’ the mechanism of action of this group of com~unds. The mechanism of action of the substituted benzamides has remained unclear despite various attempts at its elucidation. Recently, blockade of 5HT, receptors (Buchheit et al., 1985a) and activation of 5-HT, receptors lDumuis et al., f9SY; Tonini et al., 1989; Craig and Clarke, 1990) have been considered as potential prokinetic mechanisms. The availability of compounds which act as 5HT, receptor antagonists without agonist effects at 5HT, receptors, e.g. KS 205930, (Richardson et a!., 19854, and of 5-HT, receptor agonists which are - at feast in vitro - devoid of affinity
for S-HT, receptors. e.g. 5-MeOT, (Fozard. 1985; 1990; Craig and Clarke, 1990; Dumuis et al.. 3988) has enabled us to analyze the mode of action of the henzamides in the Trendelenburg preparation. Excitatory 5-HT, receptors have been identified on neurons of the myenteric plexus in the guinea pig i!cum (Mawe et al., 19861 and thus are good candidates as targets for the modulation of the peristaltic reflex. The results of the current experiments. however, argue against such an assumption. Neither ICS 205-930 (Richardson et al., 1985) nor granisetron (Sanger and Nelson, 1988k both potent antagonists at 5-HT, receptors, had any influence on the peristaltic reflex when investigated in concentrations below lo-’ M. which are specific for 5-HT, receptor blockade. Furthermore. 2-Me-5-HT. a moderately selective agonist at 5-HT, receptors (Richardson et al. 1985). did not cause any change in the peristaltic reflex. Thus there is no evidence for an involvement of 5-HT, receptors in the regulation of the peristaltic reflex in the Trendelenburg preparation, which essentially rules out the possibility that the substitued benzamides stimulate the peristaltic reflex as a consequence of their S-HT, receptor antagonist properties. This is in agreement with previous results obtained for stomach (Gullikson et al.. 19891 and small intestine (Tonini et al.. 1989; Craig and Clarke, 1990). On the other hand, 5-HT and S-MeOT are potent stimulators of i!eal motility. Since both are agonists at 5-HT, receptors (Dumuis et al.. 1988; Craig and Clarke. 1990; Fozard. 1990). the data would be consistent with a role for the 5-HT., receptor subtype in the peristaltic reflex, although neither compound is selective for 5-HT, receptors. The fact that the stimulatory effect of 5-HT is insensitive to ICS 205-930. used at a concentration of lo-’ M which is 10 times the K, concentration of ICS 205-930 for the 5-HT, receptors in this preparation (Buchheit et al., 1985bJ rules out the possibility that the effect of 5-HT is mediated by activation of 5-HT, receptors. 5-MeOT, on the other hand. is devoid of effects at 5-HT, receptors in the isolated guinea pig ileum (Fozard, 1985; 1990). However. in addition to effects at 5-HT., receptors, agonist effects of 5-MeOT have also been reported at 5-HT, and 5-HT, receptors (Hoyer, 1989; Fozard 1990). Recently results from our laboratory, however, indicate that stimulation of SHT,,, 5-HT,,. and 5-HT, receptors does not influence the peristaltic reflex (Buchheit and Buhl, 1990). A major obstacle to the characterization of S-HT, receptors is the lack of a potent and selective antagonist. Although ICS 205-930 has been shown to inhibit activation of %-IT, receptors at micromolar concentrations, we found it impossible to use such high concentrations in the present experiments since peristaltic activity was reduced by ICS 205-930 in an unpredictable way. The reason for this is unknown. Other
groups have succeeded in using ICS 305-930 at micromolar concentrations in a similar setup (Tonini et al.. 1989; Craig and Clarke. 1991). These discrepancies might be due to species differences in sensitivity to the blockade of potassium and sodium channels which has been described for ICS 205-930 (Scholtysik et al.. 1988). A plausible way to characterize a receptor in the absence of selective antagonists is to compare rank orders of potency in the system investigated and in other well defined assays. Quantitative data describing the effects of substituted benzamides at 5-HT, receptors are available from an in vitro assay that measures stimulation of CAMP in mouse colliculi neurons (Dumuis et al. 19891. Comparison of the pD, values of S-HT. 5MeOT and the substituted benzamides used in the present study for the stimulation of the peristaltic reflex with the pD2 values for the stimulation of aafenylate cyclase in mouse colliculi neurons reveals a significant correlation (P = 0.013: correlation coefficient r = 0.8583 between the two data sets (table 1). Although this by no means constitutes definite proof. it does suggest that 5-HT, receptor activation increases peristaltic activity. and that the substituted benzamides stimulate the peristaltic reflex in vitro by activation of 5HT, receptors. In conclusion. the present results suggest that the agonistic effecr. at 5-HT, receptors and not the blockade of S-HT, receptors is responsible for the prokinetic effect of substituted benzamides on the small intestine.
Acknowledgements The
authors
comments
computerized Adrian
to
are indehtrd
on the manuscript integration
Bertholrt.
Dr.
John Fozard
and to Perrr
procedure.
Jean-Louis
for his critical
Maegli for providing the
The skillful technical
nork
Runscr and Peter Wingrove
of
is grate-
fully acknwledged.
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