The in vitro pharmacological profile of prucalopride, a novel enterokinetic compound

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Abstract

Prucalopride is a novel enterokinetic compound and is the first representative of the benzofuran class. We set out to establish its pharmacological profile in various receptor binding and organ bath experiments. Receptor binding data have demonstrated prucalopride's high affinity to both investigated 5-HT4 receptor isoforms, with mean pKi estimates of 8.60 and 8.10 for the human 5-HT4a and 5-HT4b receptor, respectively. From the 50 other binding assays investigated in this study only the human D4 receptor (pKi 5.63), the mouse 5-HT3 receptor (pKi 5.41) and the human σ1 (pKi 5.43) have shown measurable affinity, resulting in at least 290-fold selectivity for the 5-HT4 receptor. Classical organ bath experiments were done using isolated tissues from the rat, guinea-pig and dog gastrointestinal tract, using various protocols. Prucalopride was a 5-HT4 receptor agonist in the guinea-pig colon, as it induced contractions (pEC50=7.48±0.06; insensitive to a 5-HT2A or 5-HT3 receptor antagonist, but inhibited by a 5-HT4 receptor antagonist) as well as the facilitation of electrical stimulation-induced noncholinergic contractions (blocked by a 5-HT4 receptor antagonist). Furthermore, it caused relaxation of a rat oesophagus preparation (pEC50=7.81±0.17), in a 5-HT4 receptor antagonist sensitive manner. Prucalopride did not cause relevant inhibition of 5-HT2A, 5-HT2B, or 5-HT3, motilin or cholecystokinin (CCK1) receptor-mediated contractions, nor nicotinic or muscarinic acetylcholine receptor-mediated contractions, up to 10 μM. It is concluded that prucalopride is a potent, selective and specific 5-HT4 receptor agonist. As it is intended for treatment of intestinal motility disorders, it is important to note that prucalopride is devoid of anti-cholinergic, anticholinesterase or nonspecific inhibitory activity and does not antagonise 5-HT2A, 5-HT2B and 5-HT3 receptors or motilin or CCK1 receptors.

Introduction

Abnormal motility of the gastrointestinal tract, such as in gastroparesis, ileus and various forms of constipation, may lead to complaints, which can be severe and socially and economically debilitating. Upper gastrointestinal tract motility disturbances can be treated with prokinetics such as metoclopramide, domperidone and cisapride. The mechanism of action of these compounds has been suggested to be peripheral dopamine D2 receptor antagonism (domperidone and metoclopramide) or 5-HT4 receptor agonism (metoclopramide, cisapride) (Briejer et al., 1995). However, these compounds seem less effective in the treatment of lower intestinal motility disturbances, e.g. constipation, although some efficacy has been reported for cisapride in a subset of patients Müller-Lissner, 1987, Longo and Vernava, 1993. This effect of cisapride suggests that stimulation of 5-HT4 receptors may be a means to influence colonic motility. Indeed, 5-HT4 receptor-mediated responses have been demonstrated in the human isolated colon Tam et al., 1994, McLean and Coupar, 1996, Prins et al., 2000a. However, cisapride is a 5-HT3 and 5-HT2A receptor antagonist as well. Both actions may be associated with constipation (Briejer et al., 1995), thus possibly counteracting the promotility effects of 5-HT4 receptor stimulation on the colon. Clearly, a more selective 5-HT4 receptor agonist could have more pronounced effects on colonic motility, and prove valuable for the treatment of slow transit constipation and post-operative atony.

Prucalopride (4-amino-5-chloro-2,3-dihydro-N-[1-(3-methoxypropyl)-4-piperidinyl]-7-benzofuran carboxamide monohydrochloride; Fig. 1) is a novel enterokinetic compound. In dogs, it induces colonic giant migrating contractions (the main drive behind distal propulsion of colonic contents; Karaus and Sarna, 1987) and a general change in colonic contractile motility patterns (Briejer et al., 1997c). Furthermore, it evokes defecation in cats as well as in human healthy volunteers and it accelerates gastric emptying in dogs Briejer et al., 1997a, Briejer et al., 1997b and humans Poen et al., 1997, Vandeplassche et al., 1997, Emmanuel et al., 1998 and colonic transit in healthy volunteers (Bouras et al., 1999). The compound is currently in clinical trials for the treatment of idiopathic chronic constipation (Coremans et al., 1999) and post-operative ileus.

This paper describes the pharmacological profile of prucalopride. Prucalopride's high specificity for 5-HT4 receptors, as determined in receptor binding studies, was confirmed in organ bath experiments that focused especially on pharmacological properties that are known to be important for modulation of gastrointestinal motility.

Section snippets

Tissue and cell membrane preparations for radioligand binding

Prucalopride was investigated for in vitro binding using membrane preparations of animal tissue or using membranes of cell lines transjected with cloned human (h) receptors. As tissue sources brain, peripheral organs, blood of animal or human origin or permanent cell lines were used. The procedures for membrane preparations were described previously (Lesage et al., 1998). The original references to the various receptor binding models are found in Lesage et al. (1998).

The culture medium

Receptor binding studies

Prucalopride (Fig. 1) was tested as a competing ligand in binding tests on a wide range of receptors including classical monoamine and peptide receptors, ion channel binding sites, lipid derived factor and neurotransmitter transporter binding sites. The resulting pIC50 and Ki-values for non-serotonergic receptors are summarised in Table 1. Prucalopride exerted low binding affinity for dopamine-D4 receptors measured in CHO cells expressing the cloned human receptor (Ki value=2.3 μM). The

Discussion

In our detailed receptor binding profile, prucalopride showed very high specificity for 5-HT4 receptors. Based on the comparison of Ki-values, prucalopride had 290- and 460-fold higher affinity for the h5-HT4(b) receptor over the hD4 receptor labelled with [3H]spiperone and the hσ1-site labelled with [3H]haloperidol, respectively. For the h5-HT4(a) receptor the selectivity was 780- and 1500-fold over the hD4 receptor and the hσ1-site, respectively. Within the 5-HT receptor family the

Acknowledgements

The statistical help of W. De Ridder is greatly appreciated. P. De Winter, P. Van Bergen and Mrs. C. Borgmans are acknowledged for their experimental assistance in performing the organ bath experiments and Mrs. D. Verkuringen for helpful assistance in preparing this manuscript.

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