Carlo A. Rizzi

Identification of serotonin 5-HT4 recognition sites in the porcine caudate nucleus by radioligand binding

Specific binding for the serotonin 5-HT4 receptor (5-HT4R) radioligand [3H]GR 113808 was identified in pig caudate nucleus and characterized by serotonin subtype selective drugs. Binding was inhibited by serotonin and by synthetic indoles, benzamides and benzimidazolones known to characterize the 5-HT4R in functional tests. Rank order of potency of 5-HT4R antagonists was: GR 125487 (Ki, 0.19 nM) > GR 113808 >> SC 53606 > SDZ 205,557 > RS 235971/190 > DAU 6285 > tropisetron > DAU 6215. GR 125487 and GR 113808 were highly selective with respect to the 5-HT3 receptor (5-HT3R). Rank order of potency of 5-HT4R agonists was: SC 53116 (Ki, 21 nM) > BIMU 1 > cisapride > BIMU 8 > serotonin > renzapride > S-zacopride > metoclopramide > R-zacopride > 5-methoxytryptamine >> 5-carboxamidotryptamine. BIMU 8, renzapride, metoclopramide and the zacopride enantiomers gave shallow competition curves. The agonists were substantially less selective than the antagonists with respect to the 5-HT3R. With only two exceptions, SCH 23390 and metergoline, which bound with sub-microM affinity to the 5-HT4R, binding was not inhibited by compounds selective for other G-protein-coupled or channel-gated receptors. Highly significant correlations in affinities of compounds for 5-HT4R in caudata of pigs, guinea pigs and humans were found suggesting no difference among mammalian species.

Pharmacological characterization of the 5-hydroxytryptamine receptor coupled to adenylyl cyclase stimulation in human brain

Recently, a 5-hydroxytryptamine (5-HT) receptor has been described, whose pharmacology was distinct from that of the already known serotonergic receptors, so that it has been called 5-HT4. Because the lack of a high affinity radioligand, the identification of this receptor depends entirely on functional pharmacological analysis. Its stimulation leads to an increase in cyclic AMP accumulation in mouse embryo colliculi neurons, in guinea pig hippocampus and in human heart. We studied the effect of two indoleamines, 5-HT and 5-methoxytryptamine (5-MeO-T), and a benzimidazolone derivative, BIMU 8, in stimulating basal adenylyl cyclase activity in human frontal cortex, and characterized the receptor subtype involved. In membranes prepared from this tissue, 5-HT, 5-MeO-T and BIMU 8 dose-dependently stimulated (13-25%) the basal enzyme activity (220 pmoles cyclic AMP/min/mg protein). 5-MeO-T behaved as a full agonist, BIMU 8 elicited about 60% of the maximal 5-HT effect. The selective 5-HT1A agonist 8-OH-DPAT, was devoid of any stimulating activity. ICS 205-930, a low affinity 5-HT4 receptor antagonist, completely reversed the effect of all three agonists at high concentrations. Therefore, the present data are consistent with the 5-HT-mediated stimulation of adenylyl cyclase in human frontal cortex resulting by the activation of a 5-HT4 receptor subtype.

Benzimidazolones and renzapride facilitate acetylcholine release from guinea-pig myenteric plexus via 5-HT4 receptors

The effects of the 5-HT4 receptor agonists BIMU 8, BIMU 1, renzapride and of the 5-HT1p receptor agonist 5-hydroxyindalpine on basal and electrically evoked outflow of tritium were studied in guinea-pig longitudinal muscle myenteric plexus preparations preincubated with [3H]choline. Muscle contractions were recorded simultaneously.BIMU 8 caused a calcium dependent and tetrodotoxin sensitive increase in basal [3H]outflow that was assumed to represent release of [3H]acetylcholine. In addition, BIMU 8 enhanced the release of [3H]acetylcholine and twitch contractions evoked by submaximal electrical stimulation. Ondansetron (1 μmol/l) did not change the effects of BIMU 8, but DAU 6285 and tropisetron (each 1 μmol/l) competitively antagonized the various facilitatory effects of BIMU 8 with pA2 values of 7.0–7.2 (DAU 6285) and 7.0–7.3 (tropisetron). The phosphodiesterase inhibitors IBMX and rolipram did not increase the effects of BIMU 8. BIMU 1 and renzapride also concentration-dependently increased basal release of acetylcholine, and release and contractions caused by submaximal stimulation. The effects of BIMU 1 and renzapride were competitively antagonized by 1 μmol/l tropisetron (pA2 6.6–7.1). The EC50 values for the increase in the evoked [3H]acetylcholine release and contractions were closely similar. 5-Hydroxyindalpine did not change basal release and slightly inhibited the evoked release of [3H]acetylcholine. Release of acetylcholine and contractions elicited by submaximal stimulation were strongly inhibited by ( + )-tubocurarine which indicates that nicotinic ganglionic transmission is involved in this kind of release.The results suggest that BIMU 8, BIMU 1 and renzapride stimulate 5-HT4 receptors at cholinergic interneurones and thereby facilitate nicotinic ganglionic transmission in the myenteric plexus. Cyclic AMP is probably not involved in the 5-HT4 receptor mediated facilitation of acetylcholine release.

Aims and Methods

Objective: Few epidemiological studies are available on Italian patients with lower urinary tract symptoms and their QoL. QUIBUS (QUality of life Investigated in BPH patients with Urinary Symptoms) is an observational longitudinal study aimed at evaluating symptoms and QoL in a large sample of Italian patients and investigating their correlation with demographic, social and clinical characteristics of BPH. Patients and Methods: Patients with lower urinary tract symptoms and prostate enlargement suggestive of BPH (both old and new diagnosis) were enrolled between November 1998 and May 1999 in 31 Italian centers of urology. This longitudinal investigation consists of an enrollment visit, in which demographic, social and clinical aspects are recorded as baseline data, and a follow-up visit after 1 year of treatment freely assigned by the investigators. Symptoms and QoL are assessed by means of IPSS, ICS-BPH (at both visits) and SF-36 (only at the follow-up visit) questionnaires. Results: 1,033 patients were enrolled. The follow-up visit is still under evaluation. In this series of papers the baseline results are presented and discussed in terms of (i) medical management, (ii) life-style, (iii) symptoms, bothersomeness and QoL, (iv) sexual function of a large and representative sample of Italian patients and (v) uroflowmetry.

5-hydroxytryptamine4 receptor agonists facilitate cholinergic transmission in the circular muscle of guinea pig ileum: antagonism by tropisetron and DAU 6285.

The effect of 5-hydroxytryptamine (5-HT), BIMU 8 (endo-N-(8-methyl-8-azabicyclo [3.2.1.] oct-3-yl)-2,3-dihydro-3-(1-methyl)ethyl-2-oxo-1H-benzimidazole-1- carboxamide hydrochloride) and cisapride was studied on the electrically-induced neurogenic cholinergic twitch contractions in the guinea pig ileum circular muscle. These compounds caused a concentration-dependent increase in the amplitude of submaximal twitch contractions with the following rank order of potency: 5-HT greater than BIMU 8 = cisapride. The effect of 5-HT was competitively antagonized by tropisetron (ICS 205-930) (apparent pA2 value: 6.4), suggesting an interaction at 5-hydroxytryptamine4 (5-HT4) receptors. The novel benzimidazolone derivative DAU 6285 (endo-6-methoxy-8-methyl-8-azabicyclo [3.2.1.] oct-3-yl-2,3-dihydro-2-oxo-1H-benzimidazole-1-carboxylate hydrochloride), antagonized the effect of 5-HT, BIMU 8 and cisapride with apparent pA2 values in the range 7.1-7.3. Our findings demonstrate that cholinergic neurones innervating the circular coat are endowed with excitatory 5-HT4 receptors. DAU 6285 is approximately 5-9-fold more potent than tropisetron as antagonist at these receptors.

Pharmacological properties of a novel class of 5-HT3 receptor antagonists

The pharmacological profile of six representative members of a novel class of 5-HT3 receptor antagonists is described. The compounds are esters and amides of benzimidazolone-1-carboxylic acid with a basic azabicycloalkyl moiety (compounds 1-3) and their respective ethyl derivatives (compounds 4-6). In isolated preparations (rabbit heart and guinea pig ileum) all compounds antagonized the 5-HT3 receptor-mediated effects of serotonin, with potencies comparable with those of the reference compounds, ICS 205.930 and GR 38032F (-log IC50 9.30-11.9 and 6.8-8.20, in heart and ileum, respectively). In the anaesthetised rat, all agents potently inhibited the Bezold-Jarisch reflex whether given i.v. or i.d. I.v. administration of compounds prevented cisplatin-induced emesis in dogs (ID50 ranging from 3.7 to 147 micrograms/kg). All agents accelerated gastric emptying of solids in rats (ED50 about 10-160 micrograms/kg i.p.). In addition, compounds 4 and 5 were able to stimulate 5-HT4 receptors in the isolated guinea pig ileum, as well as enhance contractile activity in the Heidenhain gastric pouch of dogs, showing clearcut prokinetic properties.

Italian validation of the International Consultation on Incontinence Questionnaires.

To validate the Italian version of two questionnaires for lower urinary tract symptoms (LUTS), i.e. the long (LF) and the short form (SF) of the International Consultation on Incontinence Questionnaire (ICIQ).

A comparison of the efficacy and tolerability of tamsulosin and finasteride in patients with lower urinary tract symptoms suggestive of benign prostatic hyperplasia

In this multicentre, double-blind study, patients with LUTS/BPH were randomised to 26 weeks with finasteride 5 mg once daily (n=204) or tamsulosin 0.4 mg once daily (n=199). Double-blind treatment was continued for another 26 weeks (total treatment duration: 1 y). The primary efficacy parameter was the difference in mean change in total Symptom Problem Index (SPI) from baseline to end point at week-26 in the intention-to-treat (ITT) and per protocol (PP) populations. Tamsulosin induced a greater improvement in total SPI (−5.2 points or −37%) compared to finasteride (−4.5 points or −31%) at week-26 (P=0.055 in ITT and P=0.032 in PP). Tamsulosin improved urinary symptoms (particularly the more bothersome storage symptoms) and flow more quickly than finasteride. The difference was statistically significant for the SPI from week-1 (reduction, respectively, −2.5 vs −1.8 points, P=0.043) to week-18 and for Qmax from week-1 (increase, respectively, 2.3 vs 0.7 ml/s, P=0.0007) to week-12. Both treatments were well tolerated with a comparable incidence of adverse events, including urinary retention.

Gastroprokinetic properties of the benzimidazolone derivative BIMU 1, an agonist at 5-hydroxytryptamine4 and antagonist at 5-hydroxytryptamine3 receptors

We have investigated the in vivo motor stimulating and gastroprokinetic properties of the azabicycloalkyl benzimidazolone derivative BIMU 1 (3-ethyl-2,3dihydro-N-(8-methyl-8-azabicyclo [3.2.1] oct-3-yl)-2-oxo 1H-benzimidazole-1-carboxamide hydrochloride) and its binding profile at 5-hydroxytryptamine3 and 5-hydroxytryptamine4 receptors, in an attempt to assess the serotonergic mechanism underlying its prokinetic action.BIMU 1 dose-dependently (0.01–0.3 mg/kg iv.) increased the motility of a denervated pouch of canine stomach. This excitatory action was sensitive to muscarinic blockade. A similar stimulatory effect was exerted by the benzamidic prokinetic agent cisapride (0.03–0.3 mg/kg i.v.) but not by the 5-HT3 receptor antagonist ondansetron (up to 1 mg/kg i.v ). The significance for propulsive efficacy of the motor stimulating activity of BIMU 1 was evaluated in a model of gastric emptying of liquids in the conscious dog. The emptying rate of a non-caloric liquid meal instilled through a gastric fistula was accelerated by both BIMU 1 (0.01–1 mg/kg i.v. and 0.1–3 mg/kg p.o.) and cisapride (0.03–1 mg/kgiv.and0.3–10 mg/kgp.o.).Ondansetron (1 mg/kg i.v.) did not show any effect. The activity of the 5-HT4 receptor antagonist DAU 6285 was evaluated in the gastric emptying model per se and in interaction experiments on the accelerating action of BIMU 1 (0.3 mg/kg L v.). At 1 mg/kg iv., DAU 6285 was ineffective on its own and failed to antagonize BIMU 1-induced prokinetic action;at the dose of 3 mg/kg i.v., it depressed the gastric emptying rate per se by 15% and totally abolished the accelerating effect of BIMU 1.In the binding assay, BIMU 1 exhibited an appreciable affinity for 5-HT3 receptors in NG 108-15 cells (KD: 0.8 nmol/l) and for 5-HT4 receptors in pig striatum (KD: 26.5 nmol/l). Compared to BIMU 1, cisapride bound with a similar affinity to 5-HT4 (KD: 35.2 mnol/l) and a much lower affinity to 5-HT3 receptors (KD: 155 nmol/l). By contrast, ondansetron was highly selective for 5-HT3 sites (KD: 4.7 nmol/l), being ineffective in the assay for 5-HT4 receptors (KD > 10000).Our results show that BIMU 1, like cisapride and unlike ondansetron, is an effective stimulant of gastric motility and propulsion. The action of BIMU 1 appears to depend on 5-HT4 receptor stimulation and to involve the activation of cholinergic nerve pathways.

Therapeutic potential of drugs with mixed 5-HT4 agonist/5-HT3 antagonist action in the control of emesis

Drugs interacting with serotonin (5-hydroxytryptamine, 5-HT) receptors are of value in the treatment of several gastrointestinal disturbances. Selective 5-HT3 receptor antagonists (ondansetron, granisetron, tropisetron) are widely utilized to control emesis induced by chemotherapy and radiation, while agonists at 5-HT4 receptors (cisapride, renzapride, BIMU compounds) are endowed with gastrointestinal prokinetic action. Here we overview the therapeutic potential of drugs with potent mixed 5-HT4 agonist/5-HT3 antagonist properties (i.e. BIMU 1) in the management of anticancer therapy-induced emesis and of intestinal adynamic post-operative conditions associated with vomiting. In the former situation, the agonism at 5-HT4 receptors is expected to be of benefit via two possible mechanism: (i) inhibition of 5-HT release from enterochromaffin cells; (ii) restoration of anally driven peristaltic waves in the upper gastrointestinal tract. Moreover, 5-HT4 receptor-induced prokinetic activity may counteract colonic constipation, an unwanted effect which occurs in a number of patients treated with pure 5-HT3 receptor antagonists. Additionally, the above mentioned drugs might be of value in post-operative conditions associated with intestinal adynamia and emesis sensitive to 5-HT3 receptor blockade.