Angelo Sagrada

WAL 2014 - A muscarinic agonist with preferential neuron-stimulating properties

The ability of WAL 2014 to elicit muscarinic responses was investigated in various in vitro and in vivo models. In CHO cells transfected with human m1- or m3- receptor genes, WAL 2014 was clearly more effective in stimulating the M1-mediated PI response. In isolated tissue preparations, WAL 2014 exhibited full agonist properties in the rabbit vas deferens (putative M1 receptor) and behaved like a partial agonist at M2 receptors in the atrium and M3 receptors in the ileum of guinea-pigs. In the pithed rat, in which the increase in blood pressure is mediated through a stimulation of M1 receptors in sympathetic ganglia, WAL 2014 produced a full dose response curve, whereas the reference compounds RS 86 and arecoline exhibited a bell-shaped behaviour. This is in accord with the view that WAL 2014 selectively activates M1 receptors in sympathetic ganglia, whereas conventional agonists in the same dose range stimulate both ganglionic M1 and vascular M3 receptors. The preferential neuron-stimulating properties were confirmed by EEG recording in the rabbit, in which muscarinic activation occurred at doses similar to those for ganglionic stimulation in the pithed rat. On the other hand, higher doses of WAL 2014 were needed to elicit muscarinic effects in peripheral effector organs, i.e. bradycardia, urinary bladder contraction and increase in airway resistance. It is concluded that WAL 2014 due to its preferential neuronal activity is a promising candidate for a cholinergic substitution therapy in Alzheimers disease.

DAU 6285: A novel antagonist at the putative 5-HT4 receptor

The antagonistic properties of DAU 6285, an azabicycloalkyl benzimidazolone derivative, at putative 5-hydroxytryptamine4 (5-HT4) receptors were investigated in in vitro preparations of guinea-pig ileum and human atrium, in comparison to ICS 205-930. DAU 6285 behaved as a competitive antagonist in all the preparations examined. Its affinity (pA2) ranged between 6.50 and 7.12 in the test models considered. The affinity of ICS 205-930 was 2-3 fold lower. At variance with ICS 205-930, DAU 6285 displayed a weak affinity for 5-HT3 receptors (pKi = 6.1, rat cortex; pA2 less than 5, guinea-pig ileum). In the guinea-pig ileum, DAU 6285 (10 microM) did not exert antimuscarinic, antihistaminic, antinicotinic or myolytic activity. Moreover, it did not bind to other 5-HT receptor subtypes, or to adrenergic, dopaminergic, benzodiazepine, nicotine, GABA receptors. DAU 6285 may represent a suitable tool for studies in the field of 5-HT4 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.

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.

Pharmacological characterization of muscarinic receptors involved in McN‐A‐343‐induced effects on intestinal motility and heart rate in conscious dogs

1 Intravenous injection of the muscarinic agonist, McN‐A‐343, in conscious dogs equipped with an ileal Thiry fistula produced a dose‐related inhibition of intestinal phasic contractile activity, and an increase in heart rate. 2 The inhibitory action of McN‐A‐343 on motility was antagonized with different potencies by antimuscarinic drugs. The non‐selective drug, N‐methylatropine, blocked the McN‐A‐343 effect as well as the reflex phasic activity. The M1‐selective compound, pirenzepine (1–30 μg kg−1), was a potent antagonist of the McN‐A‐343 effect, whereas the cardioselective M2‐antagonist, AF‐DX 116, and the smooth muscle selective compound, 4‐diphenylacetoxy‐N‐methyl piperidine (4‐DAMP), were completely ineffective at the doses tested. 3 The McN‐A‐343‐induced inhibition of intestinal motility was blocked by locally applied lignocaine, suggesting the involvement of a neural inhibitory pathway. The resistance to hexamethonium and (α1‐, α2‐ β−) adrenoceptor blocking drugs excluded transmission through a nicotinic synapse or release of catecholamines. 4 McN‐A‐343‐induced tachycardia was also the result of muscarinic receptor activation. It was very sensitive to antagonism by 4‐DAMP, while being completely unaffected by AF‐DX 116. Pirenzepine displayed an intermediate profile, reducing tachycardia at doses fully active in reversing the agonist‐mediated effect on intestinal motility. Propranolol partially reduced McN‐A‐343 tachycardia, suggesting catecholamine release. 5 The two McN‐A‐343 effects investigated in the present study appear to be mediated by different muscarinic receptor subtypes. While the inhibitory action on intestinal motility results from stimulation of M1‐muscarinic receptors, the tachycardia is mediated by receptors blocked selectively by 4‐DAMP.

Himbacine discriminates between putative muscarinic M1 receptor-mediated responses

This study describes the antagonistic properties of himbacine, in comparison with those of pirenzepine, at muscarinic receptors mediating the depolarization of rat superior cervical ganglion, the inhibition of electrically-induced twitch contractions of rabbit vas deferens and the contraction of dog saphenous vein, currently classified as putative muscarinic M1 sites. The affinity of himbacine for the vas deferens site (pA2 8.08) was nearly ten times higher than those for the M1 receptors of rat ganglion and dog saphenous vein (pA2 7.14 and 7.16, respectively); affinity estimates for pirenzepine were similar throughout the different preparations. The present data are consistent with the allocation of ganglion and saphenous vein receptors into the M1 subclass; the profile of the vas deferens site, conversely, appears to be different, and possibly more closely related to that reported for the M4/m4 receptor.

Cimetropium bromide: in vitro and in vivo evaluation of spasmolytic activity on human and dog colon.

The present study investigates the spasmolytic properties of cimetropium bromide, compared to atropine, on human and canine large bowel. The drug behaved as a competitive antagonist of muscarinic-mediated contractions in isolated colonic preparations from both species, with affinity values (pA2) ranging between 7.41 and 7.82. When administered intravenously to conscious dogs provided with a colonic Thiry fistula, cimetropium was a potent inhibitor of large bowel motility evoked by both exogenous and endogenous stimuli. The compound (10-100 micrograms/kg) counteracted colonic motor response to neostigmine administration with an ID50 of 27.9 micrograms/kg; both tonic and phasic components of contractile response were affected. In a comparable range of doses (3-100 micrograms/kg), the drug inhibited motor activity elicited by intraluminal distension.

Antagonistic properties of McNeil‐A‐343 at 5‐HT4 and 5‐HT3 receptors

1 This study describes the in vitro interaction of the muscarinic ligand McNeil‐A‐343 with two 5‐hydroxytryptamine (5‐HT) receptor subtypes, the 5‐HT4 and 5‐HT3 receptors, using functional as well as radioligand binding studies. 2 In the rat oesophageal muscularis mucosae, precontracted with carbachol, McNeil‐A‐343 was a competitive antagonist (pA2 6.2) of the 5‐HT4 receptor which mediates the relaxation induced by 5‐HT. The compound per se relaxed the oesophagus at high concentration only (≥ 10 μm), an effect unchanged by desensitization of the 5‐HT4 receptor with 10 μm 5‐methoxytryptamine. In the same preparation in the absence of tone, McNeil‐A‐343 displaced the carbachol concentration‐response curve to the right, yielding an apparent affinity (pA2) of 4.9 for muscarinic receptors. 3 In the rat isolated superior cervical ganglion preparation, after blockade of muscarinic and nicotinic receptors, McNeil‐A‐343 caused a concentration‐dependent depolarization that was unaffected by 100 nm ondansetron. The concentration‐fast depolarization curve to 5‐HT, mediated by the 5‐HT3 receptor, was displaced to the right by McNeil‐A‐343, which showed an apparent affinity (pA2) of 4.8 for the 5‐HT3 subtype. 4 In binding studies, McNeil‐A‐343 recognized a single population of 5‐HT4 receptors in pig caudate nucleus, with a pAi of 5.9. The binding affinity of McNeil‐A‐343 for 5‐HT3 receptors in NG 108‐15 cells was approximately four times lower (pK1 5.3). Binding affinities (pK1) for muscarinic receptor subtypes in rat tissues were 5.3 (Mb cortex), 5.2 (M2, heart) and 4.9 (M3, submandibular glands), respectively. 5 McNeil‐A‐343 is an antagonist at 5‐HT4 and 5‐HT3 receptors; the interaction of the compound with these receptor subtypes (notably the 5‐HT4) occurs in a range of concentrations which generally overlaps that relevant to the interaction with muscarinic receptors.

New pyrrolidin-, piperidin- and azepin-2-oxocarboxylic acid esters are preferential M1, M3 muscarinic antagonists. Synthesis and bronchospasmolytic activity

A series of new 3-tropanol and 3-quinuclidinol esters of phenyl-substituted pyrrolidin-, piperidin- and azepin-2-oxocarboxylic acid were synthesized and tested for antimuscarinic activity. The compounds showed a preferential in vitro activity at M1 and M3 receptor subtypes and an interesting activity profile in vivo. A potential use as selective bronchospasmolytic agents has been suggested for selected compounds.