Giovanni Battista Schiavi

Binding profile of a novel cardioselective muscarine receptor antagonist, AF-DX 116, to membranes of peripheral tissues and brain in the rat

The heterogeneity of muscarine receptors was examined in two brain regions (cerebral cortex and cerebellum) and in some parasympathetically innervated peripheral tissues (heart, salivary gland and intraorbital lacrimal gland), by in vitro binding techniques. As a tool, we used a new antimuscarinic compound, AF-DX 116 (see text for structural formula and chemical name). In competition experiments against 3H-N-methylscopolamine (3H-NMS) or 3H-pirenzepine (3H-PZ), AF-DX 116 was found to bind with high affinity to muscarine receptors in the heart and cerebellum (KDs approximately equal to 115 nM), with intermediate affinity to M1 receptors in neuronal tissue (KD = 760 nM) and with low affinity to receptors in exocrine glands (KDs approximately equal to 3200 nM). Its receptor interaction was found to be of the simple, competitive type. Thus, AF-DX 116 shows a novel cardioselective profile. On the basis of the results which demonstrate that the muscarine receptors in the heart and exocrine glands are clearly distinct, it is proposed that these receptors may be subclassified as M2 cardiac type and M2 glandular type muscarine receptors.

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.

Therapeutic potential of CNS-active M2 antagonists: Novel structures and pharmacology

Clinical trials with muscarinic agonists or acetylcholine esterase inhibitors for the treatment of Alzheimers dementia have shown disappointing or equivocal results. An alternative treatment of this disease is the development of drugs which enhance the release of acetylcholine. It is believed, that of the five muscarinic receptor subtypes so far identified in the brain, M2 receptors are located presynaptically in the cortex and hippocampus and upon stimulation inhibit the release of acetylcholine. Based on this hypothesis, we initiated a drug discovery program with the aim of identifying selective and centrally active M2 antagonists which are capable of enhancing cholinergic transmission. These efforts resulted in the successful design and synthesis of novel muscarinic antagonists able to cross the blood brain barrier. Moreover, these compounds show few peripheral effects and possess a superior M2 versus M1 selectivity. The prototype of this novel class of M2 selective compounds, BIBN 99, could be a valuable tool to test the hypothesis that lipophilic M2 antagonists show beneficial effects in the treatment of cognitive disorders.

Characterization of BIBN 99: a lipophilic and selective muscarinic M2 receptor antagonist.

The present study was designed to characterize the receptor selectivity profile of the novel muscarinic M2 receptor antagonist BIBN 99 (5,11-dihydro-8-chloro-11-[[4-[3-[(2,2-dimethyl-1- oxopentyl)ethylamino]propyl]-1-piperidinyl]acetyl]-6H- pyrido[2,3-b][1,4]benzodiazepin-6-one). In radioligand binding studies BIBN 99 showed high affinity for m2/M2 sites (pKi = 7.52/7.57), intermediate affinity for m4 sites (pKi = 6.76) and low affinity for m1/M1 (pKi = 5.97/6.17), m3/M3 (pKi = 6.11/6.04) and m5 sites (pKi = 5.84). Functional studies in vitro showed BIBN 99 to be a competitive antagonist and to have an 11- to 25-fold higher affinity for M2 receptors than for putative M1 receptors in the rabbit vas deferens or M3 receptors in guinea-pig trachea. In vivo studies revealed that BIBN 99 is able to cross the blood-brain barrier, and although showing an approximately 3-fold higher affinity for M2 binding sites BIBN 99 appeared to be 7- to 18-fold less potent than AF-DX 116 in inhibiting muscarinic agonist or vagally induced bradycardia in rats and guinea-pigs. The results show that BIBN 99 is the first lipophilic muscarinic M2 receptor antagonist to have remarkable M2 versus M1 selectivity (30-fold). In addition, BIBN 99 possesses central nervous system activity and only minor peripheral cardiac effects.

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.

Chapter 21 Pharmacological muscarinic receptor subtypes

Publisher Summary This chapter discusses the pharmacological muscarinic receptor subtypes. The introduction of several selective muscarinic receptor antagonists has permitted the classification of muscarinic receptors into three pharmacological receptor subtypes, M1, M2, and M3, with indications for a fourth type, and has played a role in prompting the discovery of the molecular forms of the receptor. The distribution of muscarinic receptor subtypes in body tissues are described by hybridization techniques and has provided insight in elucidating the pharmacologically defined receptors present in smooth muscle and striatum, using pirenzepine (PZ), AF-DX 116, hexahydrosiladifenidol (HHSiD), and methoctramine (METH). Affinity constants for equilibrium binding of PZ to muscarinic receptors in membranes from the brain and peripheral tissues are presented in the chapter. The tissue selectivity profile correlated well with the pharmacological activity of the drug determined in vivo, and in vitro on isolated organ preparations. The property of PZ to discriminate highly between the M1 and M2 receptors lead to the detection of heterogeneity of receptors in the rat striatum and to the characterization of the receptor subtypes with other selective compounds.

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.

The role of electronic and conformational properties in the activity of 5-HT3 receptor antagonists

Abstract The conformational and electronic properties of 11 5-HT 3 receptor antagonists, were investigated to rationalize their biological activity. All compounds fulfill the previously reported geometrical requirements for the pharmacophore, although they show a wide spectrum of activities. Quantitative structure-activity relationships (QSAR) models were searched using the cross-validated partial least squares technique. The results of the QSAR analysis show that a unique model can rationalize both the in vitro (affinity, K d ) and the in vivo (50% inhibitory dose, ID 50 ) activities. This model includes the value of the molecular electrostatic potential minimum above the aromatic fragment, used as an index of the π-electron density, as well as the relative population of the anti-periplanar (app, the “active”) conformation. Two other models were also obtained that include electrophilic superdelocalizability indexes. Although these models give a more satisfactory value of the cross-validated correlation coefficient ( R 2 cv ), they represent two different models for K d and ID 50 which is in contrast with the experimental evidence that a correlation exists between these two activities. It is concluded that the use of electronic and conformational properties in addition to the geometrical characteristics may better describe and predict the activity of 5-HT 3 receptor antagonists, than the use of geometrical characteristics alone.

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.