Eugenia Monferini

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.

BIMG 80, a novel potential antipsychotic drug: evidence for multireceptor actions and preferential release of dopamine in prefrontal cortex.

Abstract: In radioligand binding studies, BIMG 80, a new putative antipsychotic, displayed good affinity at certain serotonin (5‐HT1A, 5‐HT2A, 5‐HT6), dopamine (D1, D2L, D4), and noradrenergic (α1) receptors. The effect of acute subcutaneous BIMG 80, clozapine, haloperidol, risperidone, amperozide, olanzapine, and Seroquel was then investigated on dopamine release in medial prefrontal cortex, nucleus accumbens, and striatum in freely moving rats using the microdialysis technique. Four different neurochemical profiles resulted from the studies: (a) Systemic administration of BIMG 80, clozapine, and amperozide produced greater percent increases in dopamine efflux in medial prefrontal cortex than in the striatum or the nucleus accumbens. (b) Haloperidol induced a similar increase in dopamine concentrations in the striatum and nucleus accumbens with no effect in the medial prefrontal cortex. (c) Risperidone and olanzapine stimulated dopamine release to a similar extent in all brain regions investigated. (d) Seroquel failed to change significantly dopamine output both in the medial prefrontal cortex and in the striatum. Because an increase in dopamine release in the medial prefrontal cortex may be predictive of effectiveness in treating negative symptoms and in the striatum may be predictive of induction of extrapyramidal side effects, BIMG 80 appears to be a potential antipsychotic compound active on negative symptoms of schizophrenia with a low incidence of extrapyramidal side effects.

Muscarinic receptor heterogeneity in guinea pig intestinal smooth muscle: binding studies with AF-DX 116

Muscarinic receptor subtypes in longitudinal and circular smooth muscles of the guinea pig ileum were characterized with the use of the cardioselective antagonist AF-DX 116 in binding competition experiments against 0.3 nM [3H] N-methylscopolamine [( 3H]NMS). This compound recognized a heterogeneous receptor population in both smooth muscles, revealing the existence of different percentages of the cardiac (KD = 92-110 nM) and the glandular (KD = 1150-2541 nM) muscarinic receptor subtypes. These results, together with the low potency displayed by AF-DX 116 to inhibit the agonist-stimulated smooth muscle contraction and salivary secretion allow the suggestion that the glandular muscarinic receptor subtype, showing a low affinity for AF-DX 116, is involved in smooth muscle contraction.

Characterization of a novel 5-HT4 receptor antagonist of the azabicycloalkyl benzimidazolone class: DAU 6285

SummaryThree chemical classes of serotonin 5-HT4 receptor agonists have been identified so far: 5-substituted indoles (e.g. 5-HT), benzamides (e.g. renzapride) and benzimidazolones (e.g. BIMU 8). In a search for 5-HT4 receptor antagonists, we have discovered that the benzimidazolone derivative DAU 6285 (for structure see text), is 3–5 times more potent than tropisetron in blocking 5-HT, renzapride and BIMU 8 induced stimulation of adenylate cyclase activity in mouse embryo colliculi neurons. Schild plot analysis yielded Ki values of 220, 181 and 255 nmol/l, respectively. In addition, DAU 6285 showed poor activity as a 5-HT3 receptor ligand with respect to tropisetron, as demonstrated by in vitro binding studies (Ki, 322 vs 2.8 nmol/l) and by its antagonistic activity in the Bezold-Jarisch reflex test (ID50, 231 vs 0.5 μg/kg, i.v.). No significant binding (Ki>10 μmol/l) of DAU 6285 to serotonergic 5-HT1A, 5-HT1B, 5-HT1C, 5-HT1D, and 5-HT2 receptors as well as to adrenergic α1, α2, dopaminergic D1, D2 or muscarinic M1–M3 receptor subtypes was found. The data indicate that DAU 6285 has a somewhat higher affinity than tropisetron for 5-HT4 receptors, a property confirmed in functional tests, and much lower affinity than tropisetron for 5-HT3 receptors. The compound represents a new interesting tool for investigating the pharmacological and physiological properties of 5-HT4 receptors.

Azabicycloalkyl benzimidazolone derivatives as a novel class of potent agonists at the 5-HT4 receptor positively coupled to adenylate cyclase in braina

SummaryRecent experimental evidence indicates that central 5-HT4 receptors which are positively coupled to adenylate cyclase, are stimulated by a family of 2-methoxy-4-amino-5-chloro substituted benzamide derivatives. These compounds are also potent stimulants of the gastro-intestinal motility. In this study the ability of three azabicycloalkyl benzimidazolone derivatives, BIMU 1, BIMU 8, and DAU 6215 (structural formulas are given in the text), to stimulate cAMP formation in colliculi neurons in primary culture have been tested. Two of the compounds, BIMU 1 and BIMU 8, which show prokinetic activity in various animal models, were also good agonists at the 5-HT4 receptors, whereas DAU 6215, a drug devoid of prokinetic activity, was only a weak, partial agonist at 5-HT4 receptors.The rank order of their potencies as compared with those of 5-HT and cisapride was as follows: BIMU 8 = cisapride > 5-HT > BIMU 1 > DAU 6215. The efficacies of BIMU 8 and cisapride were comparable (133 ± 9% and 124 ± 8% of the maximal 5-HT efficacy, respectively), whereas BIMU 1 and DAU 6215 elicited, respectively, only 72 ± 11 % and 16 ± 4% of the maximal 5-HT effect. The activities of the azabicycloalkyl benzimidazolone derivatives and 5-HT on cAMP formation were not additive and ICS 205–930 antagonized the stimulatory effect of these compounds with low potency (pKi = 6.1–6.4), further strengthening the notion of interaction with 5-HT4 receptors. In addition, cross desensitization between the effects of 5-HT and the azabicycloalkyl benzimidazolones on adenylate cyclase was noted, another argument in favor of an interaction of these drugs on 5-HT4 receptors.

Characterization of the muscarinic receptor subtypes in the rat urinary bladder

We investigated the nature of the muscarinic receptors present in the rat urinary bladder by performing binding studies with various selective (pirenzepine, AF-DX 116, hexahydrosiladifenidol, benzhexol, 4-diphenyl-acetoxy-N-methyl piperidine methiodide, dicyclomine, secoverine) and classical (N-methylscopolamine, atropine) antagonists. Competition experiments were carried out against [3H]N-methyl scopolamine at 30 degrees C in Na+/Mg2+ HEPES buffer; non-specific binding was determined in the presence of 1 microM 3-quinuclidinyl benzilate. Of all the antagonists examined, only AF-DX 116 exhibited a heterogeneous binding profile (nH less than 1). Computer-assisted analysis showed that the data fitted best to a two-binding site model, revealing the existence of high and low affinity receptors. The affinity values of AF-DX 116, determined in binding experiments carried out in heart and gland homogenates, allowed us to classify the rat urinary bladder receptors into cardiac and glandular subtypes. We suggest that the glandular receptor subtype is involved in smooth muscle contraction, since AF-DX 116 was equally potent in inhibiting smooth muscle contraction and the secretion of saliva.

BIMT 17, a 5-HT2A receptor antagonist and 5-HT1A receptor full agonist in rat cerebral cortex

In the search for antidepressant agents with a rapid onset of action, we have found that compound BIMT 17 (1-[2-[4-(3-trifluoromethylphenyl)piperazin1-yl]ethyl]benzimidazol-[1H]-2-one) shows a good affinity for cerebral cortical 5-HT1A (pKi = 7.72) and 5-HT2A (pKi = 6.90) receptors, with no appreciable affinity for the other 5-HT receptor subtypes, including 5-HT2C. BIMT 17 reduced forskolin-stimulated cAMP accumulation in the cerebral cortex (pEC50 = 6.09) and in the hippocampus (pEC50 = 6.50), and antagonized 5-HT-induced phosphatidylinositol turnover (pKi = 6.96) in the cerebral cortex. The effect on cAMP accumulation was blocked by the 5-HT1A receptor antagonist tertatolol. Buspirone, 8-OH-DPAT and S 14671 {1-[2-(2-thenoylamino)ethyl]-4[1-(7-methoxynaphtyl)]piperazine, claimed to be 5-HT1A receptor agonists, did not reduce forskolin-stimulated cAMP formation in the cerebral cortex.On the basis of these data, it was concluded that BIMT 17 was the only compound that behaved as a full agonist with respect to the CAMP response in the cortex, while exerting concurrent agonism at 5-HT1A receptors and antagonism at 5-HT2A receptors. These characteristics might explain the peculiar behaviour of BIMT 17 in mimicking the inhibitory action of 5-HT on the basal firing rate of the cortical neurons (see accompanying paper).

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.

Characterization of muscarinic receptors in salivary and lacrimal glands of the rat.

The binding characteristics of muscarinic receptors in rat salivary and lacrimal glands were studied by means of radioligand binding techniques. In competition experiments against [3H]N-methylscopolamine, classical muscarinic antagonists ipratropium bromide, N-methylscopolamine and N-methylatropine exhibited very similar KD values in all the glands and their binding behavior was well described by a one binding site model (nH congruent to 1). The novel cardioselective antimuscarinic compound, AF-DX 116, displayed an equally low affinity in all the tissues examined. Pirenzepine and dicyclomine, two other selective muscarinic antagonists, showed a similar behaviour in all but the sublingual gland, where their binding profile indicated the presence of a heterogeneous receptor population (nH = 0.74 and 0.84, respectively). Histological studies of the sublingual-submandibular glandular complex demonstrated the presence of ganglionic structures mainly located in the hilum of the sublingual-submandibular glandular complex connected with the sublingual gland. Binding studies carried out with pirenzepine on the hilum and on a synaptosomal preparation from this region again revealed the presence of two populations of muscarinic receptors with KD values of 22-25 and 270-463 nM. These results are best explained by the presence of M1 and M2 receptors located on neuronal and glandular structures.

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.