Tiotropium bromide (Ba 679 BR), a novel long-acting muscarinic antagonist for the treatment of obstructive airways disease

Tiotropium bromide (Ba 679 BR) is a novel potent and long-lasting muscarinic antagonist that has been developed for the treatment of chronic obstructive airways disease (COPD). Binding studies with [3H]tiotropium bromide in human lung have confirmed that this is a potent muscarinic antagonist with equal affinity for M1-, M2- and M3-receptors and is approximately 10-fold more potent than ipratropium bromide. Tiotropium bromide dissociates very slowly from lung muscarinic receptors compared with ipratropium bromide. In vitro tiotropium bromide has a potent inhibitory effect against cholinergic nerve-induced contraction of guinea-pig and human airways, that has a slower onset than atropine or ipratropium bromide. After washout, however, tiotropium bromide dissociates extremely slowly compared with the dissociation of atropine and ipratropium bromide. Measurement of acetylcholine (ACh) release from guinea-pig trachea shows that tiotropium bromide, ipratropium bromide and atropine all increase ACh release on neural stimulation and that this effect is washed out equally quickly for the three antagonists. This confirms binding studies to transfected human muscarinic receptors which suggested that tiotropium bromide dissociates slowly from M3-receptors (on airway smooth muscle) but rapidly from M2 autoreceptors (on cholinergic nerve terminals). Clinical studies with inhaled tiotropium bromide confirm that it is a potent and long-lasting bronchodilator in COPD and asthma. Furthermore, it protects against cholinergic bronchoconstriction for > 24 h. This suggests that tiotropium bromide will be a useful bronchodilator, particularly in patients with COPD, and may be suitable for daily dosing. The selectivity for M3- over M2-receptors may also confer a clinical advantage.     

Acetylcholine-induced proliferation of fibroblasts and myofibroblasts in vitro is inhibited by tiotropium bromide

Acetylcholine (ACh) has been suggested to exert various pathophysiological activities in the airways in addition to vagally-induced bronchoconstriction. This archetypal neurotransmitter and other components of the cholinergic system are expressed in a number of non-neuronal cells in the airways. Non-neuronal ACh released from these cells may affect fibroblasts (Fb) as well as inflammatory cells in lung tissue. Tiotropium bromide is a once-a-day antimuscarinic drug, marketed under the brand name Spiriva, for the treatment of chronic obstructive pulmonary disease (COPD). Besides its proven direct bronchodilatory activity, recent evidence suggests that tiotropium may be able to reduce the frequency of exacerbations and attenuate the decline in lung function, thus improving the course of obstructive airway diseases. The aim of the present study was to investigate the effects of tiotropium on the ACh-induced proliferation of primary human Fb isolated from biopsies of lung fibrosis patients and myofibroblasts (MyFb) derived from these cells. A human lung Fb cell line acted as control. Expression of muscarinic receptor subtypes M1, M2 and M3 was demonstrated by RT-PCR in both cell types. Acetylcholine stimulated proliferation in all cells investigated. Tiotropium concentration-dependently inhibited the ACh-induced proliferation in both the Fb and MyFb with a maximum effect at 30 nM. These results suggest that cholinergic stimuli mediated by muscarinic receptors could contribute to remodeling processes in chronic airway disease. Tiotropium bromide may have a beneficial influence on airway remodeling processes in chronic airway diseases through antiproliferative effects on fibroblasts and myofibroblasts.     

The in vitro dissociation kinetics of (R,R)-[125I]4IQNB is reflected in the in vivo washout of the radioligand from rat brain.

We have determined the kinetics of dissociation of (R)-3-Quinuclidinyl (R)-4-[125I]Iodobenzilate ((R,R)-[125I]4IQNB) from muscarinic acetylcholine receptor preparations from the cortex, hippocampus, caudate/putamen, thalamus, pons and colliculate bodies. The dissociation curves are well described by a biexponential function and are consistent with subtype selectivity favoring slow dissociation from the M1, M3, and M4 receptors with a 20-fold faster dissociation rate for the M2 receptor. Following intravenous injection, (R,R)-[125I]4IQNB binds to receptor in the rat brain in concentrations which reflect the receptor concentration present in a structure. We determined the extent of radioligand present at two times, 2 and 24 hrs, as an indication of the relative proportions of m-AChR which exhibits rapid vs. slow dissociation of (R,R)-[125I]4IQNB. A good correlation between in vitro and in vivo results suggests that the relative populations of receptor subtypes can be imaged using in vivo pharmacokinetics of (R,R)-[125I]4IQNB.     

Effects of inflammatory cells on neuronal M2 muscarinic receptor function in the lung

In the lungs, acetylcholine released from the parasympathetic nerves stimulates M3 muscarinic receptors on airway smooth muscle inducing contraction and bronchoconstriction. The amount of acetylcholine released from these nerves is limited locally by neuronal M2 muscarinic receptors. These neuronal receptors are dysfunctional in asthma and in animal models of asthma. Decreased M2 muscarinic receptor function results in increased release of acetylcholine and in airway hyperreactivity. Inflammation has long been associated with hyperreactivity and the role of inflammatory cells in loss of neuronal M2 receptor function has been examined. There are several different mechanisms for loss of neuronal M2 receptor function. These include blockade by endogenous antagonists such as eosinophil major basic protein, decreased expression of M2 receptors following infection with viruses or exposure to pro inflammatory cytokines such as gamma interferon. Finally, the affinity of acetylcholine for these receptors can be decreased by exposure to neuraminidase.     

In vivo demonstration of M3 muscarinic receptor subtype selectivity of darifenacin in mice

A novel muscarinic receptor antagonist, darifenacin, inhibited specific binding of [N-methyl-(3)H]scopolamine ([(3)H]NMS) in the mouse bladder, submaxillary gland and heart in a concentration-dependent manner. The inhibitory effect was most potent in the submaxillary gland, followed by the bladder and heart. In addition, darifenacin inhibited specific [(3)H]NMS binding in the membranes of CHO-K1 cell lines expressing muscarinic M(2) and M(3) receptor subtypes, and the potency was significantly (22-fold) greater at the M(3) than at the M(2) subtype. At 0.5 to 12 h after oral administration of darifenacin, a significant increase in K(d) values for specific [(3)H]NMS binding was seen in the bladder, submaxillary gland and lung of mice, compared with control values. Also, there was a sustained decrease in the B(max) values in the submaxillary gland. These data suggest that muscarinic receptor binding of oral darifenacin is rapid in onset and of a long duration. On the other hand, oral darifenacin exerted only temporary or little binding of muscarinic receptors in the heart and colon. Pilocarpine-induced salivary secretion in mice was continuously suppressed by oral darifenacin. The time-course of suppression coincided well with that for the muscarinic receptor binding in the submaxillary gland. The antagonistic effect of darifenacin against the dose-response curves for pilocarpine appeared to be insurmountable. In conclusion, the present study has shown that oral darifenacin may exert a pronounced and long-lasting binding of muscarinic receptors in tissues expressing the M(3) subtype.     

The pharmacological action of MT-7

The mamba toxin MT-7 is the most selective ligand currently available for the muscarinic M1 receptor subtype. The toxin binds stably to the receptor and blocks the agonist-induced activation non-competitively. Although its mode of action on M1 receptors is not yet fully understood, some of the toxin properties support an allosteric mechanism. Thus, the toxin fails to elicit a complete inhibition of the binding of either the muscarinic antagonist [3H]N-methyl-scopolamine ([3H]NMS) or the agonist [3H]acetylcholine ([3H]ACh). When added to ligand-occupied M1 receptors, the toxin slows the dissociation rate of [3H]NMS and increases that of [3H]ACh. Site-directed mutagenesis studies have provided important information about the toxin amino acid residues which are critical for the stable binding to the receptor and for the allosteric modulation of antagonist dissociation. In vivo studies have shown that the intracerebral injection of MT-7 causes a long-lasting blockade of M1 receptor, thus providing a tool for the characterization of the functional role of this receptor subtype in discrete brain areas.     

Studies on Muscarinic Binding Sites in Human Brain Identified with [3H]Pirenzepine

Pirenzepine, a potent antimuscarinic agent with apparent selectivity for a subtype (M1) of muscarinic receptors, was used in tritiated form to characterize its binding to human brain tissue. Specific [3H]pirenzepine binding showed rapid association and dissociation. From kinetic and competitive binding experiments, its KD was 5.5 nM and 9 nM, respectively. Regional distribution of [3H]pirenzepine binding determined in parallel with [3H]quinuclidinyl benzilate binding, a nonselective muscarinic antagonist, indicated a significant correlation for the maximum number of binding sites for the two radioligands in 13 brain regions, with the highest amount of binding for each in the putamen and the least in the cerebellum. Binding for [3H]pirenzepine averaged 57% of that for [3H]quinuclidinyl benzilate, with a range of 20% (cerebellum) to 77% (frontal cortex). Most antidepressants and neuroleptics tested had affinities for [3H]pirenzepine binding sites that were not significantly different from their previously reported values obtained with the use of [3H]quinuclidinyl benzilate.     

Deoxamuscaroneoxime derivatives as useful muscarinic agonists to explore the muscarinic subsite: demox, a modulator of orthosteric and allosteric sites at cardiac muscarinic M2 receptors

A series of muscarinic agonists, straight chained, branched, cyclic alkyl and aromatic derivatives of the oxime 1 (demox) was designed with the aim of investigating their activity on muscarinic receptor subtypes. Effects on M1 receptor were assessed functionally by a microphysiometer apparatus, while M2, M3, and M4 receptor potency and affinity were studied on isolated preparations of guinea pig heart, ileum, and lung, respectively. The results suggest that the substitution of a hydrogen with a long side-chain or bulky group generally induces a decrease in potency at M1 and M3 subtypes, while a general increase in this parameter is obtained at M2 subtype. Among the agonists 2-18, compound 4 behaves as a full agonist with a preference for M3 subtype. Moreover, compound 12 is inactive at M1 and M4 receptors while it displays a full agonist activity at M2 and M3 subtypes. Since demox displays a variable response on cardiac M2 receptors regulating heart force, an in-depth inquiry of the functional behaviour of this compound was carried out at M2 receptors. In presence of 10(-11) and 10(-10) M demox, the binding of [3H]-NMS was increased by approximately 30% as a consequence of an increase of the association of [3H]-NMS to membranes; this effect was not observed in presence of a higher concentration of [3H]-NMS. Higher concentrations of demox decreased the binding of [3H]-NMS to heart atrial membranes but significantly retarded the dissociation of this radioligand. Our results suggest that demox may interact with orthosteric and allosteric sites of atrial M2 muscarinic receptor.     

Evidence for a tandem two-site model of ligand binding to muscarinic acetylcholine receptors

After short preincubations with N-[(3)H]methylscopolamine ([(3)H]NMS) or R(-)-[(3)H]quinuclidinyl benzilate ([(3)H]QNB), radioligand dissociation from muscarinic M(1) receptors in Chinese hamster ovary cell membranes was fast, monoexponential, and independent of the concentration of unlabeled NMS or QNB added to reveal dissociation. After long preincubations, the dissociation was slow, not monoexponential, and inversely related to the concentration of the unlabeled ligand. Apparently, the unlabeled ligand becomes able to associate with the receptor simultaneously with the already bound radioligand if the preincubation lasts for a long period, and to hinder radioligand dissociation. When the membranes were preincubated with [(3)H]NMS and then exposed to benzilylcholine mustard (covalently binding specific ligand), [(3)H]NMS dissociation was blocked in wild-type receptors, but not in mutated (D99N) M(1) receptors. Covalently binding [(3)H]propylbenzilylcholine mustard detected substantially more binding sites than [(3)H]NMS. The observations support a model in which the receptor binding domain has two tandemly arranged subsites for classical ligands, a peripheral one and a central one. Ligands bind to the peripheral subsite first (binding with lower affinity) and translocate to the central subsite (binding with higher affinity). The peripheral subsite of M(1) receptors may include Asp-99. Experimental data on [(3)H]NMS and [(3)H]QNB association and dissociation perfectly agree with the predictions of the tandem two-site model.     

Effect of M2 and M3 muscarinic receptors on airway responsiveness to carbachol in bronchial-hypersensitive (BHS) and bronchial-hyposensitive (BHR) guinea pigs.

The expression balance of M2 and M3 muscarinic receptor subtypes on the pathogenesis of airway hyperresponsiveness was investigated by using two congenitally related strains of guinea pigs, bronchial-hypersensitive (BHS) and bronchial-hyposensitive (BHR). CCh-induced airway responses in vivo and in vitro were investigated by comparing the effects of muscarinic receptor subtype antagonists, and the relative amounts of M2 and M3 muscarinic receptor mRNA in tracheal smooth muscle and lung tissue were investigated. After treatment with muscarinic receptor subtype antagonists, the ventilatory mechanics (VT, Raw, and Cdyn) of response to CCh aerosol inhalation were measured by the bodyplethysmograph method. The effects of these antagonists on CCh-induced tracheal smooth muscle contraction were also investigated. The effects of M2 muscarinic receptor blockade were less but the effects of M3 muscarinic receptors blockade on the airway contractile responses were greater in BHS than in BHR. In M3 muscarinic receptor blockades, CCh-induced tracheal contractions in BHS were significantly greater than those in BHR. In tracheal smooth muscle from BHS, the relative amount of M2 muscarinic receptors mRNA was less but that of M3 muscarinic receptor mRNA was more than those in BHR. These results suggest that the high ACh level as a consequence of dysfunction of M2 muscarinic autoreceptors and the excessive effect of M3 muscarinic receptors on the airway smooth muscle may play an important role in the pathogenesis of airway hyperresponsiveness.     

Pharmacologic characterization of muscarinic receptors of insect brains.

Muscarinic receptors in brain membranes from honey bees, houseflies, and the American cockroach were identified by their specific binding of the non-selective muscarinic receptor antagonist [3H]quinuclidinyl benzilate ([3H]QNB) and the displacement of this binding by agonists as well as subtype-selective antagonists, using filtration assays. The binding parameters, obtained from Scatchard analysis, indicated that insect muscarinic receptors, like those of mammalian brains, had high affinities for [3H]QNB (KD = 0.47 nM in honey bees, 0.17 nM in houseflies and 0.13 nM in the cockroach). However, the receptor concentration was low (108, 64.7, and 108 fmol/mg protein for the three species, respectively). The association and dissociation rates of [3H]QNB binding to honey bee brain membranes, sensitivity of [3H]QNB binding to muscarinic agonists, and high affinity for atropine were also features generally similar to muscarinic receptors of mammalian brains. In order to further characterize the three insect brain muscarinic receptors, the displacement of [3H]QNB binding by subtype-selective antagonists was studied. The rank order of potency of pirenzepine (PZ), the M1 selective antagonist, 11-[2-[dimethylamino)-methyl)1-piperidinyl)acetyl)-5,11- dihydro-6H-pyrido(2,3-b)-(1,4)-benzodiazepin-6 one (AF-DX 116), the M2-selective antagonist, and 4-DAMP (4-diphenylacetoxy-N-methylpiperidine methiodide) the M3-selective antagonist, was also the same as that of mammalian brains, i.e., 4-DAMP greater than PZ greater than AF-DX 116. The three insect brain receptors had 27-50-fold lower affinity for PZ (Ki 484-900 nM) than did the mammalian brain receptor (Ki 16 nM), but similar to that reported for the muscarinic receptor subtype cloned from Drosophila. Also, the affinity of insect receptors for 4-DAMP (Ki 18.9-56.6 nM) was much lower than that of the M3 receptor, which predominates in rat submaxillary gland (Ki of 0.37 nM on [3H]QNB binding). These drug specificities of muscarinic receptors of brains from three insect species suggest that insect brains may be predominantly of a unique subtype that is close to, though significantly different from, the mammalian M3 subtype.     

Molecular and pharmacological characterization of muscarinic receptors in retinal pigment epithelium: role in light-adaptive pigment movements

Muscarinic receptors are the predominant cholinergic receptors in the central and peripheral nervous systems. Recently, activation of muscarinic receptors was found to elicit pigment granule dispersion in retinal pigment epithelium isolated from bluegill fish. Pigment granule movement in retinal pigment epithelium is a light-adaptive mechanism in fish. In the present study, we used pharmacological and molecular approaches to identify the muscarinic receptor subtype and the intracellular signaling pathway involved in the pigment granule dispersion in retinal pigment epithelium. Of the muscarinic receptor subtype-specific antagonists used, only antagonists specific for M1 and M3 muscarinic receptors were found to block carbamyl choline (carbachol)-induced pigment granule dispersion. A phospholipase C inhibitor also blocked carbachol-induced pigment granule dispersion, and a similar result was obtained when retinal pigment epithelium was incubated with an inositol trisphosphate receptor inhibitor. We isolated M2 and M5 receptor genes from bluegill and studied their expression. Only M5 was found to be expressed in retinal pigment epithelium. Taken together, pharmacological and molecular evidence suggest that activation of an odd subtype of muscarinic receptor, possibly M5, on fish retinal pigment epithelium induces pigment granule dispersion.     

Different antagonist binding properties of rat pancreatic and cardiac muscarinic receptors

The antagonist binding properties of rat pancreatic and cardiac muscarinic receptors were compared. In both tissues pirenzepine (PZ) had a low affinity for muscarinic receptors labelled by (3H)N-methylscopolamine [3)NMS) (KD values of 140 and 280 nM, respectively, in pancreatic and cardiac homogenates). The binding properties of pancreatic and cardiac receptors were, however, markedly different. This was indicated by different affinities for dicyclomine, (11-([(2-[diethylamino)-methyl)-1-piperidinyl] acetyl)-5, 11-dihydro-6H-pyrido(2,3-b)(1,4) benzodiazepin-6-on) (AFDX-116), 4-diphenylacetoxy-N-methyl-piperidine methobromide (4-DAMP) and hexahydrosiladifenidol (HHSiD). Pancreatic and cardiac muscarinic receptors also showed different (3H)NMS association and dissociation rates. These results support the concept of M2 receptor heterogeneity and confirm that M2 receptor subtypes have different binding kinetic properties.     

The Cu-Zn superoxide dismutase (SOD1) inhibits ERK phosphorylation by muscarinic receptor modulation in rat pituitary GH3 cells

The Cu-Zn superoxide dismutase (SOD1) belongs to a family of isoenzymes that are able to dismutate the oxygen superoxide in hydrogen peroxide and molecular oxygen. This enzyme is secreted by many cellular lines and it is also released trough a calcium-dependent depolarization mechanism involving SNARE protein SNAP 25. Using rat pituitary GH3 cells that express muscarinic receptors we found that SOD1 inhibits P-ERK1/2 pathway trough an interaction with muscarinic M1 receptor. This effect is strengthened by oxotremorine, a muscarinic M agonist and partially reverted by pyrenzepine, an antagonist of M1 receptor; moreover this effect is independent from increased intracellular calcium concentration induced by SOD1. Finally, P-ERK1/2 inhibition was accompanied by the reduction of GH3 cell proliferation.These data indicate that SOD1 beside the well studied antioxidant properties can be considered as a neuromodulator able to affect mitogen-activated protein kinase in rat pituitary cells trough a M1 muscarinic receptor.     

Design, synthesis and antimuscarinic activity of some imidazolium derivatives

A series of imidazolium salt derivatives was prepared as part of a search for subtype-selective antimuscarinic agents. On the basis of measurements of the antimuscarinic activity and subtype-selectivity for M2 and M3 muscarinic receptors, the structure-activity relationships of these compounds are discussed.     

Exploring the potential for subtype-selective muscarinic agonists in glaucoma

Pilocarpine has been used to lower intraocular pressure (IOP) in glaucoma patients for more than 100 years. Since the identification of five muscarinic receptor subtypes, there has been an interest in separating the IOP-lowering effects from the ocular side effects of pupil constriction and lens accommodation. However, all these actions seem to be mediated by the M3 receptor. A novel muscarinic receptor agonist, AGN 199170, that has no activity on the M3 subtype was compared to pilocarpine in a monkey glaucoma model. This compound lowered IOP suggesting that muscarinic agonists targeted at muscarinic receptors other than the M3 subtype may be able to selectively lower IOP.     

Muscarinic binding to mouse brain receptor sites

In mouse brain the binding of [³H]-Atropine to the muscarinic receptor seems to be a simple mass-action determined process as gauged both by approach to equilibrium kinetics and binding at equilibrium. In contrast, using isotopic dilution technique, dissociation measurements indicate the existence of two receptor-ligand complexes. It would appear that association and dissociation rates of binding of the muscarinic antagonists atropine, scopolamine, N-methyl-4-piperidyl benzilate (4NMPB) and 3-quinuclidinyl benzilate (QNB) decrease with increasing affinity based on comparisons of kinetic binding data. The differences between the association rate constants are small whereas those between the dissociation rate constants differ markedly. This kinetic behavior is similar to the well-known time profile of antimuscarinic activity in isolated tissues. These phenomena are discussed in terms of possible isomerization of the receptor-ligand complex, as has been proposed recently for [³H]-scopolamine and [³H]-4NMPB binding.     

A novel subtype of muscarinic receptor identified by homology screening

A new member of the protein superfamily of G-protein coupled receptors has been isolated by homology screening. By virtue of its homology with other muscarinic acetylcholine receptors and its ability to bind muscarinic specific antagonists, this muscarinic receptor subtype is designated M4. The M4 mRNA is preferentially expressed in certain brain regions. The existence of multiple receptor subtypes encoded by distinct genes in the brain has functional implications for the molecular mechanisms underlying information transmission in neuronal networks.     

Binding of radioiodinated SPECT ligands to transfected cell membranes expressing single muscarinic receptor subtypes

The equilibrium dissociation constant and the kinetic rate constants were determined for the binding of (R)-[3H]3-quinuclidinyl benzilate ([3H]QNB) and [125I]3-quinuclidinyl-4-iodobenzilate ((R,R)- and (R,S)-[125I]IQNB) to transfected cell membranes expressing one single muscarinic acetylcholine receptor (mAChR) subtype. The association and dissociation kinetics for the m2 subtype were more rapid than for the m1 and m3 subtypes. The differential kinetic properties may be useful for the single photon emission computed tomographic (SPECT) evaluation of regional mAChR subtype alterations in disease states.     

MAPK pathway mediates muscarinic receptor-induced human lung fibroblast proliferation

Airway remodelling is a pathological feature of chronic inflammatory and obstructive airway diseases like asthma and COPD wherein fibroblasts contribute to structural alteration processes. We recently reported expression of multiple muscarinic receptors in human lung fibroblasts and demonstrated muscarinic receptor-induced, G(i)-mediated proliferation in these cells. We now explore the underlying intracellular signalling pathways. As a measure of cell proliferation ((3)H)-thymidine incorporation in primary human lung fibroblasts and MRC-5 fibroblasts was increased by about 2 fold in presence of the muscarinic receptor agonist carbachol (10 microM) and this effect could be prevented by the MEK inhibitor PD 98059 (30 microM). Western blot analysis revealed a rapid (within 2 min) activation of p42/44 MAPK (ERK1, ERK2) following exposure to 10 microM carbachol or oxotremorine, effects blocked by tiotropium as well as atropine. In conclusion, the proliferative response of lung fibroblasts to muscarine receptor stimulation is mediated via activation of the classical MEK-ERK MAPK cascade. It is suggested that prevention of cholinergic driven fibroblast proliferation by prolonged blockade of airway muscarinic receptors may contribute to the reported long term beneficial effects of anticholinergics.