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The Proceedings of the American Thoracic Society 2:297-304 (2005)
© 2005 The American Thoracic Society

Cholinergic Pathways in the Lungs and Anticholinergic Therapy for Chronic Obstructive Pulmonary Disease

Kristen E. Belmonte

Respiratory and Inflammation Centre of Excellence in Drug Discovery, GlaxoSmithKline, King of Prussia, Pennsylvania

Correspondence and requests for reprints should be addressed to Kristen E. Belmonte, Ph.D., Neuroscience Drug Discovery, Merck Research Laboratories, WP26A-2000, 770 Sumneytown Pike, West Point, PA 19486. E-mail: kristen_belmonte{at}merck.com


   ABSTRACT
TOP
 ABSTRACT
MUSCARINIC RECEPTORS IN THE...
 MECHANISMS OF mAChR DYSFUNCTION...
 ANTICHOLINERGIC BRONCHODILATOR...
 CONCLUSIONS
 REFERENCES
 
Abundant data from animal models and humans support the hypothesis that changes at the level of parasympathetic neuronal control of airway smooth muscle result in increased bronchoconstriction in response to vagal stimulation, leading to airway hyperresponsiveness. Neuronal inhibitory M2 muscarinic acetylcholine receptors on parasympathetic nerves are responsible for limiting acetylcholine release from these nerves. In humans with asthma, and after pulmonary inflammatory events in experimental animals, these receptors are dysfunctional, which results in airway hyperresponsiveness. Although it is unknown what mechanisms underlie airway hyperresponsiveness in chronic obstructive pulmonary disease, loss of parasympathetic control of airway smooth muscle is thought to be a contributing mechanism. As such, anticholinergic therapy is used extensively and with a high degree of success in the treatment of this condition. The future for inhaled anticholinergic compounds for the treatment of chronic obstructive pulmonary disease appears to rest in their combination with other agents, such as ß2 agonists and phosphodiesterase-4 inhibitors. Nonselective anticholinergic agents might be the best choice, because M2 muscarinic receptors on airway smooth muscle inhibit the generation and accumulation of cyclic adenosine monophosphate. Adequate concurrent blockade of M3 muscarinic receptors would be expected to counteract the enhanced acetylcholine release that would result from blockade of neuronal inhibitory M2 muscarinic receptors.

Key Words: airway hyperresponsiveness • bronchodilator therapy • muscarinic receptors • parasympathetic nerves

In humans and in the majority of mammalian species, cholinergic parasympathetic nerves provide the dominant innervation to the lungs. Release of acetylcholine from these nerves regulates airway tone (1), airway smooth muscle contraction (1), mucus secretion (2), and vasodilation (3) through interaction with muscarinic acetylcholine receptors (mAChRs) found on airway smooth muscle, glands, and pulmonary vasculature. In addition, neurotransmission through parasympathetic ganglia (4) and acetylcholine release from parasympathetic nerves (5) are regulated by mAChRs found on cholinergic ganglia and nerve endings (6, 7). Enhanced parasympathetic activity is the dominant reversible component of airway obstruction in chronic obstructive pulmonary disease (COPD) (8), and some airflow limitation in COPD is associated with hyperinflation that can be reversed by anticholinergic therapy (9, 10). Airway hyperresponsiveness to methacholine or histamine is also present in patients with COPD (11). As such, anticholinergic therapy is used extensively and with a high degree of success in the treatment of this condition. This review discusses the mechanisms whereby mAChR function may be changed in airway disease and the therapeutic potential of anticholinergics for the treatment of COPD.


   MUSCARINIC RECEPTORS IN THE AIRWAYS
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There are five subtypes of mAChRs, termed M1–M5. Each is the product of distinct genes and belongs to the superfamily of G-protein–coupled receptors that have seven transmembrane-spanning domains (12). Of these five subtypes, only M1, M2, and M3 mAChRs are expressed in the human lung and in the lungs of most mammals (6). These receptors are expressed on the airway ganglia and nerves, on airway smooth muscle, on airway mucous glands, and on pulmonary vascular endothelium.

Muscarinic Receptors on Airway Smooth Muscle
The airway smooth muscle, including human airway, contains a mixed population of M2 and M3 mAChRs (Figure 1). In the majority of species, contraction of airway smooth muscle is mediated by acetylcholine-induced activation of M3 mAChRs, which preferentially couple to the heterotrimeric G protein Gq11, resulting in stimulation of phospholipase C and an increase in intracellular calcium (12). Interestingly, despite the clear role for M3 mAChRs in airway smooth muscle contraction, the population of M3 mAChRs is quite small relative to that of M2 mAChRs. The proportion of M2 to M3 mAChRs on airway smooth muscle is approximately 4:1, varying slightly depending on the species (13). Thus, it is important to understand the role that M2 mAChRs play in the contraction of airway smooth muscle and the maintenance of smooth muscle tone.



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  		Figure 1. Muscarinic acetylcholine receptors (mAChRs) on pulmonary parasympathetic nerves and airway smooth muscle. Acetylcholine (ACh) released by parasympathetic nerves stimulates M3 mAChRs on airway smooth muscle, resulting in contraction. M2 mAChRs on airway smooth muscle facilitate M3 mAChR–mediated contraction by counteracting the cAMP-mediated relaxant pathway. Release of ACh by the nerves is tightly controlled by M2 mAChRs found on parasympathetic nerve endings. M1 mAChRs found within parasympathetic ganglia are thought to facilitate cholinergic neurotransmission that is mediated primarily by nicotinic ACh receptors (data not shown).

 
The earliest work to elucidate the function of M2 mAChRs on airway smooth muscle established that these receptors preferentially couple to the G protein G{alpha}o/i and function to counteract the ß2 receptor–mediated relaxant pathway by inhibiting the generation and accumulation of cyclic adenosine monophosphate (cAMP; Figure 2) (14, 15). It is unclear whether this activity results from an effect at the level of the adenylate cyclase enzyme (14) or from an effect on the action of calcium-activated potassium channels (also known as maxi-K or BK channels). These channels are coupled to ß2 receptors via the Gs signaling protein (16) and are functionally opposed by activation of M2 mAChRs coupled to the Gi signaling protein on airway smooth muscle (15). The result of this interaction is that M2 mAChRs could induce a degree of airway smooth muscle contraction via reversal of hyperpolarization mediated by the ß2 receptor and maxi-K channels.



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  		Figure 2. M2 mAChRs on airway smooth muscle. These receptors preferentially couple to G{alpha}o/i and function to counteract the ß2 receptor–mediated relaxant pathway by inhibiting the generation and accumulation of cAMP.

 
Evidence from M2 and M3 mAChR knockout mice has suggested that M2 mAChRs may play a more direct role than previously thought in airway smooth muscle contraction. The tracheal smooth muscle of M3 mAChR knockout mice had a 50–60% reduction in the maximal contractile response to carbachol applied ex vivo (17), whereas dual M2/M3 mAChR knockout mice had no remaining contractile responses (18).

Muscarinic Receptors on Airway Parasympathetic Nerves and Ganglia
The presence of mAChRs on airway nerves and within the cholinergic ganglia has been detected by autoradiography in various animal species, including humans, guinea pigs, rabbits, and dogs. However, the ability to distinguish between the various mAChR subtypes in these studies is limited (6, 7). The most compelling evidence in support of selective mAChR subtype expression on airway neuronal tissue comes from functional studies performed with selective mAChR antagonists, such as pirenzepine (M1 > M3 > M2) and gallamine (M2 > M3) (12).

The presence of prejunctional inhibitory M2 mAChRs on postganglionic nerves in the airways was first described in studies in which blockade of M2 mAChRs with the subtype-selective antagonist gallamine potentiated vagally induced bronchoconstriction in guinea pigs, whereas stimulation of M2 mAChRs with the selective (M2 > M3) agonist pilocarpine inhibited bronchoconstriction (5, 19). These inhibitory autoreceptors have since been identified on the airway nerves of humans and all mammalian species studied (20). Thus, activation of neuronal inhibitory M2 mAChRs with acetylcholine released from parasympathetic nerves limits further acetylcholine release, and this ability has been quantified (21). M2 mAChRs have also been demonstrated functionally within the cholinergic ganglia, where they play a role in inhibiting the slow excitatory postsynaptic potential (22).

M1 mAChRs have also been described in the cholinergic ganglia (Figure 1) of various mammalian species, such as guinea pig (23) and dog (24). In vitro they have been shown to enhance depolarization via inhibition of potassium channel activity (23, 25), thus facilitating neurotransmission (mediated primarily by nicotinic acetylcholine receptors) through the ganglia. However, the evidence supporting their presence and functional importance in human cholinergic ganglia is still somewhat ambiguous. For example, reflex bronchoconstriction induced by inhaled sulfur dioxide in atopic subjects can be significantly decreased by inhalation of the M1 mAChR-selective antagonist pirenzepine, suggesting a role for M1 mAChRs in facilitating cholinergic neurotransmission through the ganglia (4). Yet inhalation of pirenzepine at rest had only a subtle effect on resting airway tone in normal individuals (26), and it had no effect on lung function in patients with obstructive disease (presumably patients with asthma and patients with COPD, described by the authors as patients with "reversible and irreversible obstructive airway disease" as defined by spirometry) (27). Thus, although it appears that M1 mAChRs may be present in human airways, presumably within airway ganglia and on airway nerves, the significance of their function is unclear, and it is unknown whether their function or expression might be modified by disease states.

Expression of M1 mAChRs on human sympathetic neurons has also been described (28). In humans, the sympathetic nerves do not directly supply the airway smooth muscle (29). Instead, they impinge on the cholinergic ganglia and modulate noradrenaline release, thus decreasing cholinergic activity (30).

Muscarinic Receptors on Airway Submucosal Glands
The dominant control of airway secretions in humans and in all mammals studied is provided by the cholinergic nerves (2). Cholinergic receptor stimulation, either by the application of muscarinic agonist or stimulation of cholinergic nerves, results in mucus secretion, and it may also be important in the regulation of water and electrolyte balance across the epithelia (31, 32) Localization of mAChRs on submucosal glands has been demonstrated by autoradiographic (6, 7) and in situ hybridization methods (33) in various species, including humans. These mAChRs have been elucidated as the M1 and M3 mAChR subtypes (7). Although M1 mAChRs on secretory structures in the lungs appear to predominate over M3 mAChRs (1:2 ratio), the M3 mAChRs are the predominant receptors mediating mucus secretion (34), whereas M1 mAChRs are postulated to have an accessory role in electrolyte and water balance (35).

Muscarinic Receptors on Airway Vasculature
Airway vessels are dilated after stimulation of cholinergic nerves (3) and by the application of acetylcholine. In humans this action has been demonstrated to be an endothelium-dependent mechanism (36) and is thought to be mediated by M3 mAChRs (37). It is probably not the predominant mechanism for vasodilatation.


   MECHANISMS OF mAChR DYSFUNCTION IN THE LUNGS
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Role of Parasympathetic Nerves in COPD
Enhanced parasympathetic activity is the dominant reversible component of airway obstruction in COPD (8), and some of the airflow limitation in COPD is associated with hyperinflation that can be reversed by anticholinergic therapy (9, 10). Airway hyperresponsiveness to methacholine or histamine, demonstrated either as a leftward shift in concentration of aerosolized agonist needed to elicit contractile responses, or as enhanced reactivity to a given agonist (or both), is also present in patients with COPD, and it can be used as one of the diagnostic measures of this disease (11). Chronic viral infection and oxidant injury are both factors present in the lungs of individuals with COPD (38). Airway hyperresponsiveness and parasympathetic nerve dysfunction resulting from changes at the level of mAChR activity have been demonstrated in animal models and in humans after viral infection, ozone exposure, and antigen challenge. Although it is unknown what specific mechanisms underlie airway hyperresponsiveness in COPD, loss of parasympathetic control of airway smooth muscle is thought to be a contributing mechanism, because anticholinergic drugs decrease airway hyperresponsiveness and improve lung volume in COPD (8, 9, 39).

Postjunctional M3 mAChR Function in Airway Hyperresponsiveness
It would be logical to surmise that there might be significant changes in the number and/or function of M3 mAChRs on airway smooth muscle with airway hyperresponsiveness. Studies of isolated airway tissue taken from patients with asthma (40) or patients with COPD (41), or from vagotomized animals (vagal nerve cut to eliminate vagal reflex activity) after the induction of experimental airway hyperresponsiveness, have failed to demonstrate a change in the function of M3 mAChRs on airway smooth muscle in the absence of airway innervation (Figure 3) (42–44).



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  		Figure 3. Role of parasympathetic nerves in airway hyperresponsiveness. Methacholine, a muscarinic receptor agonist, produced dose-dependent bronchoconstriction in all groups tested before (A) and after (B) vagal sectioning. In the presence of intact vagal nerves, antigen-challenged rats were hyperresponsive to methacholine compared with control animals. However, after vagal sectioning, there was no difference in the responsiveness of antigen-challenged animals compared with controls, indicating that M3 mAChRs were functioning normally. Thus, antigen-induced airway hyperresponsiveness to methacholine is mediated by a change in function of the parasympathetic nervous control of airway smooth muscle. Reprinted by permission from Reference 43.

 
The population of M3 mAChRs in the airways is also unchanged in patients with airway hyperresponsiveness, such as patients with asthma, in comparison with normal subjects (45, 46). The promoter of the human M3 mAChR gene was described, and various polymorphisms in the gene were identified (47). None of the polymorphisms was significantly more frequent in patients with asthma than in healthy subjects, and there was no evidence of nonrandom transmission of any of the polymorphism haplotypes to individuals with asthma or airway hyperresponsiveness. Taken together, these data support the notion that changes in airway hyperresponsiveness exist primarily as a result of changes at the level of the parasympathetic neuronal control of airway smooth muscle, as opposed to changes in M3 mAChR–mediated cholinergic contraction of airway smooth muscle itself. Indeed, substantial evidence from animal models and human studies supports this hypothesis.

Dysfunction of Neuronal mAChRs in Airway Hyperresponsiveness
M2 mAChR dysfunction associated with airway hyperresponsiveness has been demonstrated in animals after viral infection, antigen challenge, and ozone exposure (48), as well as in subjects with asthma (49). Because this receptor is responsible for the regulation of neurotransmitter release from airway cholinergic nerves, loss of function of this receptor results in unopposed acetylcholine release after vagal stimulation, leading to enhanced contraction of airway muscle. M2 mAChR function has also been studied in patients with stable COPD, and in these patients neuronal M2 mAChRs appear to be functioning normally (50). The function of M2 mAChRs during COPD exacerbations has not been studied.

In general, neuronal inhibitory M2 mAChR dysfunction can be tied to the presence of inflammatory cells and the mediators released by these cells, in close proximity to the nerves in the airways. These mediators affect the function of the receptor directly as well as expression of the M2 mAChR gene.

Ozone-induced airway hyperresponsiveness associated with M2 MACHR DYSFUNCTION.
Exposure of healthy human subjects to ozone results in an influx of protein, inflammatory cells, and mediators into the airway, leading to airway hyperresponsiveness (51), and in COPD, high levels of environmental ozone result in exacerbation (52). Preclinically, exposure of experimental animals to ozone can be used to model an aspect of oxidative stress and airway hyperresponsiveness that is present in the lungs of individuals with COPD. In guinea pigs, exposure to ozone leads to eosinophil influx to the lungs, resulting in loss of neuronal inhibitory M2 mAChR function (53, 54) and airway hyperresponsiveness without changing the function of M3 mAChRs on airway smooth muscle. The loss of neuronal M2 mAChR function is thought to be mediated by the release of eosinophilic major basic protein (MBP), because dysfunction after ozone exposure can be prevented by the depletion of eosinophils or by pretreatment with an antibody to MBP (53). Furthermore, radioligand binding studies performed in the presence of various eosinophilic cationic proteins demonstrate that MBP itself can act as an allosteric antagonist for M2 mAChRs (55). It is unknown whether the function of M2 mAChRs on airway smooth muscle is changed by ozone exposure.

Virally induced airway hyperresponsiveness associated with neuronal M2 MACHR DYSFUNCTION.
It is thought that nearly 40% of COPD exacerbations occur because of viral infection and that viruses are associated with prolonged symptom recovery from COPD exacerbation as well as frequency of exacerbation (56). Similarly, viruses can trigger the majority of childhood asthmatic exacerbations (57) and up to 50% of exacerbations in adults with asthma (58). Even individuals with "normal" nonreactive airways have developed airway hyperresponsiveness that persists for many weeks after pulmonary viral infections (59). This airway hyperresponsiveness can be reversed with anticholinergic therapy, suggesting that it results from a dysfunction in cholinergic control of the airways (59).

The presence of virus may affect the structure of the M2 mAChR itself, leading to changes in receptor function. The M2 mAChR is highly glycosylated at three asparagine residues located on the extracellular portion of the receptor, and the carbohydrate portion makes up more than 25% of the molecular weight of the M2 mAChR (60). These glycosyl groups play an important role in agonist binding at M2 mAChRs. Neuraminidase, which is contained in the coat of influenza and parainfluenza viruses (61) and is highly expressed by infected tissues (62), has been shown to cleave key sialic acid residues from the M2 mAChR surface (63). As a result, the affinity for agonist at M2 mAChRs is decreased by an order of magnitude. Decreased agonist affinity at neuronal M2 mAChRs would be expected to lead to enhanced acetylcholine release after stimulation of parasympathetic nerves, resulting in increased M3 mAChR–mediated contraction of airway muscle.

In animal models, viruses can also induce airway hyperresponsiveness mediated by neuronal inhibitory M2 mAChR dysfunction, via activation of inflammatory cells, in particular macrophages (64) and CD8+ T lymphocytes (65). It is unknown what mediators produced by macrophages cause neuronal M2 mAChR dysfunction, but helper T-cell type 1 cytokines released from CD8+ T cells, possibly triggered by the production of double-stranded RNA during viral replication (66), may play an important role. In in vitro experiments using cultured parasympathetic nerves, interferon-{gamma} downregulates expression of the M2 mAChR gene, as does coculture with the virus itself, leading to enhanced acetylcholine release in response to electrical field stimulation (67). Viral infection does not affect the function of M3 mAChRs on airway muscle (64), and it is unknown whether the function of M2 mAChRs on airway smooth muscle is changed by viral infection.

Antigen-induced airway hyperresponsiveness associated with neuronal M2 MACHR DYSFUNCTION.
In humans, inhalation of allergen results in an influx of eosinophils to the lungs. Airway hyperresponsiveness mediated by neuronal inhibitory M2 mAChR dysfunction after antigen sensitization and challenge in guinea pigs is mediated by eosinophils (48). Blockade of eosinophil influx to the lungs via an antibody to interleukin-5 (68) or to very late antigen-4, an adhesion molecule (69) prevents antigen-induced airway hyperresponsiveness and neuronal M2 mAChR dysfunction. Eosinophils migrate to airway nerves and cluster around the ganglia after antigen challenge of rats (43) and guinea pigs (70) and release eosinophil cationic proteins, such as MBP. Eosinophil-mediated loss of M2 mAChR function and the resulting airway hyperresponsiveness can be prevented by pretreatment with an antibody to MBP, or acutely reversed by the administration of a polyanion, such as heparin (43, 71). Interestingly, clusters of eosinophils and immunolocalization of MBP surrounding the airway cholinergic nerves have been demonstrated in humans with fatal asthma (70), suggesting that eosinophils and MBP-mediated blockade of M2 mAChR function may play a similar role in human airway hyperresponsiveness. Neither antigen challenge nor eosinophil MBP has an effect on M3 mAChR function (48).


   ANTICHOLINERGIC BRONCHODILATOR THERAPY FOR COPD
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Inhaled antimuscarinics have been used as bronchodilator therapy for centuries. The plants of the Datura genus, for example, Atropa belladonna and Datura stramonium, are rich in anticholinergic alkaloids such as atropine and stramonium (72). These plants can be dried and smoked as a mechanism to deliver these agents to the airways (Figure 4) (73). This practice is discussed in detail in a treatise on asthma published in the mid-18th century (74). Interestingly, it was documented even at that time that anticholinergic therapy relieves the bronchoconstriction associated with the use of tobacco products but not asthmatic bronchoconstriction (75, 76). The trouble with Datura-derived alkaloids as bronchodilator therapy (apart from the obvious effects of smoking on the lungs) is that these agents are rapidly absorbed into the systemic circulation, where they exert anticholinergic effects on mAChRs expressed in other organs, such as heart, gut, and bladder. In addition, they easily cross the blood–brain barrier and exert potent hallucinogenic effects (77). Today, it is perhaps shocking to note that Datura-derived cigarettes were used as bronchodilator therapy well into the 1960s and 1970s (73).



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  		Figure 4. Early inhaled anticholinergic agents used to treat airway hyperresponsiveness. Image used courtesy of Mark Saunders.

 
Past and Current Inhaled Anticholinergic Therapeutics
The advent of modern inhaled anticholinergic compounds depended on the ability to prevent systemic absorption of these molecules while retaining potent anticholinergic activity. This was achieved by the quaternization of the tertiary nitrogen of the tropine moiety (of atropine) or the scopine moiety (of scopolamine). The results were highly charged quaternary ammonium salts that were poorly absorbed across membranes and, thus, had low oral and systemic bioavailability and low blood–brain barrier penetration but were still highly potent antagonists at mAChRs. However, these compounds had a relatively short residence time either in the lung or at mAChRs, resulting in a short duration of action, and thus had to be administered multiple times per day.

Ipratropium bromide arrived on the scene in the mid-1970s. Early studies with ipratropium bromide in patients with COPD or asthma provided support for the beneficial effects of anticholinergics in the treatment of COPD (but not asthma) that had been described in the 1800s (8, 78, 79). As anticholinergic therapy is effectively "antibronchoconstrictor" therapy, as opposed to "bronchodilator" therapy, the onset of activity of these agents would not be expected to be as rapid as that seen with active bronchodilator therapy through the use of an inhaled ß2 agonist. Indeed, studies with ipratropium bromide show peak bronchodilator effect 30 to 60 min after inhalation (79). The side effects of ipratropium bromide are low, making it useful for patients who cannot tolerate ß2-agonist therapy because of the cardiovascular impact. However, the activity of ipratropium bromide is sustained for only about 4 to 6 h, so it is usually administered four times per day, making it inconvenient for patients. Thus, there remained a clinical need for an inhaled anticholinergic compound that would enable once-daily therapy for COPD.

The second-generation inhaled anticholinergic was thought to be oxitropium bromide, which was introduced shortly after ipratropium bromide. However, disappointingly, oxitropium bromide had only a slightly longer duration of action in the clinic, 6 to 8 h versus 4 to 6 h with ipratropium. And so it, too, had to be administered multiple times per day (80). Oxitropium bromide was withdrawn from the market.

A true second-generation quaternary anticholinergic appeared with the launch of tiotropium bromide (81). This molecule is an order of magnitude more potent than ipratropium bromide (the KD, a measure of the "affinity" of a drug for its receptor, is 0.014 nM at cloned human M3 mAChRs vs. 0.204 nM with ipratropium bromide) (81). Tiotropium bromide has a long half-life (t1/2) at M3 mAChRs (35 h) compared with M1 mAChRs (t1/2 = 15 h) and M2 mAChRs (t1/2 = 3.6 h), as defined by radioligand dissociation studies. These results suggest a "kinetic" selectivity and as such demonstrate a long duration of action consistent with once-daily dosing (81). The mechanism allowing for the long residency of tiotropium bromide at M3 mAChRs is not completely known. However, the presence of a reactive epoxide on the tropane ring portion of tiotropium bromide could create a covalent interaction with key amino acids on M3 mAChRs but not with other mAChRs, leading to the reported "kinetic" selectivity for M3 mAChRs. Tiotropium is associated with a low incidence of cardiovascular side effects, consistent with low systemic exposure, and the major side effect reported is dry mouth (81).

Clinical Outcomes of Anticholinergic Therapy
The Global Initiative for Chronic Obstructive Lung Disease guidelines (82) recommend anticholinergic (or ß2-agonist) bronchodilator therapy as the foundational treatment for COPD. The use of inhaled anticholinergic agents also results in improvements in dyspnea, and it may have effects on health status and exacerbations.

Bronchodilation.
The use of anticholinergics in patients with COPD results in bronchoprotection from cholinergic stimuli, and protection from airway hyperresponsiveness to methacholine or histamine provocation (39). Whereas anticholinergics are similar to or better than ß2 agonists for the treatment of bronchoconstriction in COPD, the degree of improvement in lung function in older patients is often subtle. Although most drug trials use spirometrically measured parameters as endpoint measures, what may be more important to the patient with COPD are changes in symptoms, exacerbations, and exercise tolerance.

Dyspnea and hyperinflation.
Dyspnea is one of the most important symptoms in COPD. Measurements of respiratory rate, oxygen saturation, and arterial blood gases do not measure dyspnea; instead, the "gold standard" of diagnosis and assessment is the patient's self-report, as assessed by some commonly employed scales, such as the Borg scale (83) and the Transitional Dyspnea Index (84). Both ipratropium and tiotropium have been shown to reduce dyspnea as measured by either scale. For ipratropium, the effect has been shown over a short course of treatment—that is, a few days (85) or a few weeks (10). For tiotropium, the effect has been shown during treatment for many months (9, 86).

Improvement of dyspnea in COPD is probably dependent on changes in lung volume, because changes in hyperinflation (as measured by improvements in inspiratory capacity and forced vital capacity and a reduction in functional residual capacity) correlate with reduced dyspnea scores during exercise testing (9, 10). With tiotropium, improvement in forced vital capacity continues for 8 d of once-daily therapy as the drug reaches pharmacologic steady state. It is thought that this progressive improvement probably reflects incremental improvements in the ventilatory condition of the lung, as airways that had previously been closed begin to open (87). Improvements in inspiratory capacity and forced vital capacity are greater with the long-acting agent tiotropium than with ipratropium, suggesting that sustained relaxation of airway muscle is needed to produce the most significant changes in these measures. Reductions in dyspnea scores and improvements in exercise tolerance would be expected to result in improvements in patient perception of quality of life with COPD.

Health status, exacerbations, and hospitalizations.
The St. George's Respiratory Questionnaire, a commonly used measure of health status in COPD, measures three main components of the patient's overall health: symptoms, activity level, and impact (88). Treatment with anticholinergics such as ipratropium and tiotropium is effective in improving health status as measured by the St. George's Respiratory Questionnaire, and treatment with tiotropium results in greater improvement than does treatment with ipratropium (86, 89).

The assessment of exacerbations in COPD is considerably more difficult. Exacerbations generally occur about one to three times per year for a patient with COPD, making the duration of the study important. A second complication is that there is no established definition of "exacerbation." The definition can vary from "increased symptoms," meaning increased dyspnea, increased production of sputum, and more frequent cough, to a "need to utilize health care resources," such as increased use of prescription medication or visits to the hospital or physician's office. Studies performed with tiotropium lasted 12 mo and defined an exacerbation as "an increase in 2 or more respiratory symptoms for at least 3 consecutive days" (86, 89). These studies demonstrated that treatment with tiotropium reduced the incidence of exacerbations compared with placebo or ipratropium, and treatment with either ipratropium or tiotropium reduced the need for hospitalization.

Mucus secretion.
Excessive mucus secretion is a hallmark of COPD. Because mucus secretion is regulated, in part, by M1 and M3 mAChRs, many investigators have searched for an effect of anticholinergic therapy on mucus secretion. However, results from such studies are variable and generally disappointing. High doses of oral atropine (600 µg four times daily for 5 wk) reduced sputum volume in patients with obstructive disease (asthma and chronic bronchitis); however, in the same study no effect was seen when atropine was inhaled, even at high doses (1,200–2,400 µg twice daily) (90). The same group subsequently published similar results showing that 4 wk of ipratropium had no effect on sputum production in bronchially obstructed patients (91). Generally speaking, studies of inhaled quaternary anticholinergic compounds have not demonstrated an effect on mucus secretion in either normal or obstructed patients (92). However, one study of inhaled oxitropium (200 µg three times daily for 8 wk) demonstrated a slow but progressive decrease in the volume of sputum production in patients with chronic bronchitis, which started after the third week of treatment and reached statistical significance after Weeks 6 to 8 (93). Thus, it may be that sustained cholinergic blockade is necessary to obtain significant effects on mucus secretion in COPD.

Future of Anticholinergic Therapy for COPD
The theory has been advanced that an M3 mAChR–selective compound might have advantages over the current nonselective molecules. The major argument supporting this hypothesis is that such a compound might have less potential for cardiovascular side effects, because bradycardia is mediated primarily by M2 mAChRs located on the myocardium (12). As discussed above, tiotropium is considered to be a kinetically selective molecule, as it dissociates from M1 and M2 mAChRs more rapidly than it does from M3 mAChRs (92). However, anticholinergic agents that are not subtype selective, such as ipratropium, are generally well tolerated by patients with COPD at therapeutic doses routinely used in the clinic (79). Thus, demonstrating a therapeutic advantage of an M3 mAChR–selective compound over nonselective compounds would be difficult and would require large clinical trials. Indeed, tiotropium appears to be tolerated as well as ipratropium, not necessarily better tolerated, with dry mouth being the major adverse event reported for both molecules (94).

An additional argument against the need for subtype-selective inhaled anticholinergics can be made on the basis of the activity of M2 mAChRs on airway smooth muscle. Because these receptors inhibit ß2-agonist–mediated relaxation of airway smooth muscle, blockade of M2 mAChRs would be beneficial in COPD, especially if anticholinergic therapy were combined with ß2-agonist therapy. Furthermore, because M2 mAChRs on airway smooth muscle reduce the generation of cAMP, blockade of these receptors would be beneficial in COPD if anticholinergic therapy occurs in combination with therapeutics that inhibit the cAMP-metabolizing enzyme phosphodiesterase-4. Adequate concurrent blockade of M3 mAChRs would be expected to counteract the enhanced acetylcholine release that would result from blockade of neuronal inhibitory M2 mAChRs. Thus, it appears that nonselective anticholinergic agents might be the best choice for use in combination therapy for COPD.


   CONCLUSIONS
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 CONCLUSIONS
REFERENCES
 
The future for inhaled anticholinergic compounds for the treatment of COPD appears to rest in their combination with other therapeutic agents, perhaps particularly with drugs that result in elevation of cAMP, such as ß2 agonists and phosphodiesterase-4 inhibitors. These agents have already been shown to be therapeutically useful in COPD. Nonselective anticholinergic agents might offer the best opportunity for use in combination therapy for COPD, because M2 mAChRs on airway smooth muscle inhibit the generation and accumulation of cAMP. Adequate concurrent blockade of M3 mAChRs would be expected to counteract the enhanced acetylcholine release that would result from blockade of neuronal inhibitory M2 mAChRs.


   ACKNOWLEDGMENTS
 
The author thanks Dr. Michael Palovich at GlaxoSmithKline for helpful comments on this manuscript, and Mark Sanders (www.inhalatorium.com) for the pictures of asthma cigarettes and advertisements depicted in Figure 4.


   FOOTNOTES
 
Supported by the Respiratory and Inflammation Centre of Excellence in Drug Discovery, GlaxoSmithKline (King of Prussia, PA).

Conflict of Interest Statement: K.E.B. was an employee of GlaxoSmithKline at the time this manuscript was written.

(Received in original form April 22, 2005; accepted in final form May 13, 2005)


   REFERENCES
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 ABSTRACT
 MUSCARINIC RECEPTORS IN THE...
 MECHANISMS OF mAChR DYSFUNCTION...
 ANTICHOLINERGIC BRONCHODILATOR...
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