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
Abundant data from animal models and humans support the hy-
pothesis that changes at the level of parasympathetic neuronal
control of airway smooth muscle result in increased bronchocon-
striction in response to vagal stimulation, leading to airway hyper-
responsiveness. Neuronal inhibitory M2 muscarinic acetylcholine re-
ceptors 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 hyper-
responsiveness. Although it is unknown what mechanisms underlie
airway hyperresponsiveness in chronic obstructive pulmonary dis-
ease, 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 2 agonists and phosphodiesterase-4 inhibitors. Nonselective anti-
cholinergic agents might be the best choice, because M2 muscarinic
receptors on airway smooth muscle inhibit the generation and accu-
mulation of cyclic adenosine monophosphate. Adequate concurrent
blockade of M3 muscarinic receptors would be expected to counter-
act the enhanced acetylcholine release that would result from block-
ade of neuronal inhibitory M2 muscarinic receptors.
Keywords: airway hyperresponsiveness; bronchodilator therapy; mus-
carinic 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 mus-
carinic acetylcholine receptors (mAChRs) found on airway
smooth muscle, glands, and pulmonary vasculature. In addition,
neurotransmission through parasympathetic ganglia (4) and ace-
tylcholine release from parasympathetic nerves (5) are regulated
by mAChRs found on cholinergic ganglia and nerve endings
(6, 7). Enhanced parasympathetic activity is the dominant revers-
ible component of airway obstruction in chronic obstructive pul-
monary 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.
(Received in original form April 22, 2005; accepted in final form May 13, 2005)
Supported by the Respiratory and Inflammation Centre of Excellence in Drug
Discovery, GlaxoSmithKline (King of Prussia, PA).
Correspondence and requests for reprints should be addressed to Kristen E. Bel-
monte, Ph.D., Neuroscience Drug Discovery, Merck Research Laboratories,
WP26A-2000, 770 Sumneytown Pike, West Point, PA 19486. E-mail: kristen_
belmonte@merck.com
Proc Am Thorac Soc Vol 2. pp 297304, 2005
DOI: 10.1513/pats.200504-043SR
Internet address: www.atsjournals.org
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
There are five subtypes of mAChRs, termed M1M5. Each is
the product of distinct genes and belongs to the superfamily of
G-proteincoupled 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 medi-
ated by acetylcholine-induced activation of M3 mAChRs, which
preferentially couple to the heterotrimeric G protein Gq11, re-
sulting 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 popu-
lation 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.
The earliest work to elucidate the function of M2 mAChRs
on airway smooth muscle established that these receptors prefer-
entially couple to the G protein Go/i and function to counteract
the 2 receptormediated relaxant pathway by inhibiting the
generation and accumulation of cyclic adenosine monophos-
phate (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 potas-
sium channels (also known as maxi-K or BK channels). These
channels are coupled to 2 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 2 receptor and maxi-K
channels.
Evidence from M2 and M3 mAChR knockout mice has sug-
gested that M2 mAChRs may play a more direct role than pre-
viously thought in airway smooth muscle contraction. The tra-
cheal smooth muscle of M3 mAChR knockout mice had a
5060% reduction in the maximal contractile response to carba-
chol 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 cho-
linergic ganglia has been detected by autoradiography in various
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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 mAChRmediated 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).
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 bronchocon-
striction in guinea pigs, whereas stimulation of M2 mAChRs with
the selective (M2 M3) agonist pilocarpine inhibited broncho-
constriction (5, 19). These inhibitory autoreceptors have since
been identified on the airway nerves of humans and all mamma-
lian 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 nico-
tinic 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 in-
halation of the M1 mAChR-selective antagonist pirenzepine,
suggesting a role for M1 mAChRs in facilitating cholinergic neu-
rotransmission through the ganglia (4). Yet inhalation of piren-
zepine at rest had only a subtle effect on resting airway tone in
normal individuals (26), and it had no effect on lung function
PROCEEDINGS OF THE AMERICAN THORACIC SOCIETY VOL 2 2005
Figure 2. M2 mAChRs on airway smooth muscle. These receptors prefer-
entially couple to Go/i and function to counteract the 2 receptormedi-
ated relaxant pathway by inhibiting the generation and accumulation
of cAMP.
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 dis-
ease 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 noradren-
aline 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 demon-
strated by autoradiographic (6, 7) and in situ hybridization meth-
ods (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 ap-
pear to predominate over M3 mAChRs (1:2 ratio), the M3
mAChRs are the predominant receptors mediating mucus secre-
tion (34), whereas M1 mAChRs are postulated to have an acces-
sory 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 mecha-
nism (36) and is thought to be mediated by M3 mAChRs (37).
It is probably not the predominant mechanism for vasodilatation.
Belmonte: Cholinergic Pathways
Figure 3. Role of parasympathetic nerves in airway hyperresponsive-
ness. Methacholine, a muscarinic receptor agonist, produced dose-depen-
dent bronchoconstriction in all groups tested before (A ) and after (B )
vagal sectioning. In the presence of intact vagal nerves, antigen-chal-
lenged rats were hyperresponsive to methacholine compared with con-
trol 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 medi-
ated by a change in function of the parasympathetic nervous control
of airway smooth muscle. Reprinted by permission from Reference 43.
MECHANISMS OF mAChR DYSFUNCTION
IN THE LUNGS
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 hyper-
responsiveness to methacholine or histamine, demonstrated ei-
ther 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 activ-
ity have been demonstrated in animal models and in humans
after viral infection, ozone exposure, and antigen challenge. Al-
though 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 iso-
lated 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 dem-
onstrate a change in the function of M3 mAChRs on airway
smooth muscle in the absence of airway innervation (Figure 3)
(4244).
The population of M3 mAChRs in the airways is also un-
changed 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
299
described, and various polymorphisms in the gene were identi-
fied (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 primar-
ily as a result of changes at the level of the parasympathetic
neuronal control of airway smooth muscle, as opposed to changes
in M3 mAChRmediated 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 hyperrespon-
siveness 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 ani-
mals 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 hyperrespon-
siveness 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 pretreat-
ment 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 ther-
300
apy, 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. Neuramini-
dase, which is contained in the coat of influenza and parainflu-
enza 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 parasym-
pathetic nerves, resulting in increased M3 mAChRmediated con-
traction of airway muscle.
In animal models, viruses can also induce airway hyper-
responsiveness 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 dou-
ble-stranded RNA during viral replication (66), may play an
important role. In in vitro experiments using cultured parasym-
pathetic nerves, interferon- 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 aller-
gen results in an influx of eosinophils to the lungs. Airway hyper-
responsiveness 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 eosino-
phil MBP has an effect on M3 mAChR function (48).
ANTICHOLINERGIC BRONCHODILATOR THERAPY
FOR COPD
Inhaled antimuscarinics have been used as bronchodilator ther-
apy for centuries. The plants of the Datura genus, for example,
Atropa belladonna and Datura stramonium, are rich in anticho-
linergic alkaloids such as atropine and stramonium (72). These
plants can be dried and smoked as a mechanism to deliver these
PROCEEDINGS OF THE AMERICAN THORACIC SOCIETY VOL 2 2005
Figure 4. Early inhaled anticholinergic agents used to treat airway hyper-
responsiveness. Image used courtesy of Mark Saunders.
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 associ-
ated with the use of tobacco products but not asthmatic broncho-
constriction (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 bloodbrain barrier
and exert potent hallucinogenic effects (77). Today, it is perhaps
shocking to note that Datura-derived cigarettes were used as bron-
chodilator therapy well into the 1960s and 1970s (73).
Past and Current Inhaled Anticholinergic Therapeutics
The advent of modern inhaled anticholinergic compounds de-
pended 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 scopol-
amine). The results were highly charged quaternary ammonium
salts that were poorly absorbed across membranes and, thus,
had low oral and systemic bioavailability and low bloodbrain
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 multi-
ple 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 anticho-
linergics 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 2 agonist.
Indeed, studies with ipratropium bromide show peak broncho-
dilator effect 30 to 60 min after inhalation (79). The side effects
of ipratropium bromide are low, making it useful for patients who
cannot tolerate 2-agonist therapy because of the cardiovascular
Belmonte: Cholinergic Pathways
impact. However, the activity of ipratropium bromide is sus-
tained 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 com-
pound that would enable once-daily therapy for COPD.
The second-generation inhaled anticholinergic was thought
to be oxitropium bromide, which was introduced shortly after ipra-
tropium 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 ap-
peared with the launch of tiotropium bromide (81). This mole-
cule 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 2-agonist) broncho-
dilator 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 exacerba-
tions.
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 2 agonists for the treatment of bronchoconstric-
tion 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 impor-
tant symptoms in COPD. Measurements of respiratory rate,
oxygen saturation, and arterial blood gases do not measure dys-
pnea; instead, the gold standard of diagnosis and assessment
is the patients self-report, as assessed by some commonly em-
ployed 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 ipra-
tropium, the effect has been shown over a short course of treat-
mentthat is, a few days (85) or a few weeks (10). For tiotrop-
ium, 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)
301
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 dys-
pnea 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.
Georges Respiratory Questionnaire, a commonly used measure
of health status in COPD, measures three main components of
the patients 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. Georges Respiratory Questionnaire, and treatment
with tiotropium results in greater improvement than does treat-
ment 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 physicians
office. Studies performed with tiotropium lasted 12 mo and de-
fined 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 inci-
dence 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 g 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 (1,2002,400
g 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 anticholiner-
gic compounds have not demonstrated an effect on mucus secre-
tion in either normal or obstructed patients (92). However, one
study of inhaled oxitropium (200 g 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 mAChRselective
compound might have advantages over the current nonselective
molecules. The major argument supporting this hypothesis is
302
that such a compound might have less potential for cardiovascu-
lar 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 mole-
cule, 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 mAChRselective compound
over nonselective compounds would be difficult and would re-
quire large clinical trials. Indeed, tiotropium appears to be toler-
ated 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-selec-
tive inhaled anticholinergics can be made on the basis of the
activity of M2 mAChRs on airway smooth muscle. Because these
receptors inhibit 2-agonistmediated relaxation of airway
smooth muscle, blockade of M2 mAChRs would be beneficial
in COPD, especially if anticholinergic therapy were combined
with 2-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 coun-
teract 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
The future for inhaled anticholinergic compounds for the treat-
ment of COPD appears to rest in their combination with other
therapeutic agents, perhaps particularly with drugs that result in
elevation of cAMP, such as 2 agonists and phosphodiesterase-4
inhibitors. These agents have already been shown to be therapeu-
tically 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 concur-
rent blockade of M3 mAChRs would be expected to counteract
the enhanced acetylcholine release that would result from block-
ade of neuronal inhibitory M2 mAChRs.
Conflict of Interest Statement : K.E.B. was an employee of GlaxoSmithKline at the
time this manuscript was written.
Acknowledgment : 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.
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