Aclidinium bromide, a new, long-acting, inhaled muscarinic antagonist: In vitro plasma inactivation and pharmacological activity of its main metabolites

Article abstract of DOI:10.1016/j.ejps.2010.01.004

Aclidinium bromide is a novel, long-acting inhaled muscarinic antagonist drug in Phase III clinical trials for chronic obstructive pulmonary disease (COPD). The aims of this study were to evaluate the in vitro stability of the ester drug aclidinium in plasma from various species, and the in vitro and in vivo pharmacological activity of its hydrolysis metabolites. Following incubation of aclidinium in pooled samples of human, rat, guinea pig or dog plasma, the rate of hydrolysis was quantified by reversed phase ultra performance liquid chromatography and mass spectrometry. Tiotropium and ipratropium were used as comparators. The in vitro biochemical profile of the hydrolysis metabolites of aclidinium was assessed in human M₁ to M₅ muscarinic receptors and in a standard selectivity panel (85 G protein-coupled receptors [GPCRs], ion channels and enzymes). The bronchodilator activity of the metabolites of aclidinium bromide was studied in guinea pigs after acetylcholine-induced bronchoconstriction. Aclidinium was rapidly hydrolysed into carboxylic acid and alcohol derivatives in guinea pig, rat, human and dog plasma with half-lives of 38, 11.7, 2.4 and 1.8 min, respectively. In contrast, ≥70% of tiotropium and ipratropium remained unchanged in the plasma after 60 min of incubation. The carboxylic acid and alcohol metabolites had no significant affinity for any of the muscarinic receptors, other GPCRs, ion channels or enzymes studied and showed no relevant antibronchoconstrictory activity in vivo. These results suggest that aclidinium may have a reduced systemic exposure and therefore less propensity for class-related systemic side effects in the clinical setting.

Full text of DOI:10.1016/j.ejps.2010.01.004

European Journal of Pharmaceutical Sciences 39 (2010) 283–290
Contents lists available at ScienceDirect
European Journal of Pharmaceutical Sciences
journal homepage: www.elsevier.com/locate/ejps
Review
Aclidinium bromide, a new, long-acting, inhaled muscarinic antagonist: In vitro
plasma inactivation and pharmacological activity of its main metabolites
Sonia Sentellas, Israel Ramos, Joan Albertí, Miquel Salvà, Francisca Antón,
Montserrat Miralpeix, Jorge Beleta, Amadeu Gavaldà∗
Almirall, R&D Center, Sant Feliu de Llobregat, Barcelona, Spain
a r t i c l e i n f o
a b s t r a c t
Article history:
Aclidinium bromide is a novel, long-acting inhaled muscarinic antagonist drug in Phase III clinical trials for
chronic obstructive pulmonary disease (COPD). The aims of this study were to evaluate the in vitro stability
of the ester drug aclidinium in plasma from various species, and the in vitro and in vivo pharmacological
activity of its hydrolysis metabolites. Following incubation of aclidinium in pooled samples of human,
rat, guinea pig or dog plasma, the rate of hydrolysis was quantified by reversed phase ultra performance
liquid chromatography and mass spectrometry. Tiotropium and ipratropium were used as comparators.
The in vitro biochemical profile of the hydrolysis metabolites of aclidinium was assessed in human M1
to M5 muscarinic receptors and in a standard selectivity panel (85 G protein-coupled receptors [GPCRs],
ion channels and enzymes). The bronchodilator activity of the metabolites of aclidinium bromide was
studied in guinea pigs after acetylcholine-induced bronchoconstriction.
Received 16 September 2009
Received in revised form 8 January 2010
Accepted 11 January 2010
Available online 20 January 2010
Keywords:
Aclidinium bromide
Plasma hydrolysis
Plasma esterases
Metabolites pharmacology
Aclidinium was rapidly hydrolysed into carboxylic acid and alcohol derivatives in guinea pig, rat, human
and dog plasma with half-lives of 38, 11.7, 2.4 and 1.8 min, respectively. In contrast, ≥70% of tiotropium
and ipratropium remained unchanged in the plasma after 60 min of incubation. The carboxylic acid and
alcohol metabolites had no significant affinity for any of the muscarinic receptors, other GPCRs, ion
channels or enzymes studied and showed no relevant antibronchoconstrictory activity in vivo. These
results suggest that aclidinium may have a reduced systemic exposure and therefore less propensity for
class-related systemic side effects in the clinical setting.
© 2010 Elsevier B.V. All rights reserved.
Contents
1.
2.
Introduction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 284
Materials and methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 284
2.1.
2.2.
2.3.
2.4.
Materials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 284
Animals. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 285
Animal and human plasma samples . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 285
Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 285
2.4.1.
2.4.2.
2.4.3.
2.4.4.
2.4.5.
2.4.6.
2.4.7.
In vitro plasma stability . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 285
Non-enzymatic hydrolysis of aclidinium bromide . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 285
HPLC–UV and UPLC–MS/MS analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 285
Identification of hydrolysis metabolites of aclidinium bromide . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 285
Affinity for the human M1 to M5 muscarinic receptor subtypes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 286
Biochemical profile for the main aclidinium metabolites . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 286
In vivo effects on acetylcholine inducing bronchoconstriction in anaesthetised guinea pigs. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 287
2.5.
Data analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 287
∗
Corresponding author at: Laboratorios Almirall, S.A., Laureà Miró, 408-410, 08980 Sant Feliu de Llobregat, Barcelona, Spain. Tel.: +34 93 291 3818;
fax: +34 93 291 2827.
E-mail address: amadeu.gavalda@almirall.com (A. Gavaldà).
0928-0987/$ – see front matter © 2010 Elsevier B.V. All rights reserved.
doi:10.1016/j.ejps.2010.01.004

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S. Sentellas et al. / European Journal of Pharmaceutical Sciences 39 (2010) 283–290
3.
Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 287
3.1.
3.2.
3.3.
3.4.
3.5.
3.6.
In vitro plasma stability . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 287
Identification of metabolites . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 287
Non-enzymatic hydrolysis of aclidinium . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 288
Affinity studies for human muscarinic receptor subtypes, M1 to M5 at equilibrium . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 288
Selectivity profile. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 288
In vivo effects on the acetylcholine induced bronchoconstriction in anaesthetised guinea pigs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 288
4.
Discussion. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 288
Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 290
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 290
1. Introduction
glycolic acid, sodium salt]) and tiotropium bromide were
synthesised at the Department of Medicinal Chemistry, Lab-
oratorios Almirall (Barcelona, Spain). Chemical structures of
aclidinium bromide, LAS34823 and LAS34850 are depicted in
Fig. 1. Ipratropium bromide, atropine sulphate, acetylcholine
chloride, urethane and phosphate-buffered saline (PBS) with
calcium and magnesium were purchased from Sigma Chem-
icals (Madrid, Spain) and sodium pentobarbital was obtained
from Industrial Kern (Barcelona, Spain). Membrane preparations
expressing human M1 to M5 receptors (obtained from transfected
Chinese hamster ovary CHO-K1 cells) were purchased from Mem-
brane Target Systems, PerkinElmer Life and Analytical Sciences
(Boston, MA, USA). 1-[N-methyl-3H] scopolamine methyl chloride
(3H-NMS) was obtained from PerkinElmer Life and Analytical
Sciences.
Stock solutions of aclidinium, ipratropium and tiotropium for
plasma stability were prepared at a final concentration of 1 mg/ml
(expressed as free base) by dissolving each compound in a 0.1 N
hydrochloric acid:acetonitrile (20:80, v/v) mixture. All equilibrium
binding studies were performed in 96-well plates (Nunc, Thermo
Fischer Scientific, Roskilde, Denmark), with all stock solutions of
the compounds being prepared at 10−2 M in dimethyl sulphoxide
(DMSO). In the anaesthetised guinea pig studies, aclidinium and its
hydrolysis metabolites were dissolved in 0.2–1% (v/v) HCl in the
presence of polyethylene glycol 300. Ipratropium and tiotropium
were dissolved in distilled water.
Chronic obstructive pulmonary disease (COPD) is a preventable
and treatable lung disease characterised by airflow limitation that
is not fully reversible (Rabe et al., 2007). It is estimated that COPD
caused approximately 2.7 million deaths worldwide in 2002 (Lopez
et al., 2006). In 2001, COPD represented the sixth most common
cause of mortality and the disease is expected to be the third
most common cause of mortality worldwide by 2020 (Lopez et al.,
2006).
The major reversible component of airway obstruction in COPD
is believed to be cholinergic (Gross and Skorodin, 1984a,b; Gross,
1988). Inhaled anti-cholinergics, which increase bronchodilation
by blocking muscarinic receptors on airway smooth muscle, are
established treatment options for COPD (Barnes, 2004). Currently,
only two inhaled muscarinic antagonists are available for the
management of symptomatic COPD patients: ipratropium, a short-
acting agent requiring up to 4 doses per day and tiotropium,
a long-acting, once-daily treatment (Barnes, 2004; Gross, 2006;
Hanania and Donohue, 2007). Certain systemic anti-cholinergic
effects are associated with these compounds, including dry mouth
(reported in 11.6% of patients treated with tiotropium bromide),
glaucoma, constipation, increased heart rate and urinary retention
(Kesten et al., 2006; Lee et al., 2008; Singh et al., 2008). There-
fore, a compound with similar or improved bronchodilatory effects
and lower systemic availability resulting in fewer unwanted anti-
cholinergic effects would be attractive.
Aclidinium bromide is a novel, long-acting muscarinic antag-
onist in Phase III clinical trials for the maintenance treatment of
COPD. Aclidinium is a potent muscarinic receptor antagonist with
a long residence half-life at human M3 receptors and shorter resi-
dence time at human M2 receptor, resulting in kinetic selectivity of
the M3 versus the M2 receptor subtype (Gavaldà et al., 2009). These
pharmacological characteristics of aclidinium result in its long-
lasting antibronchoconstrictory effect with a fast onset of action in
both in vitro and in vivo animal models (Gavaldà et al., 2009). Acli-
dinium undergoes rapid hydrolysis in human plasma, contributing
to a low and transient systemic exposure and a reduced potential
for class-related systemic side effects in the clinical setting (Prat et
al., 2009).
This study evaluated the contribution of in vitro enzymatic and
non-enzymatic hydrolysis to the cleavage of the ester bond of acli-
dinium, and characterised the in vitro and in vivo pharmacological
activity of the two metabolites resulted from aclidinium hydrolysis.
2. Materials and methods
2.1. Materials
Aclidinium bromide, its alcohol metabolite (LAS34823 [3(R)-
hydroxy-1-(3-phenoxy-propyl)-1-azonia-bicyclo[2.2.2]octane,
bromide]), its carboxylic acid metabolite (LAS34850 [dithienyl-
Fig. 1. Hydrolytic cleavage of the ester moiety of aclidinium bromide into its alcohol
(LAS34823) and carboxylic acid (LAS34850) derivatives.

S. Sentellas et al. / European Journal of Pharmaceutical Sciences 39 (2010) 283–290
285
2.2. Animals
Male Dunkin-Hartley guinea pigs (400–600 g at the time of
and during HPLC–UV analysis to prevent aclidinium ester cleav-
age.
experimental procedures) were obtained from Harlan (Interfauna
Ibérica, Sant Feliu de Codines, Spain or Horst, The Netherlands).
Guinea pigs were housed in groups of four or five at 20–24 ◦C under
a 12-h light/dark cycle. Food (maintenance diet for guinea pigs with
vitamin C [SAFE 114, SAFE, France]) and water were available ad
libitum. Male Beagle dogs (9–20 kg at the time of experimental pro-
cedures) were supplied by guaranteed commercial suppliers. Dogs
were housed at 15–21 ◦C, 40–70% humidity, under a 12-h light/dark
cycle and fed on a maintenance diet (Harlan TEKLAD 2021, Madison,
WI, USA) with free access to water. All experiments were carried
out with the approval of the Animal Ethical Committee of Almirall,
Barcelona, Spain.
2.4.3. HPLC–UV and UPLC–MS/MS analysis
The determination of aclidinium bromide and its hydrolysis
metabolites in incubation samples from stability studies conducted
in phosphate buffer at different pH values was performed using a
Waters Alliance 2790 HPLC system (Waters, Milford, MA, USA) with
UV detection (Waters 2996PDA) at 219 nm (LAS34823) and 238 nm
(aclidinium and LAS34850). Aclidinium and its metabolites were
separated on a Waters Symmetry C18 (250 mm × 4.6 mm, 5 ␮m)
protected by a Tracer Spherisorb ODS-2CN (10 mm × 4.6 mm) and
eluted using a gradient with (solvent A) 10 mM sodium phosphate
containing 5 mM 1-octanesulphonic acid (pH 2.8) and (solvent B)
acetonitrile. The initial mobile phase composition was 35% B and
progressed linearly to 50% B in 12 min. The mobile phase was
returned to the starting solvent mixture in 0.1 min and the sys-
tem equilibrated for 8 min between runs. A constant flow rate
of 1 ml/min was employed. The injection volume was 90 ␮l and
the retention times were approximately 4.2, 6.8 and 13 min for
LAS34823, LAS34850 and aclidinium, respectively (Fig. 2A).
The determination of aclidinium, tiotropium and ipratropium
in incubation samples from plasma stability studies were con-
ducted by using UPLC with mass spectrometry detection. Gradient
chromatographic conditions (maximum 3.5 min run time) were
employed using a Waters Acquity C18 (2.1 mm × 50 mm, 1.7 ␮m)
on a UPLC Acquity system (Waters, Milford, MA, USA). The mobile
phase used for analysis was (solvent A) 10 mM formic acid:2.5 mM
ammonia (pH 3.0) and (solvent B) acetonitrile at a flow-rate
of 0.4 ml/min (aclidinium and ipratropium) and 0.35 ml/min
(tiotropium). Different linear gradient programmes were used:
from 30% to 90% solvent B in 1 min (aclidinium), from 5% to 50% sol-
vent B in 2 min (tiotropium), and from 5% to 50% solvent B in 1.2 min
(ipratropium). The retention times were 0.9, 1.7 and 1.1 min for
aclidinium, tiotropium and ipratropium, respectively. A final step
of column washout with 90% solvent B for approximately 1 min
was also conducted in all analysis before re-equilibration at starting
eluent conditions.
2.3. Animal and human plasma samples
Plasma samples from male Dunkin-Hartley guinea pigs (n = 10)
and male Beagle dogs (n = 4) were obtained by centrifugation of
blood samples taken into sodium heparin tubes (2000 × g at 4 ◦C)
for 10 min. Plasma samples from six human volunteers (three male
and three female) were similarly obtained. Male Wistar rat plasma
was obtained from RCC Cida (Barcelona, Spain) using sodium hep-
arin as an anticoagulant. The resulting plasma from each species
was pooled and stored at −80 ◦C until use.
2.4. Methods
2.4.1. In vitro plasma stability
Plasma samples from each species were incubated in tripli-
cate for 5 min at 37 ◦C before the addition of the test compound
to initiate the reaction (final concentration: 40 ng/ml, expressed
as free base). The final molar concentrations were 83 nM, 102 nM
and 120 nM for aclidinium bromide, tiotropium bromide and
ipratropium bromide, respectively. The final concentration of ace-
tonitrile in the incubations was below 0.5%.
Aliquots of the incubation mixtures (100 ␮l) were taken at pre-
determined time points during the incubation, up to 90 min for
aclidinium and up to 6 h for tiotropium and ipratropium. Each
aliquot was transferred to a vessel containing 300 ␮l of cold ace-
tonitrile:1N hydrochloric acid (90/10, v/v) to stop the reaction.
Samples were then centrifuged at 2500 × g for 10 min at 4 ◦C. Blank
incubations in the absence of the test compound were also per-
formed.
Detection was performed on
a Quattro Premier triple
quadrupole mass spectrometer (Waters Micromass) with electro-
spray ion source operating in positive ion mode. The capillary
voltage was set to 3.0 kV, the source block temperature was 120 ◦C,
and the desolvation gas (nitrogen) was heated to 350 ◦C and deliv-
ered at a flow rate of 550 l/h. The cone gas (nitrogen) was set
to 50 l/h. Quantification was performed using multiple reaction
monitoring (MRM) for the transitions 484 → 262 (aclidinium; cone
voltage, 50 V; collision energy, 35 eV), 392 → 152 (tiotropium; cone
voltage, 45 V; collision energy, 30 eV), and 332 → 166 (ipratropium;
cone voltage, 25 V; collision energy, 50 eV). Data acquisition and
processing were performed with MassLynx version 4.1 soft-
ware (Waters). Fig. 2B shows the multiple reaction monitoring
(MRM) chromatograms obtained from the UPLC–MS/MS analysis
of 40 ng/ml aclidinium, 40 ng/ml tiotropium bromide and 40 ng/ml
ipratropium bromide in human plasma.
The analyses of test compounds were performed by ultra per-
formance liquid chromatography (UPLC) with mass spectrometry
detection.
2.4.2. Non-enzymatic hydrolysis of aclidinium bromide
Incubation mixtures containing 50 mM sodium phosphate
(pH 2.0, 4.0, 6.0, 7.0, 7.4, 8.0 and 9.0) and aclidinium (50 ␮M)
in 0.5 ml (final volume) were pre-incubated at 37 ◦C for 3 min.
Reactions were initiated by the addition of 25 ␮l of 1 mM acli-
dinium bromide prepared in 0.01N HCl:acetonitrile (80:20, v/v).
The final concentration of acetonitrile in the incubation mix-
tures was 1%. At pre-defined times of 30, 60, 120 and 180 min,
100 ␮l aliquots of the incubation mixtures were taken and the
reactions stopped by the addition of 100 ␮l of cold acetoni-
trile:0.2N hydrochloric acid (20:80, v/v) followed by centrifugation
at 2500 × g for 10 min at 4 ◦C. Samples corresponding to time
zero were prepared manually separately. All the incubations were
carried out in duplicate. The amount of aclidinium and the acid
and alcohol hydrolysis products in the clear supernatant fraction
were determined. All samples were kept at +4 ◦C once obtained
2.4.4. Identification of hydrolysis metabolites of aclidinium
bromide
The identification of the aclidinium bromide hydrolysis metabo-
lites formed in incubations was conducted by comparison of the
electrospray-ionisation mass spectra and the chromatographic
retention times of the metabolites with those of authentic stan-
dards of LAS34823 (alcohol derivative) and LAS34850 (carboxylic
acid derivative) using a Quattro Premier triple quadrupole mass
spectrometer (Waters Micromass, Manchester, UK) with electro-
spray ion source operating in positive and negative ion mode.

286
S. Sentellas et al. / European Journal of Pharmaceutical Sciences 39 (2010) 283–290
Fig. 2. (A) LC–UV (220 nm) chromatograms of a standard solution of 50 ␮M aclidinium, 50 ␮M LAS34823 and 50 ␮M LAS34850 in 50 mM phosphate buffer and (B) represen-
tative LC–MSMS MRM chromatograms of aclidinium, tiotropium and ipratropium bromide in human plasma. Plasma samples contained test substances at a concentration
of 40 ng/ml.
2.4.5. Affinity for the human M1 to M5 muscarinic receptor
subtypes
aliquots of the reaction were transferred to GF/C filter plates (Mil-
lipore, Barcelona, Spain) pre-treated for 1 h with wash buffer (Tris
50 mM, NaCl 100 mM, pH 7.4) containing 0.05% polyethylenimine.
Bound and free 3H-NMS were then separated by rapid vacuum
filtration followed by four washes with ice-cold wash buffer. Fil-
ters were then dried for 30 min before addition of 30 ␮l OptiPhase
Supermix (PerkinElmer Life and Analytical Sciences, Boston, MA,
USA) and radioactivity was quantified using a MicroBeta Trilux
microplate scintillation counter (PerkinElmer Life and Analytical
Sciences, Boston, MA, USA).
The affinity of aclidinium and its main metabolites for the
different human muscarinic receptor subtypes was determined
by measuring their ability to displace bound 3H-NMS in cell
membrane preparations expressing one of the human muscarinic
receptor subtypes at equilibrium.
Protein concentrations were 8.1, 10.0, 4.9, 4.5 and 5.0 ␮g/well
for M1, M2, M3, M4 and M5 receptor membrane preparations,
respectively. The 3H-NMS concentration was 0.3 nM for the M1
and M4 assays and 1 nM for the M2, M3, and M5 assays simi-
lar to the radioligand equilibrium dissociation constant (Kd) for
the different muscarinic receptor subtypes. A range of compound
concentrations (10−14 to 10−5 M) were tested in duplicate to gen-
erate competition curves. Non-specific binding was determined in
the presence of atropine (1 ␮M). Assay reagents were dissolved in
assay binding buffer (PBS with calcium and magnesium) to a total
volume of 200 ␮l. After a 2- (M1 to M4) or 6- (M5) h incubation
period at room temperature in 96-well microtiter plates to ensure
that equilibrium was achieved for all compounds tested, 150 ␮l
2.4.6. Biochemical profile for the main aclidinium metabolites
The in vitro selectivity profile of the main metabolites of acli-
dinium bromide was assessed in a standard selectivity panel
of 85 receptor and enzyme assays including these receptors:
A1, A2A, A3, ␣1 (non-selective), ␣2 (non-selective), ␤1, ␤2, AT1,
AT2, BZD (central), B1, B2, CB1, CB2, CCKA, CCKB, CRF1, D1, D2,
D3, D4, ETA, ETB, GABA (non-selective), AMPA, kainate, NMDA,
H1 (central), H2, H3, I1 (peripheral), I2 (central), LTB4, LTD4, MC4,
M (non-selective), NK1, NK2, NK3, Y (non-selective), N (neuronal),

S. Sentellas et al. / European Journal of Pharmaceutical Sciences 39 (2010) 283–290
287
Table 1
opiate (non-selective), ORL1, PCP, P2X, P2Y, 5-HT (non-selective),
Apparent half-lives of aclidinium, ipratropium and tiotropium in Wistar rat, Dunkin
␴ (non-selective), glucocorticoid, oestrogen, progesterone, andro-
Hartley guinea pig, Beagle dog and human plasma (37 ◦C). Mean values (n = 3).
+
+
gen, TRH, V1, V2, Ca2 channel (L, DHP site), Ca2 channel
(L, diltiazem site), Ca2 channel (L, verapamil site), K+
chan-
+
Compound
Half-life (h)
Rat
ATP
nel, K+ channel, SK+ channel, Na+ channel (site2), Cl− channel,
V
Ca
Guinea pig
Dog
0.03 (1.8)
Human
NE transporter, DA transporter, GABA transporter, choline trans-
porter, 5-HT transporter. Enzymes included in this study were:
PDE1, PDE2, PDE3, PDE4, PDE5, adenylyl cyclase (basal), guany-
lyl cyclase (basal), PKC, COMT, GABA transaminase, glutamate
decarboxilase, MAO-A, MAO-B, phenylethanolamine N-methyl
transferase, tyrosine hydroxylase, ATPase (Na+/K+) and acetyl-
cholinesterase.
The effects of the acid and alcohol metabolites of aclidinium
(10 ␮M each) on the assay signal (specific binding or enzyme activ-
ity) were determined and tested in duplicate and the percentage
inhibition calculated. A reference compound appropriate for each
individual assay was also tested in duplicate at a minimum of
seven different concentrations. The IC50 values obtained from the
dose–response curves were validated by comparison with those in
the literature. Assays were performed by CEREP (Celle L’Evescault,
France).
Aclidiniuma
Tiotropium
Ipratropium
0.19 (11.7)
1.2
>6
0.64 (38.3)
0.04 (2.4)
1.9
>6
1.2
>6
1.6
>6
a
In brackets, half-lives are expressed in minutes.
stant k and the half-life (t1/2 = −0.693/k) were calculated from the
slope of the linear regression line in the elimination phase of the
semi logarithmic plot using WinNonlin software (version 5.0.1,
Pharsight Corp., USA).
Affinity at equilibrium for muscarinic receptors subtypes was
determined as IC50 for aclidinium. IC50 values were obtained from
at least three independent curves of 10 antagonist concentra-
tions run in duplicate. Affinities of LAS34823 and LAS34850 were
expressed as % inhibition at the maximum concentration tested
(10 ␮M). All adjustments were performed using Prism (GraphPad
software, San Diego, CA, USA).
2.4.7. In vivo effects on acetylcholine inducing
bronchoconstriction in anaesthetised guinea pigs
3. Results
Guinea pigs were anaesthetised with an intraperitoneal injec-
tion of 1 g/kg urethane and 20 mg/kg sodium pentobarbital.
Additional anaesthetic was administered after 60 min as required.
The trachea was cannulated and the lungs artificially ventilated
with a small rodent ventilator (Ugo Basile, Biological Research
Apparatus, Comerio-Varese, Italy) at a rate of 60 strokes/min and a
tidal volume of 10 ml/kg. Animals were maintained at 37 ◦C with a
homeothermic blanket throughout the experiment.
3.1. In vitro plasma stability
The comparative hydrolytic cleavage of the ester compounds
aclidinium, tiotropium and ipratropium in plasma samples from
different species was evaluated at 37 ◦C. The plasma hydrolytic
cleavage of aclidinium, tiotropium and ipratropium exhibited a
mono-exponential decline in all conditions evaluated. Aclidinium
was hydrolysed in plasma samples from all species studied, with
apparent half-lives at 37 ◦C of 11.7, 38.3, 1.8 and 2.4 min in rat,
guinea pig, dog and human plasma, respectively (Table 1, Fig. 3).
In contrast, tiotropium and ipratropium were relatively resistant
to ester hydrolysis in plasma. The plasma hydrolysis of tiotropium
was similar in all species with an apparent plasma half-life between
1 and 2 h. No significant hydrolysis of ipratropium was observed in
plasma at 37 ◦C for up to 6 h of incubation in all species evaluated.
Blood pressure was measured in a cannulated carotid artery
and acetylcholine was administered via a cannulated jugular
vein. Intrapulmonary pressure and blood pressure were measured
by blood pressure transducers (MLT0699, ADInstruments-Panlab,
Barcelona, Spain) connected to a bridge amplifier (PowerLab/8sp,
ADInstruments-Panlab, Barcelona, Spain). The data were recorded
using Chart 5 software (ADInstruments-Panlab, Barcelona, Spain).
After induction of anaesthesia and preparation, animals were
allowed to stabilise for 10 min before bronchoconstriction was
induced by an intravenous (i.v.) bolus of acetylcholine. Acetyl-
choline was administered at a dose (10–60 ␮g/kg) that approxi-
mately doubled the basal intrapulmonary pressure. Repeated bolus
injections of acetylcholine at the selected dose were administered
until two reproducible responses were obtained; the mean of the
two final responses before the addition of the antagonists corre-
sponded to the baseline response to acetylcholine and was used to
evaluate the antibronchoconstrictor effect of the antagonists.
Five minutes after the last administration of acetylcholine used
to calculate maximal bronchoconstriction, the test antagonist was
administered via a nebuliser (5 s duration; Mumed ultrasonic
nebuliser, Mumed Systems Ltd., London, UK). Compounds were
delivered at a concentration of 1 mg/ml. Acetylcholine doses were
then administered 5, 10, 15, 20, 25, 30, 60 and 120 min after
the administration of the compounds to evaluate their antibron-
choconstrictor effects. The effect was expressed as a percentage of
the baseline response to acetylcholine. Data were analysed using
Microsoft Excel software.
3.2. Identification of metabolites
Hydrolysis of the ester compound aclidinium bromide occurred
in aqueous solutions and plasma samples to yield the carboxylic
acid derivative (LAS34850) and alcohol derivative (LAS34823).
The identification of these two metabolites was confirmed by
comparing the MS and MS/MS spectra with those of the authentic
standards. The MS and the MS/MS spectra of both metabolites
are shown in Fig. 4. LAS34823 yielded a molecular ion of m/z 262
in positive mode. The main fragments in the product ion mass
2.5. Data analysis
In plasma stability studies, the percentage of the remaining
unaltered compound (the sample concentration at time 0 was
determined as 100%) was calculated for each time point. The con-
Fig. 3. Rat, guinea pig, dog and human plasma stability time course of aclidinium
bromide (semi-logarithmic plot). Mean values SD (n = 3).

288
S. Sentellas et al. / European Journal of Pharmaceutical Sciences 39 (2010) 283–290
Fig. 4. MS and MS/MS spectra of the hydrolysis metabolites of aclidinium bromide: (A) (+)-ESI-MS spectrum of LAS34823 (alcohol metabolite); (B) (+)-ESI-MS/MS spectrum
of LAS34823 (alcohol metabolite); (C) (−)-ESI-MS spectrum of LAS34850 (acid metabolite); (D) (−)-ESI-MS/MS spectrum of LAS34850 (acid metabolite).
placement assay using 3H-NMS and membranes of CHO-KI cells
expressing the human M1 to M5 receptor subtypes.
LAS34823 (alcohol metabolite) and LAS34850 (acid metabo-
lite) at concentrations up to 10 ␮M, were devoid of any significant
affinity for any of the muscarinic subtypes studied. In contrast, acli-
dinium, potently blocked the specific binding of 3H-NMS to human
M1 to M5 receptors in a concentration-dependent manner (Table 2).
spectrum were at m/z 168, 140, 135, 128, 124 and 107, which
were consistent with the proposed structure. LAS34850 showed,
in negative mode, a molecular ion at m/z 239 and a prominent
fragment ion at m/z 195 in the MS and MS/MS spectra, respectively.
3.3. Non-enzymatic hydrolysis of aclidinium
The stability of aclidinium (50 ␮M) in 50 mM phosphate buffer
was evaluated in vitro at different pHs at 37 ◦C. At basic and neutral
pH, the concentration of compound decreased exponentially with
time, indicating that the hydrolysis followed first order kinetics.
The hydrolysis rate increased with pH, with a hydrolysis half-life of
1.2 h at physiological pH 7.4 (Fig. 5). The compound was essentially
stable at pH ≤4.
3.5. Selectivity profile
Neither of the two aclidinium main metabolites had any rel-
evant affinity for the receptors or enzymes represented in the
standard selectivity panel described in the methods section (data
not shown). Maximal effect of aclidinium metabolites at all the
receptors, enzymes, ion channels and transporters was below
50% at the 10 ␮M concentration tested. This implies a separation
with respect to the activity of aclidinium for muscarinic recep-
tors of more than 10,000-fold. It is therefore considered to be
highly unlikely that the metabolites described could exert any
relevant pharmacological activity through the panel of targets eval-
uated.
3.4. Affinity studies for human muscarinic receptor subtypes, M1
to M5 at equilibrium
The affinity of the two aclidinium main metabolites for human
muscarinic receptors was assessed in a radioligand binding dis-
3.6. In vivo effects on the acetylcholine induced
bronchoconstriction in anaesthetised guinea pigs
To evaluate the potential in vivo bronchoprotective effect of
inhaled aclidinium and its main metabolites, 1000 ␮g/ml of each
compound was administered to different groups of anaesthetised
guinea pigs prior to acetylcholine-induced bronchoconstriction.
Aclidinium produced a potent and sustained bronchoprotection
(72.0–88.4%) over the 120-min study period (Fig. 6). In contrast,
neither LAS34823 nor LAS34850 reversed the acetylcholine-
induced bronchoconstriction to any relevant extent.
4. Discussion
Aclidinium bromide is a novel, long-acting, inhaled muscarinic
antagonist currently in development for the maintenance treat-
ment of COPD. It was selected from a series of quaternised
(3R)-quinuclidinol esters as a potent muscarinic M3 antagonist,
Fig. 5. Aclidinium stability and apparent half-lives (t1/2) in 50 mM phosphate buffer
(37 ◦C) at different pH values. Aclidinium bromide was also stable at pH 2.0 (results
not shown). Each experimental point represents the mean of duplicate samples from
independent experiments.

S. Sentellas et al. / European Journal of Pharmaceutical Sciences 39 (2010) 283–290
289
Table 2
Affinity of aclidinium and its main metabolites LAS34823 and LAS34850 for human M1, M2, M3, M4 and M5 receptors.
M1
0.2 0.0
M2
0.3 0.1
10.6 3.4
−0.4 1.4
M3
M4
M5
Aclidinium IC50 (nM)
LAS34823 (% at 10 ␮M)
LAS34850 (% at 10 ␮M)
0.4 0.1
2.6 1.7
1.8 1.8
0.2 0.0
5.6 3.0
9.2 3.6
0.4 0.0
6.0 3.2
3.2 2.4
−0.2 2.0
9.3 2.8
Affinities for aclidinium bromide are reported as IC50 (nM) values for the displacement of 3H-NMS from the corresponding receptor preparations. In the case of LAS34823
and LAS34850, the residual affinity is expressed as maximal percentage of binding displacement at 10 ␮M. Data are expressed as mean standard error of three independent
experiments.
with long duration of action as a bronchodilator in guinea pigs and
rapid human plasma hydrolysis (Prat et al., 2009).
metabolite was at least 100-fold higher than aclidinium (Jansat et
al., 2009). In contrast, the systemic exposure of the alcohol metabo-
lite was much lower and, between 2- and 5-fold that of aclidinium.
The relatively high plasma concentration of the acid metabolite
is mainly due to differences in oral absorption, which is higher
with the acid metabolite than with aclidinium and the alcohol
metabolite (Jansat et al., 2009). In addition, other minor oxidative
metabolites of aclidinium have been observed in vitro and in vivo
in animal species and humans (data not shown).
This study characterised the in vitro biological and chemical
stability of aclidinium bromide, and the in vitro and in vivo phar-
macological profile of its two main metabolites. Plasma hydrolysis
of aclidinium bromide was studied in samples from rat, guinea
pig, dog and humans. Aclidinium was rapidly hydrolysed in dog
and human plasma (t1/2 <5 min) but slightly more stable in rat
(t1/2 = 11.7 min) and guinea pig (t1/2 = 38.3 min). A similar result for
aclidinium hydrolysis in human plasma was obtained in a previous
study (Prat et al., 2009). These results suggest a rapid hydrolytic
cleavage of the ester moiety of aclidinium bromide once it reaches
systemic circulation, especially in dogs and humans. The contribu-
tion of enzymatic hydrolysis would be much more important in
dog and human plasma compared with rat and guinea pig plasma.
Additionally, aclidinium showed non-enzymatic hydrolysis of
its ester bond at neutral and basic pH. Acid-catalysed hydrolysis
was not observed below pH 4 and changes in pH near 7.4 caused
an important change in the rate of hydrolysis, confirming that acli-
dinium hydrolysis is very sensitive to pH in the physiological range.
The marked difference in the rate of aclidinium hydrolysis observed
in plasma across the species (t1/2 = 0.03–0.64 h) and in phosphate
buffer at pH 7.4 (t1/2 = 1.2 h) suggests that enzymatic hydrolysis
plays a major role in the overall hydrolysis of aclidinium. Under the
experimental conditions used in this study, the tiotropium plasma
half-lives were higher than that of aclidinium and were similar in
all species evaluated (1–2 h).
In healthy subjects, pharmacokinetic studies of aclidinium up
to 6000 ␮g by the inhaled route have shown rapid elimination
from systemic circulation (t1/2 <1.5 h), consistent with a rapid in
vivo plasma hydrolysis of aclidinium (Ferrer et al., 2008; Jansat
et al., 2009). Biological and chemical hydrolysis of the ester bond
of aclidinium in vitro results in two major compounds, the alco-
hol (LAS34823) and the carboxylic acid (LAS34850) metabolites.
Both metabolites have also been detected in human plasma after
an inhaled dose of aclidinium (400 ␮g and 800 ␮g), likely as a result
of a rapid hydrolysis in plasma, where the major circulating com-
pound was the acid derivative. The systemic exposure of the acid
Systemic exposure of anti-cholinergics has been associated with
unwanted physiological effects such as tachycardia, dry mouth, uri-
nary retention and constipation (Lieberman III, 2004). Aclidinium
undergoes rapid hydrolysis in plasma, resulting in low and tran-
sient systemic exposure to the active drug. The in vitro and in vivo
characterisation of both metabolites was performed to determine
the potential pharmacological contribution of these metabolites. In
radioligand displacement studies carried out at equilibrium using
3H-NMS, aclidinium demonstrated high subnanomolar affinity for
all human muscarinic receptor subtypes analysed. In contrast, the
two major metabolites showed no significant affinity for human
muscarinic receptors up to 10 ␮M, with displacement being lower
than 10% in all cases. The data obtained for aclidinium are consis-
tent with those previously described (Gavaldà et al., 2009).
The in vitro pharmacological selectivity profile studies were per-
formed with both aclidinium metabolites. The alcohol (LAS34823)
and carboxylic acid metabolites (LAS34850) have no relevant affin-
ity for any of the receptors and enzymes tested. This, together with
the high selectivity for the muscarinic receptors reported previ-
ously for aclidinium (Gavaldà et al., 2009) and its low systemic
exposure, suggests that aclidinium and its main metabolites may
have reduced potential in triggering unwanted anti-cholinergic
side effects. This is supported by the results from the safety stud-
ies of aclidinium in rats, guinea pigs and dogs, where aclidinium
bromide has demonstrated reduced effects on the cardiovascular,
gastric and renal systems compared with tiotropium and iprat-
ropium (Gras et al., 2008a,b; Montero et al., 2008). Additionally,
inhaled aclidinium in healthy subjects receiving up to 6000 ␮g
also was shown to have a favourable safety and tolerability profile
(Ferrer et al., 2008; Jansat et al., 2009).
The effect of LAS34823 and LAS34850 on the acetylcholine-
induced bronchoconstriction model in guinea pigs was also
assessed in comparison with aclidinium bromide. The two main
aclidinium metabolites did not show any relevant bronchodila-
tory activity in vivo. In contrast, a potent bronchodilatory activity
was observed after inhaled administration of aclidinium, in concor-
dance with results reported previously (Gavaldà et al., 2009). These
data demonstrate that the activity observed in vivo corresponds
exclusively to aclidinium bromide without contribution from its
hydrolysis products.
In summary, the data reported here show that aclidinium is
rapidly hydrolysed in plasma, likely due to the enzymatic cleav-
age of its ester, to yield its alcohol and carboxylic acid derivatives
in all species studied. These main metabolites of aclidinium are
devoid of any significant affinity for the muscarinic receptors, or a
wide panel of selected G-protein coupled receptors, ion channels or
Fig. 6. Effect of aerosolised aclidinium and its metabolites on the acetylcholine-
induced bronchoconstriction in anaesthetised guinea pigs. Reversal of bron-
chospasm values are represented as mean SEM. Data are calculated from an
average of three or four animals.

290
S. Sentellas et al. / European Journal of Pharmaceutical Sciences 39 (2010) 283–290
enzymes studied, and did not show any relevant bronchodilatory
activity in vivo. The rapid cleavage to inactive metabolites and the
low systemic exposure of aclidinium, together with its long res-
idence time at the muscarinic M3 receptor, confer this molecule
a unique mechanism of action profile that supports a favourable
safety profile and benefit-to-risk ratio in the clinical practice.
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We would like to acknowledge the significant technical support
provided by Francisco Jiménez, Julian Prieto and Enric Villanova
(Almirall). This work was funded by Laboratorios Almirall S.A.,
Spain.
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