Identification of mepenzolate derivatives with long-acting bronchodilatory activity

Article abstract of DOI:10.3389/fphar.2018.00344

The standard treatment for chronic obstructive pulmonary disease is a combination of anti-inflammatory drugs and bronchodilators. We recently found that mepenzolate bromide (MP), an antagonist for human muscarinic M3 receptor (hM₃R), has both anti-inflammatory and short-acting bronchodilatory activities. To obtain MP derivatives with longer-lasting bronchodilatory activity, we synthesized hybrid compounds based on MP and two other muscarinic antagonists with long-acting bronchodilatory activity glycopyrronium bromide (GC) and aclidinium bromide (AD). Of these three synthesized hybrid compounds (MP-GC, GC-MP, MP-AD) and MP, MP-AD showed the highest affinity for hM₃R and had the longest lasting bronchodilatory activity, which was equivalent to that of GC and AD. Both MP-GC and MP-AD exhibited an anti-inflammatory effect equivalent to that of MP, whereas, in line with GC and AD, GC-MP did not show this effect. We also confirmed that administration of MP-AD suppressed elastase-induced pulmonary emphysema in a mouse model. These findings provide important information about the structure-activity relationship of MP for both bronchodilatory and anti-inflammatory activities.

Full text of DOI:10.3389/fphar.2018.00344

ORIGINAL RESEARCH
published: 10 April 2018
doi: 10.3389/fphar.2018.00344
Identification of Mepenzolate
Derivatives With Long-Acting
Bronchodilatory Activity
Ken-Ichiro Tanaka1†, Naoki Yamakawa2†, Yasunobu Yamashita3, Teita Asano4,
Yuki Kanda1, Ayaka Takafuji1, Masahiro Kawahara1, Mitsuko Takenaga4,
Yoshifumi Fukunishi3 and Tohru Mizushima5
*
1 Research Institute of Pharmaceutical Sciences, Faculty of Pharmacy, Musashino University, Nishi-Tokyo, Japan, 2 School of
Pharmacy, Shujitsu University, Okayama, Japan, 3 Molecular Profiling Research Center for Drug Discovery, National Institute
of Advanced Industrial Science and Technology, Tokyo, Japan, 4 Institute of Medical Science, School of Medicine,
St. Marianna University, Kawasaki, Japan, 5 LTT Bio-Pharma Co., Ltd., Tokyo, Japan
The standard treatment for chronic obstructive pulmonary disease is a combination
of anti-inflammatory drugs and bronchodilators. We recently found that mepenzolate
bromide (MP), an antagonist for human muscarinic M3 receptor (hM3R), has both
anti-inflammatory and short-acting bronchodilatory activities. To obtain MP derivatives
with longer-lasting bronchodilatory activity, we synthesized hybrid compounds based
on MP and two other muscarinic antagonists with long-acting bronchodilatory activity
glycopyrronium bromide (GC) and aclidinium bromide (AD). Of these three synthesized
hybrid compounds (MP-GC, GC-MP, MP-AD) and MP, MP-AD showed the highest
affinity for hM3R and had the longest lasting bronchodilatory activity, which was
equivalent to that of GC and AD. Both MP-GC and MP-AD exhibited an anti-
inflammatory effect equivalent to that of MP, whereas, in line with GC and AD,
GC-MP did not show this effect. We also confirmed that administration of MP-AD
suppressed elastase-induced pulmonary emphysema in a mouse model. These findings
provide important information about the structure-activity relationship of MP for both
bronchodilatory and anti-inflammatory activities.
Edited by:
Weien Yuan,
Shanghai Jiao Tong University, China
Reviewed by:
Thiago J. Borges,
Universidade La Salle-Canoas, Brazil
Michela De Bellis,
Università degli Studi di Bari Aldo
Moro, Italy
Erzsébet Bartolák-Suki,
Boston University, United States
*Correspondence:
Tohru Mizushima
t.mizushima@ltt.co.jp
†These authors have contributed
equally to this work.
Keywords: chronic obstructive pulmonary disease, anti-inflammatory, long-acting bronchodilatory activity,
mepenzolate
Specialty section:
This article was submitted to
Translational Pharmacology,
a section of the journal
Frontiers in Pharmacology
INTRODUCTION
Received: 28 September 2017
Accepted: 26 March 2018
Published: 10 April 2018
Chronic obstructive pulmonary disease (COPD) is a serious health problem and the world’s fourth
leading cause of death (Mannino and Kiriz, 2006). This disease state is defined by a progressive and
not fully reversible airflow limitation and an abnormal inflammatory response (Rabe et al., 2007;
Vestbo et al., 2013; Vogelmeier et al., 2017). For the clinical treatment of COPD, it is therefore
important not only to improve airflow limitations by inducing bronchodilation, but also to hinder
disease progression by suppressing inflammatory responses.
The standard treatment for COPD is to use bronchodilators such as β2-agonists and muscarinic
receptor antagonists, with this approach being effective in overcoming airflow limitations (Barnes
and Stockley, 2005; Vestbo et al., 2013; Vogelmeier et al., 2017). Short-acting β2-agonists (SABAs)
and muscarinic antagonists (SAMAs) were previously used for COPD treatment; however, long-
acting β2-agonists (LABAs) and muscarinic antagonists [LAMAs, of which tiotropium bromide
Citation:
Tanaka K-I, Yamakawa N,
Yamashita Y, Asano T, Kanda Y,
Takafuji A, Kawahara M,
Takenaga M, Fukunishi Y and
Mizushima T (2018) Identification
of Mepenzolate Derivatives With
Long-Acting Bronchodilatory Activity.
Front. Pharmacol. 9:344.
doi: 10.3389/fphar.2018.00344
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Tanaka et al.
Identification of Mepenzolate Derivatives
(TP), glycopyrronium bromide (GC), and aclidinium bromide precursor of hybrid compound (pre-Hyb) was synthesized
(AD) are common examples] have shown superior efficacy over by the condensation of carboxylic acid (BA or CA) and
the short-acting bronchodilators for the treatment of COPD amino-alcohol (PR, PP, or QC). The each final quaternary
(Vestbo et al., 2013; Vogelmeier et al., 2017). Thus, LABAs and salt of hybrid compounds (MP-GC, GC-MP, MP-AD) was
LAMAs (or combinations of the two) are now used as first-line prepared as described previously (Yamashita et al., 2014), with
drugs for COPD treatment.
some modifications (see below). Column chromatography was
On the other hand, the inflammatory responses associated performed using Silica gel 60N (Kanto Chemical, Co., Tokyo,
with COPD are generally treated with steroids; however, their Japan).
impact on reducing disease progression and mortality (Barnes
Melting point (mp) data were recorded on Yanaco MP-
and Stockley, 2005; Calverley et al., 2007; Tashkin et al., 2008) is J3 micro-melting point apparatus. 1H-NMR spectra and 13C-
attenuated because COPD-associated inflammation often shows NMR spectra were measured at 400 and 100 MHz, respectively,
resistance to steroid treatment (Alsaeedi et al., 2002; Calverley on a VARIAN 400-MR spectrometer. High resolution mass
et al., 2007). Thus, it is important for new types of anti- spectra were detected with an ESI-TOF mass spectrometer
inflammatory compounds to be developed for the treatment of (Bruker MicroTOF, Bruker, Bremen, Germany) in the positive
COPD.
mode. HPLC data were recorded on a Waters 2695 Alliance
Based on the aforementioned, we previously screened a separation module and a Waters 2996 photodiode array detector
library of approved medicines to identify compounds capable (Waters, Milford, MA, United States) with a reverse-phase
of preventing elastase-induced pulmonary emphysema in mice column (TSKgel Super-ODS, 150 mm × 4.6 mm, 2 µm,
(an animal model of COPD), and selected mepenzolate bromide Tosoh, Co., Tokyo, Japan). CH3CN/20 mM KH2PO4 buffer
(MP) (Tanaka et al., 2013), which is an orally administered (3:7) was used as the mobile phase at a flow rate of
muscarinic receptor antagonist used to treat gastrointestinal 0.3 ml/min, with detection performed at an optical density of
disorders (Long and Keasling, 1954; Buckley et al., 1957; Chen, 210 nm.
1959). We showed that MP not only exerts an anti-inflammatory
General Procedure for the Preparation of
pre-Hyb
effect via a muscarinic receptor-independent mechanism, but also
possesses a short-acting bronchodilatory effect via a muscarinic
receptor-dependent mechanism [via its binding to human
muscarinic M3 receptor (hM3R)] (Tanaka et al., 2013, 2014;
Kurotsu et al., 2014). The anti-inflammatory activity of MP,
which was more potent than that of steroids (Tanaka et al.,
2012, 2013), is mediated via a decreased level of reactive oxygen
species (ROS) in the lung due to the inhibition of NADPH
oxidase activity and the induction of superoxide dismutase (SOD)
and glutathione-S-transferase (GST) expression (Tanaka et al.,
2013).
To a solution of 2-alkyl-2-hydroxy-2-phenylacetic acid (BA or
CA) (1.0 eq.) in DMF, carbonyldiimidazole (1.6 eq.) was added
and the mixture was stirred for 15 min at room temperature.
A solution of N-alkyl amino alcohol (PR, PP, or QC) (1.2 eq.)
in DMF was added dropwise into the mixture at 80◦C and the
mixture was stirred for 18 h at that temperature. The reaction was
quenched with H2O at room temperature and organic materials
were extracted with EtOAc. The combined extract was washed
with brine, dried over Na2SO4 and concentrated in vacuo. The
residue was then applied to a short silica gel column, eluted
with EtOAc and concentrated in vacuo to give title compound
(pre-Hyb), which was used for the next step without further
purification.
In the present study, we examined MP derivatives that
have both anti-inflammatory and longer-acting bronchodilatory
activities, as these could be beneficial for the treatment of
COPD. To this end, we synthesized hybrid compounds from
MP and long-acting muscarinic antagonists (GC and AD) and
identified one compound that showed both a longer duration
of bronchodilatory activity (equivalent to GC and AD) together
with anti-inflammatory properties consistent with those of MP.
(±)-3-(2-Hydroxy-2,2-diphenylacetoxy)-1,1-
dimethylpyrrolidinium Bromide
(MP-GC)
2.0 g (8.7 mmol) of benzilic acid (BA) and 1.3 mL (11 mmol)
of 1-methylpyrrolidin-3-ol (PR) were used to give pre-Hyb. To
a solution of pre-Hyb (2.2 g, 7.0 mmol) in CH3CN (12 mL),
CH3Br (2.0 M in THF, 18 mL) was added and stirred for 5 h at
room temperature. The precipitate was filtered off and the filtrate
MATERIALS AND METHODS
Chemistry
Muscarinic M3 receptor antagonists were purchased from re-crystallized with CH3CN and diethyl ether to give the title
commercial sources; mepenzolate bromide (MP) was from compound as a white solid (2.0 g, 69% 2 steps). mp: 211–212◦C;
Sigma-Aldrich (St. Louis, MO, United States); tiotropium 1H-NMR (DMSO-d6): δ = 7.29–7.38 (m, 10H), 6.81 (s, 1H), 5.51
bromide (TP) and glycopyrronium bromide (GC) were from (brs, 1H), 3.84–3.89 (m, 1H), 3.52–3.70 (m, 3H), 3.6 (s, 3H),
Tokyo Chemical Industry, Co., Ltd. (Tokyo, Japan); aclidinium 2.93 (s, 3H), 2.62–2.74 (m, 1H), 2.08–2.14 (m, 1H); 13C-NMR
bromide (AD) was from Toronto Research Chemicals (Toronto, (DMSO-d6): d = 172.3, 142.9, 127.9, 127.7, 127.0, 80.6, 73.3, 69.3,
ON, Canada). All other solvents and reagents for organic 64.0, 52.7, 51.9, 40.2, 38.9, 29.9; HRMS: calcd. for C20H24NO3
syntheses were purchased from Tokyo Chemical Industry, (M+): 326.1751; found: m/z = 326.1755; HPLC: tR (min) = 3.12
Co., Ltd. (Tokyo, Japan) or Wako Pure Chemical Industries (98%). This is a known compound, but its spectrum data are not
(Tokyo, Japan) and used without further purification. The covered by patents or non-patent literature documents.
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Identification of Mepenzolate Derivatives
described here were carried out in accordance with the Guide
for the Care and Use of Laboratory Animals as adopted
and promulgated by the National Institutes of Health, and
were approved by the Animal Care Committee of Musashino
University (Approval NOs. 16004 and 17006).
(±)-3-(2-Cyclopentyl-2-hydroxy-2-phenylacetoxy)-
1,1-dimethylpiperidinium Bromide
(GC-MP)
1.5 g (6.8 mmol) of 2-cyclopentyl-2-hydroxy-2-phenylacetic
acid (CA) and 1.0 mL (8.1 mmol) of 1-methylpiperidin-3-
ol (PP) were used to give pre-Hyb. To a solution of pre-
Hyb (0.41 g, 1.3 mmol) in CH3CN (5 mL), CH3Br (2.0 M
in THF, 6.4 mL) was added and stirred for 5 h at room
temperature. The precipitate was filtered off and the filtrate
re-crystallized with CH3CN and diethyl ether to give the title
compound as a white solid (0.38 g, 22% 2 steps). mp: 164–
165◦C; 1H-NMR (DMSO-d6): δ = 7.56–7.59 (d, J = 7.5,
2H), 7.25–7.37 (m, 3H), 5.14 (brs, 1H), 3.37–3.62 (m, 4H),
2.89–3.06 (m, 7H), 1.23–1.79 (m, 12H); 13C-NMR (DMSO-d6):
δ = 173.0, 142.2, 142.7, 128.0, 127.3, 125.7, 79.3, 66.1, 61.5,
60.8, 46.4, 40.1, 38.9, 26.6, 26.0, 25.6, 25.4, 16.4; HRMS calcd.
for C20H30NO3 (M+): 332.2220; found: m/z = 332.2224; HPLC:
tR (min) = 4.94 (98%). This is a known compound, but its
spectrum data are not covered by patents or non-patent literature
documents.
Treatment of Mice With PPE and Each
Drug
Mice maintained under anesthesia with isoflurane were
administered one intratracheal bolus of PPE (15 or 20 U/kg), MP
(3.8, 37.5 µg/kg), TP (8.4 µg/kg), GC (7.1, 35.5 µg/kg), AD (10.1,
50.4 µg/kg), MP-GC (3.6, 36.2 µg/kg), GC-MP (3.7, 36.8 µg/kg),
and MP-AD (4.9, 49.3 µg/kg) in 0.9% NaCl via micropipette
(P200). For control mice, 0.9% NaCl alone was administered by
the same procedure.
Figure 3; MP, GC, AD, MP-AD, MP-GC, or GC-MP were
administered intratracheally. After 1, 24, 48, or 72 h, mice were
exposed to nebulized methacholine 5 times and airway resistance
was determined after each methacholine challenge.
Figure 4 and Table 2; MP, TP, GC, AD, MP-AD, MP-
GC, and GC-MP were administered intratracheally once only.
After 1 h, mice were treated with or without (control) PPE
(20 U/kg) once only. Six (Figure 4) or twenty-four (Table 2)
hours after the PPE administration, BALF was prepared and
the total cell number and the number of neutrophils were
determined.
Figure 5; MP-AD was administered intratracheally once
daily for 16 days (from day 0 to day 15). 1 h after the first
administration, mice were treated with or without (control)
PPE (15 U/kg) once only on day 0. 21 days after the PPE
administration, lung mechanics was monitored, and sections of
pulmonary tissue were prepared.
(±)-3-(2-Hydroxy-2,2-diphenylacetoxy)-1-(3-
phenoxypropyl)-1-azoniabicyclo[2.2.2]octane
Bromide (MP-AD)
1.6 g (0.60 mmol) of benzilic acid (BA) and 1.0 g (7.9 mmol)
of quinuclidin-3-ol (QC) were used to give pre-Hyb. To a
solution of pre-Hyb (0.10 g, 0.30 mmol) in 1,4-dioxane (5 mL)
(3-bromopropoxy)benzene (0.11 g, 11 mmol) was added and
stirred for 6 days at room temperature. The precipitate was
filtered off and the filtrate re-crystallized with CH3CN and diethyl
ether to give the title compound as a white solid (50 mg, 30%
2 steps). mp: 177–178◦C; 1H-NMR (DMSO-d6): δ = 7.23–
7.42 (m, 12H), 6.86–6.89 (m, 3H), 6.78 (s, 1H), 5.22 (brs,
1H), 3.94–4.04 (m, 2H), 3.85–3.88 (m, 1H), 3.25–3.44 (m, 5H),
3.01–3.07 (m, 1H), 1.45–2.25 (m, 7H); 13C-NMR (DMSO-d6):
δ = 173.0, 158.6, 143.6, 143.5, 130.1, 128.5, 128.4, 128.2, 127.6,
127.5, 121.5, 115.0, 81.3, 69.1, 65.1, 61.1, 60.0, 54.3, 53.2, 24.0,
22.2, 20.9, 18.1; HRMS calcd. for C30H34NO4 (M+): 472.2482;
found: m/z = 472.2488; HPLC: tR (min) = 9.26 (99%). This
is a known compound whose 1H-NMR spectrum was similar
to that previously reported as (R)-MP-GC (Yamashita et al.,
2014).
Preparation of Bronchoalveolar Lavage
Fluid (BALF) and Cell Count Method
BALF was collected from mice by cannulating the trachea
and lavaging the lung with 1 ml of sterile PBS containing
50 U/ml heparin (2 times). About 1.8 ml of BALF was routinely
recovered from each animal. The total cell number in the BALF
was counted using a hemocytometer and a manual counter.
Cells were stained with Diff-Quik reagents after centrifugation
with CytospinꢀR 4 (Thermo Electron Corporation, Waltham,
MA, United States), and differential counts of more than 50
inflammatory cells were done in each of four or more separate
sections of smear using fluorescence microscopy (Olympus
DP71). The ratio of number of neutrophils to total cell number
was determined by counting the number of neutrophils. This cell
counting was conducted by an investigator blinded to the study
protocol.
Chemicals
Porcine pancreatic elastase (PPE) and methacholine chloride
(methacholine) were obtained from Sigma-Aldrich (St. Louis,
MO, United States). Novo-Heparin for injection was from
Mochida Pharmaceutical, Co. (Tokyo, Japan). Chloral hydrate
was from Nacalai Tesque (Kyoto, Japan). Diff-Quik was from
Sysmex, Co. (Kobe, Japan). Formalin neutral buffer solution
was from WAKO Pure Chemicals (Tokyo, Japan). Mayer’s
hematoxylin, 1% eosin alcohol solution and malinol were from
MUTO Pure Chemicals (Tokyo, Japan).
Measurement of Lung Mechanics and
Airway Resistance
Lung mechanics and airway resistance were monitored with
Animals
a
computer-controlled small-animal ventilator (FlexiVent,
ICR mice (6–7 weeks old, male) were purchased from Charles SCIREQ, Montreal, QC, Canada), as described previously
River (Yokohama, Japan). The experiments and procedures (Shalaby et al., 2010; Tanaka et al., 2012). Mice were anesthetized
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TABLE 2 | Effect of intratracheal administration of compounds on PPE-induced
inflammatory responses.
with chloral hydrate (500 mg/kg), a tracheotomy was performed,
and an 8 mm section of metallic tube was inserted into the
trachea. Mice were mechanically ventilated at a rate of 150
breaths/min, using a tidal volume of 8.7 ml/kg and a positive
end-expiratory pressure of 2–3 cmH2O.
Total respiratory system compliance was measured by the
snapshot. All data were analyzed using FlexiVent software
(version 5.3; SCIREQ, Montreal, QC, Canada).
Mice were exposed to nebulized methacholine (5 mg/ml) five
times for 20 s with a 40 s interval, and airway resistance was
measured after each methacholine challenge by the snapshot
technique. All data were analyzed using FlexiVent software.
Total cells (×105 cells)
Neutrophils (×105 cells)
Control
1.26 ± 0.25
9.66 ± 0.54∗∗
6.30 ± 0.90##
10.00 ± 1.27
10.05 ± 0.57
10.20 ± 0.93
9.09 ± 1.09
11.88 ± 2.01
9.75 ± 1.12
0.12 ± 0.07
7.46 ± 0.57∗∗
4.11 ± 0.68##
6.81 ± 1.03
7.31 ± 0.22
7.34 ± 0.92
6.25 ± 0.83
9.06 ± 1.63
6.86 ± 0.88
PPE
PPE + MP (37.5 µg/kg)
PPE + TP (8.4 µg/kg)
PPE + TP (42.1 µg/kg)
PPE + GC (7.1 µg/kg)
PPE + GC (35.5 µg/kg)
PPE + AD (10.1 µg/kg)
PPE + AD (50.4 µg/kg)
Mice were treated with or without (control) PPE (20 U/kg) once only on day 0.
The indicated doses (µg/kg) of each compound were administered intratracheally
once only on day 0. Twenty-four hours after the PPE administration, BALF was
prepared and the total cell number and the number of neutrophils were determined
as described in Section “Materials and Methods.” Values represent mean ± SEM
(n = 4–8). ∗∗P < 0.01 (versus control); ##P < 0.01 (versus PPE only). Data are
representative of two independent experiments.
Histopathological Analysis
Twenty one days after PPE treatment, mice were euthanized
with an excess of isoflurane. Lung tissue samples were fixed
in 10% formalin neutral buffer solution for 24 h at a pressure
of 25 cmH2O, and then embedded in paraffin before being
cut into 4 µm-thick sections. Sections were stained first
with Mayer’s hematoxylin and then with 1% eosin alcohol
solution (H&E staining). Samples were mounted with malinol
and scanned using a NanoZoomer-XR digital slide scanner
(Hamamatsu Photonics, Shizuoka, Japan). All images were
analyzed using NDP.view2 software (Hamamatsu Photonics,
Shizuoka, Japan).
To determine the mean linear intercept (MLI) (an indicator of
airspace enlargement), 20 lines (800 µm) were drawn randomly
on the image of a section and intersection points with alveolar
walls were counted to determine the MLI, as described previously
(Weibel, 1963). This morphometric analysis was conducted by an
investigator blinded to the study protocol.
Filter-Binding Assay
The filter-binding assay was performed as described previously
(Yamashita et al., 2014), with some modifications. Membrane
fractions prepared from Chinese Hamster Ovary (CHO)-K1 cells
expressing human muscarinic M2 receptor (hM2R) or hM3R
(Membrane Target Systems, Perkin-Elmer Life and Analytical
Sciences, Boston, MA, United States; protein concentration,
8 µg/well) were incubated with 2 nM [3H]NMS (N-methyl-
[3H]-scopolamine methylchloride) (85.5 Ci/mmol) at room
temperature for 2 h in 200 µL PBS in the presence of each
compound. A range of concentrations (10−11 to 10−6 M) for
each compound was tested in triplicate to generate competitive
binding curves. Non-specific binding was determined in the
presence of atropine (2.5 µM). The samples were passed
through a GF/C filter (Filtermat A, PerkinElmer Life and
Analytical Sciences, Boston, MA, United States) pre-incubated
for 1 h with 1.0% polyethylenimine, and washed four times
with ice-cold wash buffer [50 mM Tris/HCl (pH 7.4), 100 mM
NaCl, 0.05% Tween-80]. Filters were then dried for 30 min
before attachment to a MeltiLex A melt-on scintillation
sheet (PerkinElmer Life and Analytical Sciences, Boston, MA,
United States). The radioactivity remaining on the filter was
monitored with a MicroBeta Trilux microplate scintillation
counter (PerkinElmer Life and Analytical Sciences, Boston, MA,
FIGURE 1 | Structure of clinically-used muscarinic antagonists.
TABLE 1 | Binding of compounds to hM2R or hM3R.
Compound
Ki (nM)
hM3R
hM2R
Mepenzolate (MP)
Tiotropium (TP)
Glycopyrronium (GC)
Aclidinium (AD)
MP-GC
4.6
0.22
0.7
1.8
0.64
1.0
0.34
1.1
0.39
0.79
0.53
0.64
GC-MP
1.2
MP-AD
0.72
Membrane fractions prepared from CHO-K1 cells expressing hM2R or hM3R were
incubated with 2 nM [3H]NMS (85.5 Ci/mmol) in the presence of each compound.
A range of concentrations (10−11 to 10−6 M) for each compound was tested in
triplicate to generate competitive binding curves to determine Ki values. The shown
value is an average of at least two independent experiments.
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FIGURE 2 | Structure of hybrid compounds. The chemical structure of each compound [mepenzolate (MP), glycopyrronium (GC), and aclidinium (AD)] was
separated into parts left and right of the ester bond. These parts were then used to synthesize the shown hybrid structures.
SCHEME 1 | Synthesis of compounds MP-GC, GC-MP, and MP-AD.
United States). Affinities at equilibrium were determined as three independent curves. All curve-fitting procedures were
antagonist dissociation constant (Ki) values after correcting the performed using Prism (GraphPad Software, Inc., San Diego, CA,
experimentally determined IC50 values with the experimentally United States).
determined Kd value of NMS for hM2R or hM3R and the
Statistical Analysis
concentration of NMS, as described previously (Yamashita
et al., 2014). Shown values are an average of at least two All values are expressed as the mean ± SEM. Homoscedasticity
independent experiments. The Ki value was obtained from of data were verified by Levene test, then, data were examined
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using one-way ANOVA followed by the Tukey test or Games-
Howell test for unpaired results was used to evaluate differences
between three or more groups. Differences were considered to be
statistically significant for values of P < 0.05.
RESULTS
Affinity for hM3R and Anti-inflammatory
Activities of MP, TP, GC, and AD
We first compared the binding of MP, TP, GC, and AD to hM3R
and hM2R (Figure 1). The binding affinity of each compound
to each receptor was determined by carrying out [3H]NMS
displacement studies on each receptor. A higher Ki value means
a lower affinity for hM3R, because Ki value is related to the
concentration of each compound that causes 50% inhibition of
NMS-binding to hM3R. As shown in Table 1, MP showed the
highest Ki value (by a factor of at least five compared with TP,
GC, and AD), thus indicating it had the lowest affinity for hM3R.
Similar results were observed for hM2R (Table 1).
The total number of leucocytes and the individual number of
neutrophils in BALF are indicators of pulmonary inflammatory
responses; these increased after PPE treatment in a manner that
could be partially suppressed by the simultaneous intratracheal
administration of MP (Table 2), as described previously (Tanaka
et al., 2013, 2014). Because compared to neutrophils, neither the
macrophage nor eosinophil number was so apparently increased
in BALF after PPE treatment (Tanaka et al., 2013, 2014), we
did not count them here. As none of TP, GC, and AD showed
anti-inflammatory activity equivalent to that of MP (Table 2),
we attempted to identify MP derivatives that had an affinity
for hM3R equivalent to that of TP, GC, and AD and an anti-
inflammatory activity equivalent to that of MP.
Synthesis of Hybrid Compounds and
Their Affinities for hM3R
We synthesized hybrid compounds based on MP and LAMAs
used in the clinical setting (GC and AD). The structures of
MP and these LAMAs were separated into two parts with
a view to synthesizing hybrid compounds by substitution of
each of the parts (Figure 2). We synthesized mepenzolate-
glycopyrronium (MP-GC), glycopyrronium-mepenzolate (GC-
MP) and mepenzolate-aclidinium (MP-AD) (Figure 2). The
synthetic route for these compounds are outlined in Scheme 1.
First, the ester compound (pre-Hyb) was synthesized by the
condensation of an appropriate carboxylic acid (BA or CA)
and aminoalcohol (PR, PP, or QC). The precursor of target
compound (pre-Hyb) was quaternized with the appropriate
methyl bromide or phenoxy alkyl bromide to give target
compounds (MP-GC, GC-MP, and MP-AD). These compounds
are known and were identified by their melting points, NMR
spectra, and mass spectra. On the other hand, we could
not synthesize a hybrid compound based on aclidinium-
mepenzolate.
FIGURE 3 | Effect of compounds on methacholine-induced airway
constriction. Indicated doses of MP (A), GC, AD, and MP-AD (B) or MP-GC
or GC-MP (C) were administered intratracheally. After the indicated period,
mice were exposed to nebulized methacholine 5 times and airway resistance
was determined after each methacholine challenge as described in Section
“Materials and Methods.” Values represent mean ± SEM. ∗P < 0.05;
∗∗P < 0.01; n.s., not significant. The control data is the same in all graphs.
Data are representative of two independent experiments.
As shown in Table 1, all of the hybrid compounds had a lower
Ki value for hM3R than MP. Of the target compounds, MP-AD
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FIGURE 4 | Effect of compounds on PPE-induced inflammatory responses. Mice were treated with or without (control) PPE (20 U/kg) once only on day 0. The
indicated doses (µg/kg) of MP and MP-AD (A) or MP, GC, MP-GC, or GC-MP (B) were administered intratracheally once only. Six hours after the PPE
administration, BALF was prepared and the total cell number and the number of neutrophils were determined as described in Section “Materials and Methods.”
Values represent mean ± SEM. ∗∗P < 0.01 (versus control); #P < 0.05, ##P < 0.01 (versus PPE only); n.s., not significant. Data are representative of two
independent experiments.
showed the lowest Ki value for hM3R (highest affinity for hM3R), examined the duration of bronchodilation for MP-GC and GC-
while GC-MP showed the lowest Ki value for hM2R.
MP. Both of these compounds showed bronchodilatory activity
at 1 and 24 h, but not at 48 h (Figure 3C), suggesting that MP-
GC and GC-MP exhibit bronchodilatory activity intermediate to
that of MP and MP-AD.
Bronchodilation Activities of Hybrid
Compounds
As shown in Figure 3A, the intratracheal administration of MP
induced bronchodilation in a dose-dependent manner, which is
consistent with our previously reported findings (Tanaka et al.,
2013, 2014). We also confirmed that the bronchodilatory activity As shown in Figure 4A, the anti-inflammatory activity of MP-
of MP disappeared within 24 h (Figure 3A), suggesting that its AD was similar to that of MP. We also found that MP-GC
effects were short-acting.
Anti-inflammatory and Anti-emphysemic
Activities of Hybrid Compounds
exhibited anti-inflammatory activity, but as for GC, GC-MP did
We then compared these results with the duration of not (Figure 4B). These results suggest that the double phenyl
bronchodilatory activity of GC, AD, and MP-AD. As shown rings in MP are important for its anti-inflammatory activity.
in Figure 3B, these compounds all exhibited dose-dependent
We next examined the effect of the long-term (up to
bronchodilatory activity within 1 h of their administration. 15 days) administration of MP-AD on PPE-induced lung
Compared to baseline, significantly increased bronchodilation injury. Histopathological analysis revealed that while PPE
(reduced airway resistance) was observed 48 h (but not 72 h) after administration damaged the alveolar walls and increased MLI,
the administration, suggesting that MP-AD has a long-lasting this effect could be partially suppressed by the administration of
bronchodilatory activity, similar to that of GC and AD. We also MP-AD (Figures 5A,B). We could not find any adverse effects
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Identification of Mepenzolate Derivatives
FIGURE 5 | Effect of MP-AD on PPE-induced pulmonary damage. Mice were treated with or without (control) PPE (15 U/kg) once only on day 0. The indicated
doses of MP-AD were administered intratracheally once daily for 16 days (from day 0 to day 15). Sections of pulmonary tissue were prepared on day 21 and
subjected to histopathological examination (H&E staining) (scale bar: 2.5 mm). (A) Airspace size was estimated by determining the MLI as described in Section
“Materials and Methods.” (B) Total respiratory system compliance was determined on day 21 as described in Section “Materials and Methods.” (C) Values represent
mean ± SEM. ∗∗P < 0.01 (versus control); #P < 0.05 (versus PPE only). Data are representative of two independent experiments.
(such as decrease in body weight or behavior abnormalities) GC or AD. Among the compounds identified (MP-GC, GC-MP,
in mice subjected to the long-term administration of MP-AD. and MP-AD), MP-AD showed the highest affinity for hM3R and
Treatment of mice with PPE increased total respiratory system the longest bronchodilatory activity. It should be noted that the
compliance (Figure 5C), as described previously (Hamakawa duration of bronchodilation associated with MP-AD is equivalent
et al., 2011). We here found that treatment of mice with MP-AD to that of GC and AD, which are clinically used LAMAs. Based on
restored total respiratory system compliance (Figure 5C). Based these results, we speculate that the N-substituted 3-quinuclidinyl
on these results, we suggest that MP-AD suppresses PPE-induced ester of AD is important for its long-acting bronchodilatory
emphysema through its anti-inflammatory activity.
properties. On the other hand, both MP-GC and MP-AD showed
anti-inflammatory effects similar to MP, whereas GC-MP, like
GC and AD, did not exhibit this effect. Based on these results, we
speculate that the double phenyl rings in MP are important for its
anti-inflammatory activity. Our findings thus provide important
information concerning the structure-activity relationship for
MP, which underlies its bronchodilatory and anti-inflammatory
DISCUSSION
To obtain MP derivatives with longer-acting bronchodilatory
activity, we synthesized hybrid compounds based on MP and
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Tanaka et al.
Identification of Mepenzolate Derivatives
activities. We consider that in silico drug design should be with MP-AD restored total respiratory system compliance
done for this target, because such trial for other diseases was (Figure 5C).
successfully done for other studies (Corbo et al., 2007; De Luca
In this study, we used intratracheal administration of each
et al., 2012). Such structure-activity investigation may also help drug, because this method is convenient for experiments.
to identify compounds with better toxicological profile avoiding However, it is possible that the compound does not distribute
off target effects (De Bellis et al., 2017).
evenly through this method (Tanaka et al., 2010; Sato et al.,
As for the mechanism underlying these effects, the results 2015). The patchy appearance of airspace enlargement on the
from this study support the notion that the bronchodilatory histological section following treatment with MP-AD may be
effect is mediated through an antagonistic action toward explained by this uneven distribution of the drug. Thus, we
hM3R. Concerning the anti-inflammatory effects seen here, should examine the distribution of this drug in the lung after
we previously showed that MP decreases ROS levels in intratracheal administration in future.
the lung by inhibiting NADPH oxidase activity and by
We also confirmed here that MP-AD suppressed elastase-
inducing the expression of SOD and GST, both of which are induced pulmonary damage and emphysema in a COPD mouse
endogenous anti-oxidants (Tanaka et al., 2013). Furthermore, model. Taken together, we propose that MP-AD could serve
we previously described inhibitory effects of MP on nuclear as a new drug candidate for the treatment of COPD, given its
factor-κB and histone deacetylase 2, both of which are long-lasting bronchodilatory and anti-inflammatory activity.
involved in the pathogenesis of COPD (Tanaka et al., 2013).
Although we did not examine the effect of MP-AD on
these inflammation-related proteins, a similar mechanism may
underlie the anti-inflammatory properties of MP-AD. In order
AUTHOR CONTRIBUTIONS
to confirm this, immunohistochemical experiments would be K-IT, NY, YY, YF, and TM: participated in research design. K-IT,
useful.
NY, YY, TA, YK, and AT: conducted experiments. MK, MT, and
We measured airway resistance in mice exposed to nebulized YF: contributed new reagents or analytic tools. K-IT, NY, YY, TA,
methacholine, which is the most representative method for and TM: performed data analysis. K-IT, NY, YY, TA, YK, AT,
monitoring bronchodilatory effects (Ather et al., 2016; Murad MK, MT, YF, and TM: wrote or contributed to the writing of the
et al., 2017). Although this method is convenient, it seems manuscript.
to be an asthmatic-like condition rather than COPD-like
condition. Therefore, the effect of each drug on airway
resistance should be examined in COPD animal model in future.
FUNDING
Furthermore, in emphysema, the most important parameter
is a compliance of pulmonary alveolus or total respiratory This work was supported by Grants-in-Aid for Scientific
system. The compliance correlates best with lung structure Research from the Ministry of Health, Labour and Welfare of
(structural parameters) (Hamakawa et al., 2011). We here Japan. This research was also supported by the Suzuken Memorial
showed that treatment of mice with PPE increased total Foundation, the Pharmacological Research Foundation Tokyo,
respiratory system compliance and that treatment of mice and the Takeda Science Foundation.
action on the sphincter of Oddi, colon and urinary bladder of the dog. Arch. Int.
Pharmacodyn. Ther. 121, 78–84.
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Conflict of Interest Statement: LTT Bio-Pharma Co., Ltd. is developing MP-AD-
related compounds (such as MP) as drugs for the treatment of COPD. TM is
Chairman of LTT Bio-Pharma Co., Ltd. TA and MT are employees of an endowed
research division of LTT Bio-Pharma Co., Ltd.
Tanaka, K., Ishihara, T., Sugizaki, T., Kobayashi, D., Yamashita, Y., Tahara, K., et al.
(2013). Mepenzolate bromide displays beneficial effects in a mouse model of
chronic obstructive pulmonary disease. Nat. Commun. 4:2686. doi: 10.1038/
ncomms3686
The other authors declare that the research was conducted in the absence of any
commercial or financial relationships that could be construed as a potential conflict
of interest.
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et al. (2014). Superiority of pulmonary administration of mepenzolate bromide
over other routes as treatment for chronic obstructive pulmonary disease. Sci.
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00019.2012
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