Chemical modification-mediated optimisation of bronchodilatory activity of mepenzolate, a muscarinic receptor antagonist with anti-inflammatory activity

Article abstract of DOI:10.1016/j.bmc.2019.06.016

The treatment for patients with chronic obstructive pulmonary disease (COPD) usually involves a combination of anti-inflammatory and bronchodilatory drugs. We recently found that mepenzolate bromide (1) and its derivative, 3-(2-hydroxy-2, 2-diphenylacetoxy)-1-(3-phenoxypropyl)-1-azoniabicyclo[2.2.2]octane bromide (5), have both anti-inflammatory and bronchodilatory activities. We chemically modified 5 with a view to obtain derivatives with both anti-inflammatory and longer-lasting bronchodilatory activities. Among the synthesized compounds, (R)-(–)-12 ((R)-3-(2-hydroxy-2,2-diphenylacetoxy)-1-(3-phenylpropyl)-1-azoniabicyclo[2.2.2]octane bromide) showed the highest affinity in vitro for the human muscarinic M3 receptor (hM₃R). Compared to 1 and 5, (R)-(–)-12 exhibited longer-lasting bronchodilatory activity and equivalent anti-inflammatory effect in mice. The long-term intratracheal administration of (R)-(–)-12 suppressed porcine pancreatic elastase-induced pulmonary emphysema in mice, whereas the same procedure with a long-acting muscarinic antagonist used clinically (tiotropium bromide) did not. These results suggest that (R)-(–)-12 might be therapeutically beneficial for use with COPD patients given the improved effects seen against both inflammatory pulmonary emphysema and airflow limitation in this animal model.

Full text of DOI:10.1016/j.bmc.2019.06.016

Bioorganic&MedicinalChemistryxxx(xxxx)xxx–xxx
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Chemical modification-mediated optimisation of bronchodilatory activity of
mepenzolate, a muscarinic receptor antagonist with anti-inflammatory
activity
Yasunobu Yamashitaa,1, Ken-ichiro Tanakab,1, Naoki Yamakawac,1, Teita Asanod, Yuki Kandab,
Ayaka Takafujib, Masahiro Kawaharab, Mitsuko Takenagad, Yoshifumi Fukunishie,
⁎
Tohru Mizushimaf,
a Technology Research Association for Next-Generation Natural Products Chemistry, 2-3-26 Aomi, Koto-ku, Tokyo 135-0064, Japan
b Department of Pharmaceutical Sciences, Musashino University, 1-1-20 Shinmachi, Nishi-Tokyo 202-8585, Japan
c Shujitsu University School of Pharmacy, 1-6-1, Nishigawara, Naka-ku, Okayama 703-8516, Japan
d Institute of Medical Science, St. Marianna University School of Medicine, 2-16-1, Sugao, Miyamae-ku, Kawasaki 216-8512, Japan
e Molecular Profiling Research Center for Drug Discovery (molprof), National Institute of Advanced Industrial Science and Technology (AIST), 2-3-26, Aomi, Koto-ku,
Tokyo 135-0064, Japan
f LTT Bio-Pharma Co., Ltd, Shiodome Building 3F, 1-2-20 Kaigan, Minato-ku, Tokyo 105-0022, Japan
A R T I C L E I N F O
A B S T R A C T
Keywords:
The treatment for patients with chronic obstructive pulmonary disease (COPD) usually involves a combination of
anti-inflammatory and bronchodilatory drugs. We recently found that mepenzolate bromide (1) and its deri-
vative, 3-(2-hydroxy-2, 2-diphenylacetoxy)-1-(3-phenoxypropyl)-1-azoniabicyclo[2.2.2]octane bromide (5),
have both anti-inflammatory and bronchodilatory activities. We chemically modified 5 with a view to obtain
derivatives with both anti-inflammatory and longer-lasting bronchodilatory activities. Among the synthesized
compounds, (R)-(–)-12 ((R)-3-(2-hydroxy-2,2-diphenylacetoxy)-1-(3-phenylpropyl)-1-azoniabicyclo[2.2.2]oc-
tane bromide) showed the highest affinity in vitro for the human muscarinic M3 receptor (hM3R). Compared to 1
and 5, (R)-(–)-12 exhibited longer-lasting bronchodilatory activity and equivalent anti-inflammatory effect in
mice. The long-term intratracheal administration of (R)-(–)-12 suppressed porcine pancreatic elastase-induced
pulmonary emphysema in mice, whereas the same procedure with a long-acting muscarinic antagonist used
clinically (tiotropium bromide) did not. These results suggest that (R)-(–)-12 might be therapeutically beneficial
for use with COPD patients given the improved effects seen against both inflammatory pulmonary emphysema
and airflow limitation in this animal model.
Chemical modification
Mepenzolate
COPD
Inflammatory
Long-acting muscarinic antagonist
1. Introduction
important to suppress the inflammatory processes associated with this
disease.
Chronic obstructive pulmonary disease (COPD) is a serious health
problem that is defined by a progressive and not fully reversible airflow
limitation and an abnormal inflammatory response.1–3 To treat COPD
and to concomitantly elicit improved airflow by bronchodilation, it is
Both β2-agonists and muscarinic receptor antagonists are used
clinically to achieve bronchodilation in the treatment of COPD.2–4
Short-acting bronchodilators were previously used; however, in recent
times long-acting -agonists (LABAs) and muscarinic receptor
Abbreviations: aclidinium, aclidinium bromide; BALF, bronchoalveolar lavage fluid; CDI, carbonyldiimidazole; COPD, chronic obstructive pulmonary disease;
FEV0.1, forced expiratory volume in 0.1 s; FVC, forced vital capacity; glycopyrronium, glycopyrronium bromide; GST, glutathione S-transferase; hM2R, human
muscarinic M2 receptor; hM3R, human muscarinic M3 receptor; HDAC2, Histone deacetylase 2; IFN, interferon; IL, interleukin; KC, keratinocyte chemoattractant;
LABA, long-acting β2-agonist; LAMA, long-acting muscarinic antagonist; mepenzolate, mepenzolate bromide; MLI, mean linear intercept; [3H]NMS, N-methyl-[3H]-
scopolamine methylchloride; PPE, porcine pancreatic elastase; QNB, 3-quinuclidinyl benzilate; ROS, reactive oxygen species; SOD, superoxide dismutase; tiotropium,
tiotropium bromide; TNF, tumor necrosis factor
⁎ Corresponding author.
E-mail address: t.mizushima@ltt.co.jp (T. Mizushima).
1 These authors contributed equally to this work.
https://doi.org/10.1016/j.bmc.2019.06.016
Received 7 May 2019; Received in revised form 6 June 2019; Accepted 6 June 2019
0968-0896/©2019PublishedbyElsevierLtd.
Pleasecitethisarticleas:YasunobuYamashita,etal.,Bioorganic&MedicinalChemistry,https://doi.org/10.1016/j.bmc.2019.06.016

Y. Yamashita, et al.
Bioorganic&MedicinalChemistryxxx(xxxx)xxx–xxx
antagonists (LAMAs) have replaced the shorter-acting ones due to their
superior efficacy.2,3 Tiotropium bromide (tiotropium), glycopyrronium
bromide (glycopyrronium), and aclidinium bromide (aclidinium) are
representative LAMAs used in the clinical setting.
The inflammation associated with COPD is typically treated with
steroids; however, COPD disease progression is not fully suppressed by
steroid treatment4–6 because of resistance in part of the inflammatory
processes to steroid treatment.6,7 Histone deacetylase 2 (HDAC2) seems
to play an important role in the inflammatory responses associated with
COPD.8–10 Because corticosteroids use HDAC2 to suppress inflamma-
tion,8,11,12 the inhibitory effect of cigarette smoke on HDAC2 may be
responsible for the reduced sensitivity of COPD patients to steroid
treatment.8 It is therefore important for new types of anti-inflammatory
compounds to be developed to treat COPD.
From a library of medicines approved in Japan, we screened for
compounds that suppress porcine pancreatic elastase (PPE)-induced
pulmonary emphysema and inflammation in mice, and selected me-
penzolate bromide (mepenzolate),13 an orally administered muscarinic
receptor antagonist used for the treatment of gastrointestinal dis-
orders.14–16 Mepenzolate exhibits not only anti-inflammatory effects via
a muscarinic receptor-independent mechanism, but also provides short-
acting bronchodilatory effects via a muscarinic receptor-dependent
mechanism.13,17,18 We previously reported that mepenzolate showed
anti-inflammatory activity in PPE-induced pulmonary emphysema in
mice.13 We also showed that steroids and other anti-inflammatory
drugs did not demonstrate anti-inflammatory activity in the same PPE
models,19 suggesting that the anti-inflammatory activity of mepenzo-
late is more potent than that of steroids. The anti-inflammatory activity
of mepenzolate is mediated via a decreased level of reactive oxygen
species (ROS) in the lung due to the NADPH oxidase activity and the
induction of superoxide dismutase (SOD) and glutathione S-transferase
(GST) expression.13
Fig. 1. Structure of clinically-used muscarinic antagonists 1–4 and hybrid
compound 5.
dioxane provided the desired corresponding target compounds 6–27.
The enantiomers (R)-(–)-12 and (S)-(+)-12 were synthesized in the
same manner as that for (R)-(–)-1 and (S)-(+)-1 with enantiomerically
pure 3-quinuclidinol instead of racemic 3-quinuclidinol.21 All of the
new target compounds were characterized by NMR and HRMS. Proto-
cols for the synthesis and characterization of final compounds 6–27 and
intermediates 29–50 are described in the supporting information.
2.2. Chemical modifications to mepenzolate
The binding affinity of each compound to hM3R was determined by
carrying out N-methyl-[3H]-scopolamine methylchloride ([3H]NMS)
displacement studies on this receptor. The binding affinities of me-
penzolate (1), tiotropium (2), glycopyrronium (3), aclidinium (4) and
compound 5 (Fig. 1) are shown in Table 1 (data for 1–5 are from our
previous report20). The affinity of 5 for hM3R was higher than that of 1
but lower than that of 2.20
We previously postulated that mepenzolate derivatives possessing
both anti-inflammatory and long-acting bronchodilatory activities
might be beneficial for the treatment of COPD. To obtain such deriva-
tives, we synthesized hybrid compounds based on mepenzolate and
glycopyrronium or aclidinium, and found that one of these hybrid
compounds (3-(2-hydroxy-2, 2-diphenylacetoxy)-1-(3-phenoxypropyl)-
1-azoniabicyclo[2.2.2]octane bromide (5) showed both a longer-acting
bronchodilatory activity (equivalent to glycopyrronium and aclidi-
nium) and possessed anti-inflammatory properties (equivalent to me-
penzolate).20 Based on these results, in the present study we chemically
The total number of leucocytes and the individual number of neu-
trophils in bronchoalveolar lavage fluid (BALF) are indicators of pul-
monary inflammatory responses; we previously showed that increases
in these numbers after PPE treatment were partially suppressed by the
simultaneous intratracheal administration of 1 and 5.13,17,20 On the
other hand, none of 2–4 showed anti-inflammatory activity.20 Based on
these findings, we attempted to identify derivatives of 5 that have an
affinity for hM3R similar to that of 2 and an anti-inflammatory activity
similar to that of 1 and 5. Because the high affinities of drugs for hM2R
are associated with adverse effects on cardiac function,22 compounds
that showed a higher affinity for hM3R than for hM2R were preferred
and thus the focus of the selection process used in this study.
modified
5 to obtain derivative compounds with both anti-in-
flammatory and even longer-acting bronchodilatory activities. As for
synthesized hybrid compounds based on mepenzolate bromide (MP),
glycopyrronium bromide (GC), and aclidinium bromide (AD), both MP-
GC and MP-AD showed anti-inflammatory effects similar to MP,
whereas GC-MP, like GC and AD alone, did not.20 Based on these re-
sults, we speculated here that the double phenyl rings in MP (Fig. 1) are
important for its anti-inflammatory activity and therefore, we avoided
modifying this part. Instead, we chemically modified the terminal
phenyl ring combined with the nitrogen atom of quinuclidine. Among
the derivatives obtained, (R)-(–)-12 showed the highest affinity for
hM3R in vitro and the longest bronchodilatory activity in vivo, with an
anti-inflammatory effect similar to that of 5 and mepenzolate. These
results suggest that (R)-(–)-12 might be therapeutically beneficial in a
clinical setting for COPD patients.
The strategy used for making chemical modifications to 5 is shown
in Fig. 2. We modified the linker moiety of 5 (Scheme 1), first short-
ening (6 and 7) or extending (8 and 9) the linker length of 5. We also
changed the linker moiety of 5 into a carbon chain without a het-
eroatom (10–14), with 12 found to have the highest affinity for hM3R
(Table 1). As for hM2R, 12 showed a slightly lower affinity (Table 1).
A variety of substituents were then introduced into the terminal
phenyl ring combined with the linker of 12 to obtain derivatives thereof
(15–27) (Scheme 1). However, the simplest analogue 12, which had no
substituent on the terminal phenyl ring, showed the highest affinity for
hM3R. Based on these results, 12 was selected for further analysis.
2. Results
2.1. Chemistry
2.3. Synthesis and activity of enantiomers of mepenzolate derivatives
The route followed to synthesize target compounds 6–14 and 15–27
is outlined in Scheme 1. QNB (3-quinuclidinyl benzilate) (28) was the
key compound used for synthesizing all target compounds. Subsequent
quaternization of 28 with an appropriate alkyl bromide (29–50) in 1,4-
As for 1, 12 has one asymmetric carbon atom, enabling it to exist in
two enantiomeric forms (Fig. 2), for which a racemic mixture of these
two enantiomers was used in the experiments described above. We
recently reported that although anti-inflammatory activity was
2

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Bioorganic&MedicinalChemistryxxx(xxxx)xxx–xxx
Scheme 1.
indistinguishable between enantiomers (R)- (–)-mepenzolate (R)-(–)-1
and (S)-mepenzolate (S)-(+)-1, the binding affinity of (R)-(–)-1 to
hM3R in vitro and its bronchodilatory activity in vivo were superior to
administration (Fig. 5A, B), suggesting that (R)-(–)-12 exhibits long-
acting bronchodilatory activity, similar to that of 2.
An examination of anti-inflammatory activity revealed that 1 and
(R)-(–)-12 significantly suppressed the PPE-induced increase in the total
number of leucocytes and the individual number of neutrophils in BALF
(Fig. 4B). We recently reported a similar anti-inflammatory effect for 5,
but not for 2-4.20
that of (S)-(+)-121
.
We have synthesized (R)-(–)-12 and (S)-(+)-12, and examined their
biochemical and pharmacological activities. As shown in Table 1, (R)-
(–)-12 exhibited a higher affinity for hM3R than (S)-(+)-12. We also
compared their bronchodilatory activities. As shown in Fig. 3A, at a
dose of 48 µg/kg, 12 and (R)-(–)-12 but not (S)-(+)-12 showed
bronchodilatory activity. These in vivo results were consistent with the
in vitro results for hM3R affinity (Table 1). On the other hand, the
measured anti-inflammatory activity was indistinguishable among 12
and its enantiomers ((R)-(–)-12 and (S)-(+)-12) (Fig. 4A). Taken to-
gether, these results suggest that (R)-(–)-12 may be preferable to (S)-
(+)-12, and that the higher affinity for hM3R of the (R)-enantiomer
compared with the (S)-enantiomer was a general property of me-
penzolate derivatives.
We next examined the mechanism underlying (R)-(–)-12′s anti-in-
flammatory activity. As shown in Fig. 4C, PPE-induced an increase in
the mRNA expression of tumor necrosis factor (TNF)-α, interleukin (IL-
6), interferon (IFN)-γ, and keratinocyte chemoattractant (KC); this ex-
pression was suppressed by the administration of (R)-(–)-12 in all cases.
Furthermore, administration of (R)-(–)-12 to healthy mice increased
GST activity in the lung, suggesting that this effect is involved in the
anti-inflammatory activity of (R)-(–)-12, as in the case of 1.13
2.5. Effect of 2 and R-12 on PPE-induced lung injury and dysfunction
2.4. Bronchodilatory and anti-inflammatory activities of R-12
The effects of the long-term administration of 2 and (R)-(–)-12 on
lung injury and dysfunction induced by PPE were compared.
Histopathological analysis revealed that PPE induced damage to the
alveolar walls and an increase in the mean linear intercept (MLI; an
indicator of airspace enlargement). These effects could be partly sup-
pressed by the administration of (R)-(–)-12 but not by 2 (Fig. 6A and B).
PPE-induced lung dysfunction can be monitored by using the
FEV0.1/FVC19,23. While the PPE treatment decreased the FEV0.1/FVC
ratio, administration of (R)-(–)-12 but not 2 significantly restored the
towards control values (Fig. 6C). The results presented in Fig. 6 suggest
that the long-term administration of (R)-(–)-12, but not of 2, suppress
PPE-induced lung injury and dysfunction.
We next compared the bronchodilatory activities of 1–5 and (R)-
(–)-12, beginning with their dose–response profiles. A dose of 38 µg/kg
for 1 corresponds to 42, 36, 50, 49 and 48 µg/kg for 2, 3, 4, 5, and (R)-
(–)-12, respectively, based on their molecular weights. As shown in
Fig. 3B, 1 h after administration of each drug, complete suppression of
the methacholine-induced increase in airway resistance was observed
with 2 and (R)-(–)-12 at doses of 4.2 and 4.8 µg/kg, respectively. We
recently reported that complete suppression of the methacholine-in-
duced increase in airway resistance was observed with 1, 3, 4, and 5 at
doses of 38, 3.6, 5.0 and 4.9, respectively.20 These results suggest that
(R)-(–)-12 has a strong bronchodilatory activity, similar to that of 2
which is currently used in the clinical setting.
3. Discussion
We subsequently compared the duration of bronchodilatory activity
exhibited by the derivative compounds. We previously found that the
bronchodilatory activity of 1 disappeared within 24 h of its adminis-
tration.20 We also showed that the bronchodilatory activities of 3 and 4
(clinically used LAMAs) were still present 48 h after their administra-
tion, but had disappeared by 72 h.20 As shown in Fig. 5A, bronchodi-
latory activity with 3, 4, and 5 was not observed up to 96 h after drug
administration. On the other hand, (R)-(–)-12 and 2 still showed
bronchodilatory activity at 96 or 120 h, respectively, after
We have chemically modified 5 to produce derivatives with both
anti-inflammatory and longer-lasting bronchodilatory activities.
Although a drug’s affinity for hM3R is not always correlated with its
duration of action, we selected this index for the initial screening of
drugs given that hM3R affinity can be rapidly determined for large
panels of compounds. Of all the derivatives synthesized, 12 showed the
highest affinity for hM3R. Compound 12 has an asymmetric carbon
atom, with (R)-(–)-12 exhibiting a higher affinity for hM3R in vitro and
3

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Table 1
ring) is an important parameter determining a compound’s affinity for
hM3R, and that chemical modification of the terminal phenyl ring is not
an effective measure to increase affinity for hM3R. It was reported that
the mode of interaction of 2 with the muscarinic receptors is unique,24
with the results in this study suggesting that (R)-(–)-12 has a similar
interaction mode to that of tiotropium. Further study is needed to ex-
amine the nature of (R)-(–)-12′s mode of interaction with hM3R. In
addition, investigation of interaction of (R)-(–)-12 with the other
muscarinic receptor subtypes is also important in order to understand
the molecular mechanism of its pharmacological action.
Binding of compounds to hM2R or hM3R. 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 to generate competition curves
to determine Ki values. At the initial screening, we examined affinity values
from triplicate measurements and determined mean values. For some com-
pounds that showed a relatively high affinity at the initial screening, we re-
examined the affinity in independent experiments and confirmed the results.
Mean values of triplicate data for each one experiments are shown in the table.
Compounds whose affinity values were re-examined in additional experiments
are denoted by an asterisk (*). Data for 1–5 are from our previous report [20].
Previously, we examined the effect of intratracheal administration
of 2 on increased fecal pellet output induced by restraint stress. As a
result, compared to the protective effect of 2 against PPE-induced lung
injury, more than 100 times higher dose of 2 was required to affect fecal
pellet output.17 We assumed that this is due to the low efficiency of
intratracheally administered 2 to transfer to blood, because we showed
that the blood concentration of 2, 1 min after the intratracheal ad-
ministration (10 mg) was 3 µg/ml, whereas that of intravenous ad-
ministration (7.5 mg) was 40 µg/ml.17 Based on these results, we
speculate that intratracheally administered (R)-(–)-12 also has less ef-
fect on organs (such as colon) other than lung and bronchi, compared to
its respiratory effects.
Values represent mean
Compound
S.E.M.
Ki (nM)
hM3R
hM2R
1
5.3
2*
2.1
0.49
1.6
0.43
0.68
—
2*
2
0.20
0.81
0.56
0.88
2.8
0.02*
0.2*
3
0.2*
0.3*
0.1*
0.08
0.4*
0.7*
4
0.04*
5
0.1*
0.2*
6
7
0.65
41
0.40
12
8
21
0.2
Comparison of the activities of (R)-(–)-12 and of clinically used
LAMAs (2–4) revealed that the duration of bronchodilation by (R)-
(–)-12 was longer than that of 3–5 and similar to that of 2. The long-
term intratracheal administration of (R)-(–)-12, but not of 2, also sup-
pressed PPE-induced pulmonary emphysema and respiratory dysfunc-
tion. These results suggest that (R)-(–)-12 might be therapeutically
beneficial for the treatment of COPD patients given that (R)-(–)-12
appears to have beneficial effects against both inflammation-induced
pulmonary emphysema and airflow limitation. While we selected the
elastase model for the initial evaluation of candidate drugs given the
rapid and acute effects seen therein, further studies with other models,
such as the cigarette smoke model,25 would be important to further
characterize the properties of candidate drugs. Patent applications have
been submitted for (R)-(–)-12 and related compounds.
9
42
12
39
12
1
10
81
21
3*
11
0.86
0.44
4.7
0.4
2.3
0.72
12
0.2*
12
0.04*
0.01
13
0.6*
0.5
2
14
17
0.4
0.3
2
7.1
0.69
2.1
9.2
—
15
1.1
0.04
16
6.5
1*
2
17
20
16
2
18
3.8
19
1.9
0.6
4
1.4
—
0.5
20
19
21
1.7
0.2
0.49
0.11
1.6
1.1
0.18
0.91
2.6
1.0
68
0.2
0.1
0.3
0.5
0.1
0.7
22
0.51
2.6
0.1*
23
0.4
24
0.86
2.3
0.4*
25
2
26
7.8
2
27
1.5
0.1
0.3
4. Materials and methods
(R)-(–)-12
(S)-(+)-12
0.40
184
0.06*
0.2*
30*
28*
More detailed descriptions concerning the methods are provided in
the Supplementary Information.
—, not tested.
* , more than two independent experiments were performed.
4.1. Synthesis of targeted compounds and purity analyses.
longer bronchodilatory activity in vivo than (S)-(+)-12. Compound (R)-
(–)-12 was thus selected for further study of its pharmacological ac-
tivities compared to those of clinically used LAMAs.
Methods for the synthesis of targeted compounds are described in
the Supplementary Information. HPLC analysis was used for de-
termining the purity of targeted compounds, with a purity of greater
than 95% purity confirmed for all cases.
The results in this study suggest that the length of the linker moiety
(between the nitrogen atom of quinuclidine and the terminal phenyl
Fig. 2. Strategy used for making chemical modifications to 5 and structures of enantiomers of 1 ((R)-(–)-1 and (S)-(+)-1) and 12 ((R)-(–)-12 and (S)-(+)-12)..
4

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Fig. 3. Dose-response curves for compounds
12, (R)-(–)-12, and (S)-(+)-12 (A) or 2 and
(R)-(–)-12 (B) for methacholine-induced
airway constriction. Indicated doses were
administered intratracheally. After 1 h, mice
were exposed to nebulized methacholine
five times and airway resistance was de-
termined after each methacholine challenge
as described in the Materials and Methods.
Values
represent
mean
S.E.M.
*P < 0.05. Control data are the same in all
graphs. Data are representative of two in-
dependent experiments.
Fig. 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 (A, B, C). The
indicated doses (µg/kg) of compounds were administered intratracheally once only (A, B, C). 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 the Materials and Methods (A, B). Twenty-four hours after the PPE admin-
istration, total RNA was extracted from lung tissue and subjected to real-time RT-PCR using a specific primer set for each gene. Values were normalized to Hprt1 and
expressed relative to the Control (C). Male ICR mice were administered intratracheally with indicated doses (µg/kg) of (R)-(–)-12 or vehicle (saline) once only.
Twenty-four hours after the (R)-(–)-12 administration, lung homogenates were prepared. GST activity was determined as described in the Materials and Methods (D).
Values represent mean
S.E.M. **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.
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Fig. 5. Time-course of effect of compounds
2, 3, 4, 5 and (R)-(–)-12 (A) or 2 and (R)-
(–)-12 (B) on methacholine-induced airway
constriction. Indicated doses were adminis-
tered intratracheally. After 96 h (A) or 120 h
(B), mice were exposed to five cycles of
nebulized methacholine and airway re-
sistance was determined after each metha-
choline challenge as described in the
Materials and Methods. Values represent
mean
S.E.M. **P < 0.01; n.s., not sig-
nificant. The control data are the same as in
Fig. 6A. Data are representative of two in-
dependent experiments.
4.2. Treatment of mice with PPE and test drugs
4.5. Measurement of airway resistance and FEV0.1/FVC
Mice were sedated with 2% isoflurane and then administered in-
tratracheally with PPE (15 or 20 U/kg) followed by the test compound
(various doses) contained in a vehicle (0.9% NaCl; 1 ml/kg) via mi-
cropipette (P200). For control mice, the 0.9% NaCl vehicle alone was
administered by the same procedure.
Respiratory function and airway resistance were monitored with a
computer-controlled small-animal ventilator (FlexiVent, SCIREQ,
Montreal, Canada), as described previously.19,23 Mice were anesthe-
tized 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.
4.3. Preparation of BALF and cell count method
For measurement of methacholine-induced increases in airway re-
sistance, mice were exposed to nebulized methacholine (5 mg/ml) five
times for 20 sec with a 40 sec interval, and airway resistance was
measured after each methacholine challenge by the snapshot technique.
All data were analysed using FlexiVent software.
BALF was collected 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 was counted using a hemocytometer. Cells were
stained with Diff-Quik reagents after centrifugation in a Cytospin® 4
(Thermo Electron Corporation, Waltham, MA), and the ratio of number
of neutrophils to total cell number was determined to calculate the
number of neutrophils.
Determination of the FEV0.1/FVC (forced expiratory volume in the
first 0.1 s to forced vital capacity) ratio was performed with the same
computer-controlled small-animal ventilator connected to a negative
pressure reservoir (SCIREQ, Montreal, Canada), as described pre-
viously.19,23 Mice were tracheotomised and ventilated as described
above. The lung was inflated to 30 cmH2O over one second and held at
this pressure. After 0.2 sec, the pinch valve (connected to ventilator)
was closed and after a further 0.3 sec, the shutter valve (connected to
negative pressure reservoir) was opened for exposure of the lung to the
negative pressure. The negative pressure was held for 1.5 sec to ensure
complete expiration. The FEV0.1/FVC ratio was determined using
FlexiVent software.
4.4. Histopathological analysis
Lung tissue samples were fixed in 10% formalin neutral buffer so-
lution for 24 h at a pressure of 25 cmH2O, and then embedded in par-
affin 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 inspected
with the aid of an Olympus BX51 microscope (Tokyo, Japan).
To determine the MLI, 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.26 This morphometric analysis was con-
ducted by an investigator blinded to the study protocol.
6

Y. Yamashita, et al.
Bioorganic&MedicinalChemistryxxx(xxxx)xxx–xxx
Fig. 6. Effect of 2 and (R)-(–)-12 on PPE-induced pulmonary damage and dysfunction. Mice were treated with or without (control) PPE (15 U/kg) once only on day 0.
The indicated doses of 2 or (R)-(–)-12 were administered intratracheally once daily for 14 days (from day 0 to day 13) (A-C). Sections of pulmonary tissue were
prepared on day 14 and subjected to histopathological examination (H & E staining) (scale bar, 500 µm) (A). Airspace size was estimated by determining the MLI as
described in the Materials and Methods (B). FEV0.1/FVC values were determined on day 14 as described in the Materials and Methods (C). Values represent
mean
S.E.M. **P < 0.01 (versus control); #P < 0.05 (versus PPE only). Data are representative of two independent experiments.
4.6. Real-time reverse transcription polymerase chain reaction (RT-PCR)
analysis.
controls. To normalize the amount of total RNA present in each reac-
tion, hypoxanthine phosphoribosyltransferase 1 (HPRT1) cDNA was
used as an internal standard. Primers were designed using Primer-
BLAST websites. Primers sequences will be provided upon request.
Total RNA was extracted from lung tissue using an RNeasy kit ac-
cording to the manufacturer’s protocol. Samples were reverse-tran-
scribed using the PrimeScript® kit described above. The synthesized
cDNA was used in real-time PCR experiments with SsoAdvanced
4.7. Measurement of GST activity
Universal SYBR Green Supermix and analyzed with
a Bio-Rad
Left lung homogenates were prepared 24 h after (R)-(–)-12 admin-
istration, GST activity in the homogenates was measured with the aid of
a GST assay kit according to the manufacturer’s protocol. GST activity
was expressed relative to activity in the control sample.
(Hercules, CA) CFX96™ real-time system and CFX Manager™ software.
Specificity was confirmed by electrophoretic analysis of reaction pro-
ducts and by the inclusion of template- or reverse transcriptase-free
7

Y. Yamashita, et al.
Bioorganic&MedicinalChemistryxxx(xxxx)xxx–xxx
4.8. Filter-binding assay
doi.org/10.1016/j.bmc.2019.06.016.
The filter-binding assay was performed as described previously21
with some modifications. Membrane fractions prepared from Chinese
Hamster Ovary (CHO)-K1 cells expressing human muscarinic M2 re-
ceptor (hM2R) or human muscarinic M3 receptor (hM3R) (Membrane
Target Systems, Perkin-Elmer Life and Analytical Sciences, Boston, MA;
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) of each compound was tested
in triplicate to generate competition 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 Ana-
lytical Sciences, Boston, MA) that was 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 MeltiLex A (melt-on
scintillation sheet; PerkinElmer Life and Analytical Sciences, Boston,
MA). The radioactivity remaining on the filter was monitored with a
MicroBeta Trilux microplate scintillation counter (PerkinElmer Life and
Analytical Sciences, Boston, MA). Affinities at equilibrium were de-
termined as equilibrium dissociation constant (Ki) values after cor-
recting the experimentally determined IC50 values with the experi-
mentally determined NMS Kd value for hM2R or hM3R and the
concentration of NMS, as described previously.21 All adjustments were
performed using Prism (GraphPad Software, Inc., San Diego, CA).
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This work was supported by Grants-in-Aid for Scientific Research
from the Ministry of Health, Labour, and Welfare of Japan, as well as
the Japan Science and Technology Agency and Grants-in-Aid for
Scientific Research from the Ministry of Education, Culture, Sports,
Science and Technology, Japan. This research was also supported by
the Pharmacological Research Foundation Tokyo, the Takeda Science
Foundation, and the Suzuken Memorial Foundation.
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Participated in research design: YY, KT, NY, YF, TM.
Conducted experiments: YY, KT, NY, TA, YK, AT.
Contributed new reagents or analytic tools: MK, MT, YF.
Performed data analysis: YY, KT, NY, TA, TM.
Wrote or contributed to the writing of the manuscript:
YY, KT, NY, TA, YK, AT, MK, MT, YF, TM.
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Declaration of Competing Interest
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Tohru Mizushima is Chairman of LTT Bio-Pharma Co., Ltd. Teita
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division of LTT Bio-Pharma Co., Ltd.
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Appendix A. Supplementary data
Supplementary data to this article can be found online at https://
8