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\begin{document}

\title{Therapeutic effects of eperisone on pulmonary fibrosis via preferential
suppression of fibroblast activity}



\author[1]{Ken-ichiro Tanaka}%
\author[1]{Mikako Shimoda}%
\author[1]{Toshifumi  Sugizaki}%
\author[1]{Maki Ikeda}%
\author[1]{Ayaka Takafuji}%
\author[1]{Masahiro Kawahara}%
\author[2]{NAOKI YAMAKAWA}%
\author[3]{Tohru Mizushima}%
\affil[1]{Musashino University}%
\affil[2]{Shujitsu University}%
\affil[3]{LTT Bio-Pharma Co., Ltd}%


\vspace{-1em}



  
  \date{May 26, 2024}


\begingroup
\let\center\flushleft
\let\endcenter\endflushleft
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\endgroup





\selectlanguage{english}
\begin{abstract}
Background and purpose: Although the exact pathogenesis of idiopathic
pulmonary fibrosis (IPF) is still unknown, the transdifferentiation of
fibroblasts into myofibroblasts and the production of extracellular
matrix components such as collagen, triggered by alveolar epithelial
cell injury, are important mechanisms of IPF development. In the lungs
of IPF patients, apoptosis is less likely to be induced in fibroblasts
than in alveolar epithelial cells, and this process is involved in the
pathogenesis of IPF. Experimental approach: We used a library containing
approved drugs to screen for drugs that preferentially reduce cell
viability in LL29 cells (lung fibroblasts from an IPF patient) compared
with A549 cells (human alveolar epithelial cell line). Key results:
After screening, we selected eperisone, a central muscle relaxant used
in clinical practice. Eperisone showed little toxicity in A549 cells and
preferentially reduced the percentage of viable LL29 cells, while
pirfenidone and nintedanib did not have this effect. Eperisone also
significantly inhibited transforming growth factor-\selectlanguage{greek}β\selectlanguage{english}1-dependent
transdifferentiation of LL29 cells into myofibroblasts. In an in vivo
study using ICR mice, eperisone inhibited bleomycin (BLM)-induced
pulmonary fibrosis, respiratory dysfunction, and fibroblast activation.
In contrast, pirfenidone and nintedanib were less effective than
eperisone in inhibiting BLM-induced pulmonary fibrosis under this
experimental condition. Finally, we showed that eperisone did not induce
adverse effects in the liver and gastrointestinal tract in the
BLM-induced pulmonary fibrosis model. Conclusion and implications:
Considering these results, we propose that eperisone may be safer and
more therapeutically beneficial for IPF patients than current therapies.%
\end{abstract}%



\sloppy


\textbf{Therapeutic effects of eperisone on pulmonary fibrosis via
preferential suppression of fibroblast activity}

\textbf{Ken-ichiro Tanaka} \textsuperscript{1, *}\textbf{, Mikako
Shimoda} \textsuperscript{1}\textbf{, Toshifumi Sugizaki}
\textsuperscript{1}\textbf{, Maki Ikeda} \textsuperscript{1}\textbf{,
Ayaka Takafuji} \textsuperscript{1}\textbf{, Masahiro Kawahara}
\textsuperscript{1}\textbf{, Naoki Yamakawa}
\textsuperscript{2}\textbf{, and Tohru Mizushima}
\textsuperscript{3}\textbf{.}

\textsuperscript{1}Laboratory of Bio-Analytical Chemistry, Research
Institute of Pharmaceutical Sciences, Faculty of Pharmacy, Musashino
University, 1-1-20 Shinmachi, Nishi-Tokyo 202-8585,
Japan;\textsuperscript{2}Shujitsu University School of Pharmacy, Okayama
703-8516, Japan; \textsuperscript{3} LTT Bio-Pharma Co., Ltd, Shiodome
Building 3F, 1-2-20 Kaigan, Minato-ku, Tokyo 105-0022, Japan.

\textbf{Correspondence}

Dr. Ken-ichiro Tanaka, Laboratory of Bio-Analytical Chemistry, Research
Institute of Pharmaceutical Sciences, Musashino University, 1-1-20
Shinmachi, Nishitokyo-shi, Tokyo 202-8585, Japan. TEL \& FAX:
81-42-468-9335, E-mail:\emph{k-tana@musashino-u.ac.jp}

\textbf{Running title} : Eperisone and pulmonary fibrosis

\textbf{Keywords:} bleomycin, idiopathic pulmonary fibrosis, fibroblast,
eperisone, muscle relaxant.

\par\null

Abbreviations

\selectlanguage{greek}α-\selectlanguage{english}SMA, \selectlanguage{greek}α-\selectlanguage{english}smooth muscle actin; BLM, bleomycin; DAPI,
4,6-diamidino-2-phenylindole; DMBA, 4-dimethylaminobenzaldehyde; DMEM,
Dulbecco's modified Eagle's medium; FBS, fetal bovine serum; FoxM1,
forkhead box M1; FVC, forced vital capacity; GAPDH,
glyceraldehyde-3-phosphate dehydrogenase; HED, human equivalent dose;
IPF, idiopathic pulmonary fibrosis; TCA, trichloroacetic acid; Drebrin,
developmentally regulated brain protein.

\textbf{What is already known}

\textbf{*} Idiopathic pulmonary fibrosis (Lee, Grabb, Zipfel, \& Choi)
is a lung disease of unknown etiology with a

poor prognosis.

\textbf{*} Transdifferentiation of fibroblasts into myofibroblasts and
extracellular matrix component production are important IPF development
mechanisms.

\textbf{What this study adds}

\textbf{*} Eperisone preferentially suppressed lung fibroblast
activity\emph{in vitro} .

\textbf{*} Eperisone inhibited bleomycin-induced pulmonary fibrosis
without

causing adverse effects.

\textbf{Clinical significance}

\textbf{*} Eperisone may be safer and more therapeutically beneficial
for IPF patients

than current therapies.

ABSTRACT

\textbf{Background and purpose:} Although the exact pathogenesis of
idiopathic pulmonary fibrosis (IPF) is still unknown, the
transdifferentiation of fibroblasts into myofibroblasts and the
production of extracellular matrix components such as collagen,
triggered by alveolar epithelial cell injury, are important mechanisms
of IPF development. In the lungs of IPF patients, apoptosis is less
likely to be induced in fibroblasts than in alveolar epithelial cells,
and this process is involved in the pathogenesis of IPF.

\textbf{Experimental approach:} We used a library containing approved
drugs to screen for drugs that preferentially reduce cell viability in
LL29 cells (lung fibroblasts from an IPF patient) compared with A549
cells (human alveolar epithelial cell line).

\textbf{Key results:} After screening, we selected eperisone, a central
muscle relaxant used in clinical practice. Eperisone showed little
toxicity in A549 cells and preferentially reduced the percentage of
viable LL29 cells, while pirfenidone and nintedanib did not have this
effect. Eperisone also significantly inhibited transforming growth
factor-\selectlanguage{greek}β\selectlanguage{english}1-dependent transdifferentiation of LL29 cells into
myofibroblasts. In an \emph{in vivo} study using ICR mice, eperisone
inhibited bleomycin (BLM)-induced pulmonary fibrosis, respiratory
dysfunction, and fibroblast activation. In contrast, pirfenidone and
nintedanib were less effective than eperisone in inhibiting BLM-induced
pulmonary fibrosis under this experimental condition. Finally, we showed
that eperisone did not induce adverse effects in the liver and
gastrointestinal tract in the BLM-induced pulmonary fibrosis model.

\textbf{Conclusion and implications:} Considering these results, we
propose that eperisone may be safer and more therapeutically beneficial
for IPF patients than current therapies.

\textbf{INTRODUCTION}

Idiopathic pulmonary fibrosis (IPF) is a lung disease of unknown
etiology with a poor prognosis that is characterized by chronic
development of severe fibrosis, resulting in a honeycomb lung (Kim,
Collard, \& King, 2006; Raghu et al., 2015). Steroids and
immunosuppressive drugs have long been used to treat IPF, but in many
cases, these drugs do not show therapeutic efficacy against IPF
progression (Luppi, Cerri, Beghe, Fabbri, \& Richeldi, 2004; Raghu et
al., 2015; Walter, Collard, \& King, 2006). The primary etiology of IPF
is thought to be chronic pulmonary fibrosis triggered by chronic injury
to airway and alveolar epithelial cells. Therefore, pirfenidone and
nintedanib, antifibrotic agents that have been shown to significantly
improve the reduction of forced vital capacity (FVC) in large clinical
trials, are being used to treat IPF in clinical practice (Noble et al.,
2011; Raghu et al., 2015; Richeldi et al., 2014). However, in some
cases, these drugs have not shown efficacy and have been reported to
induce adverse effects such as elevation of liver damage markers,
diarrhea, and indigestion (Noble et al., 2011; Richeldi et al., 2014).
Thus, safer drugs eliciting a therapeutic effect equal to or greater
than that of these two approved drugs are necessary.

Although the exact cause of IPF is unknown, it is thought to be
triggered by damage to the lung epithelium as a result of increased
oxidative stress and the repair and remodeling processes, such as
collagen synthesis, that are induced to manage this damage. In other
words, this process is overstimulated, resulting in abnormal wound
repair and remodeling characterized by collagen deposition, which leads
to the development and exacerbation of pulmonary fibrosis (Kinnula \&
Myllarniemi, 2008; Sheppard, 2006). The cells that play the greatest
role in this fibrosis-promoting process are myofibroblasts.
Peribronchial and perivascular fibroblasts transdifferentiate (activate)
into myofibroblasts in response to various stimuli, especially TGF-\selectlanguage{greek}β\selectlanguage{english}1,
and accumulate extracellular matrix components, especially collagen
fibers, which are involved in fibrosis (Hinz et al., 2007; Kisseleva \&
Brenner, 2008). Furthermore, the ''apoptosis paradox'' is also a
possible mechanism of abnormal fibrosis in IPF patients. Apoptosis is
preferentially observed in alveolar epithelial cells in the lungs of IPF
patients, while little apoptosis occurs in fibroblasts. This produces a
relative imbalance resulting in increased fibroblasts in the lungs of
IPF patients, which is thought to be involved in the pathogenesis of IPF
(Maher et al., 2010; Thannickal \& Horowitz, 2006). Thus, it is
important to identify compounds that inhibit transdifferentiation of
fibroblasts into myofibroblasts or inhibit activation of myofibroblasts.
In addition, compounds that are not toxic to alveolar epithelial cells
but exert their effects preferentially on lung fibroblasts are promising
candidates for IPF therapy.

On the basis of these requirements, we implemented an innovative
research strategy (drug repositioning) to identify and develop new IPF
therapeutics by screening drugs currently in clinical use to treat other
diseases (Mizushima, 2011; Pushpakom et al., 2019). The major advantage
of this strategy is that the clinical safety of the drugs screened is
already understood, and the risk of unexpected adverse effects in humans
can be greatly reduced when these drugs are applied to treat other
diseases (Mizushima, 2011; Pushpakom et al., 2019). Using this strategy,
we screened drugs not only for IPF but also chronic obstructive
pulmonary disease, functional dyspepsia, and irritable bowel syndrome
from a library of approved drugs, identified effective drugs for each
disease, and analyzed the mechanisms by which these drugs exert their
efficacy (Asano et al., 2017; Sugizaki et al., 2019; K. Tanaka et al.,
2013; K. I. Tanaka et al., 2017). Recently, other research groups have
used this strategy to develop novel therapeutics for coronavirus
infection 2019, and candidate drugs have been discovered, including the
influenza virus treatment drug remdesivir and the antiparasitic drug
ivermectin (Alam et al., 2021). Therefore, we suggest that this strategy
is useful for discovering new candidates for treatment of human
diseases.

In our previous study, we screened compounds capable of more potently
inhibiting the growth of lung fibroblasts (LL29 cells) than that of lung
alveolar epithelial cells (A549 cells) and identified idebenone, which
has previously been used clinically as a brain metabolic stimulant, from
a library of medications already in clinical use. In addition,
intratracheal administration of idebenone to mice inhibited bleomycin
(BLM)-induced pulmonary fibrosis and decreased FVC (Sugizaki et al.,
2019). Furthermore, in our previous screening, we found that, in
addition to idebenone, the central muscle relaxant eperisone also acts
preferentially on lung fibroblasts. No studies have been conducted on
eperisone to determine its effects on fibroblasts or pulmonary fibrosis.
Therefore, in this study, we investigated the effect of eperisone, which
preferentially induces fibroblast cell death, on BLM-induced pulmonary
fibrosis. In addition, we examined its adverse effects by analyzing
plasma markers and the gastrointestinal mucosal status when eperisone
was administered to BLM-induced pulmonary fibrosis model mice.



\textbf{Materials and Methods}

\textbf{Chemicals and animals}

Eperisone hydrochloride, tolperisone hydrochloride, isoflurane,
Dulbecco's modified Eagle's medium (DMEM), L-hydroxyproline, sodium
acetate, trichloroacetic acid (TCA), perchloric acid, and formalin
neutral buffer solution were obtained from Fujifilm Wako Pure Chemical
Corporation (Tokyo, Japan). Tizanidine hydrochloride, baclofen,
methocarbamol and pirfenidone were from Tokyo Chemical Industry (Tokyo,
Japan), and nintedanib was from Cayman Chemical (Ann Arbor, MI).
Inaperizone hydrochloride and lanperizone hydrochloride were synthesized
in our laboratory. Details of the synthesis method are described in the
Supplementary Materials and Methods. BLM was from Nippon Kayaku (Tokyo,
Japan). Chloramine T and 4-dimethylaminobenzaldehyde (DMBA) were
obtained from Sigma (St. Louis, MO). Fetal bovine serum (FBS) was
purchased from BioWest (Nuaille, France), and Ham's F-12K (Kaighn's
modification) medium was from Thermo Fischer Scientific (Waltham, MA).
An antibody against \selectlanguage{greek}α-\selectlanguage{english}smooth muscle actin (\selectlanguage{greek}α-\selectlanguage{english}SMA) was from Abcam
(Cambridge, Cambridgeshire), and Alexa Fluor 594 goat anti-rabbit
immunoglobulin G were from Invitrogen (Carlsbad, CA). Recombinant human
TGF-\selectlanguage{greek}β\selectlanguage{english}1 were from R\&D Systems (Minneapolis, MN), and mounting medium for
immunohistochemical analysis (VECTASHIELD) was purchased from Vector
Laboratories (Burlingame, CA). Mayer's haematoxylin, 1\% eosin alcohol
solution, primary mordant agent, secondary mordant agent, 0.75\% Orange
G solution, Masson's staining solution B, 2.5\% phosphotungstic acid
solution, aniline blue solution, mounting medium for histological
examination (malinol) and Weigert's iron haematoxylin were from MUTO
Pure Chemicals (Tokyo, Japan). 4,6-diamidino-2-phenylindole (DAPI) was
purchased from Dojindo (Kumamoto, Japan). The RNeasy kit was obtained
from Qiagen (Valencia, CA), PrimeScript II 1\textsuperscript{st} strand
cDNA Synthesis kit was from TAKARA Bio (Ohtsu, Japan), and THUNDERBIRD\selectlanguage{ngerman}®
SYBR qPCR Mix was from Bio-Rad (Hercules, CA). ICR mice (6-7 weeks old,
male) were purchased from Charles River (Yokohama, Japan). The
experiments and procedures 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.

\textbf{Cell culture}

A549 cells (human lung epithelial cell line) or LL29 cells (lung
fibroblasts from an IPF patient) were cultured in DMEM supplemented with
10\% FBS or Ham's F-12K (Kaighn's Modification) medium supplemented with
15\% FBS, respectively, in a humidified atmosphere of 95\% air with 5\%
CO\textsubscript{2} at 37°C.

Viable cells were measured as previously described (Nakano et al., 2020;
K. I. Tanaka et al., 2019). Briefly, dissociated A549 or LL29 cells were
added to 96-well culture plates at a concentration of 1 ×
10\textsuperscript{4} cells per well in 200 \selectlanguage{greek}µ\selectlanguage{english}l of culture medium. After
a 24-h incubation, cells were treated with various reagents added to the
medium. After 24 h, the percentage of viable cells was quantified using
CellTiter-Glo\selectlanguage{ngerman}® 2.0 (Promega Corporation, Madison, WI, USA). The
cytotoxicity in LL29 cells after eperisone addition was measured every
hour using CellTox\selectlanguage{english} Green Dye (Promega Corporation, Madison, WI, USA)
and a microplate reader (Tecan, Kawasaki, Japan; excitation: 485 nm,
emission: 530 nm).

\textbf{Real-time RT-PCR analysis}

Total RNA was extracted from LL29 cells using an RNeasy kit (Qiagen,
Hilden, Germany) according to the manufacturer's protocol. Using a
PrimeScript\selectlanguage{english} II 1\textsuperscript{st} strand cDNA Synthesis kit, samples
were reverse-transcribed and THUNDERBIRD(r) SYBR qPCR Mix, Bio-Rad's
CFX96\selectlanguage{english} Real-time system, and CFX Manager\selectlanguage{english} software (Hercules, CA) were
used for real-time RT-PCR experiments. Electrophoretic analysis of
reaction products was done to confirm specificity.
Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) cDNA was used as an
internal standard. Primers were designed using either Primer3 or
Primer-BLAST. Primer sequences will be provided upon request.

\textbf{Treatment of mice with BLM, eperisone, and other reagents}

Mice were anesthetized with isoflurane and intratracheally administered
BLM (1 mg/kg, once) in sterile saline via a single channel pipette
(P200). Ten days after BLM administration, eperisone (15 or 50 mg/kg),
tolperisone (15 mg/kg), pirfenidone (200 mg/kg), and nintedanib (30
mg/kg) were administered orally for a total of 9 days from day 10 to day
18. Various analyses were then performed on day 20.

In the adverse effect study, 10 days after BLM administration, 250 mg/kg
of eperisone was orally administered once, which was five times the dose
that showed efficacy. Twenty-four hours after eperisone administration,
the fecal condition of the mice was visually examined. In addition,
plasma samples and stomach and colon tissues were collected from the
mice. Analysis of the plasma samples was performed by TRANS GENIC INC.
(https://www.transgenic.co.jp/).

\textbf{Histological and immunohistochemical analyses}

Tissue samples were fixed in 10\% formalin neutral buffer solution for
24 h, and then embedded in paraffin before being cut into 4 \selectlanguage{greek}µ\selectlanguage{english}m-thick
sections.

For staining of collagen (Masson's trichrome staining), sections were
treated sequentially with primary mordant agent, Weigert's iron
haematoxylin, secondary mordant agent, 0.75\% Orange G solution,
Masson's staining solution B, 2.5\% phosphotungstic acid solution, and
finally with aniline blue solution. Samples were mounted with malinol,
and inspected with a fluorescence microscope (Olympus DP71) or scanned
using a NanoZoomer-XR digital slide scanner. Image J software (National
Institutes of Health, Bethesda, MD) was used to calculate the percentage
of collagen positive area.

For immunohistochemical analysis of \selectlanguage{greek}α-\selectlanguage{english}SMA, sections were blocked with
2.5\% goat serum for 10 min, incubated for 12 h with an antibody against
\selectlanguage{greek}α-\selectlanguage{english}SMA (1:100 dilution) in the presence of 2.5\% bovine serum albumin,
and then incubated with Alexa Fluor 594 goat anti-rabbit immunoglobulin
G (1:500 dilution) and DAPI (5 \selectlanguage{greek}µ\selectlanguage{english}g/ml) for 1 h. Samples were mounted with
VECTASHIELD and inspected with a fluorescence microscope (Olympus DP71).
Image J software (National Institutes of Health, Bethesda, MD) was used
to calculate the percentage of \selectlanguage{greek}α-\selectlanguage{english}SMA positive area.

For histological examination, sections were stained first with Mayer's
haematoxylin and then with 1\% eosin alcohol solution (H\&E staining).
Samples were mounted with malinol and inspected with a fluorescence
microscope (Olympus DP71).

\textbf{Measurement of lung mechanics and FVC}

Measurement of lung mechanics and FVC was performed with a
computer-controlled small-animal ventilator connected to a negative
pressure reservoir (FlexiVent; SCIREQ, Montreal, Canada), as previously
described (K. Tanaka, Azuma, Miyazaki, Sato, \& Mizushima, 2012). Mice
were anaesthetised with three types of mixed anesthetic agents (0.75
mg/kg medetomidine, 4.0 mg/kg midazolam, and 5.0 mg/kg butorphanol), a
tracheotomy was performed, and an 8 mm-long section of metallic tube
(outer and inner diameters of 1.27 mm and 0.84 mm, respectively) 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 cmH\textsubscript{2}O.

Total respiratory system elastance and tissue elastance were measured by
snap shot and forced oscillation techniques, respectively. For
determination of FVC, lungs were inflated to 30 cmH\textsubscript{2}O
over one second and held at this pressure. After 0.2 sec, the pinch
valve (connected to the ventilator) was closed, and after 0.3 sec, the
shutter valve (connected to the negative pressure reservoir) was opened,
exposing the lung to the negative pressure, which was held for 1.5 sec
to ensure complete expiration. All data were analysed using FlexiVent
software (version 5.3; SCIREQ, Montreal, Canada).

\textbf{Hydroxyproline determination}

Hydroxyproline content was determined as previously described (Woessner,
1961). Briefly, the lung was removed and homogenised in 1.0 ml of 5\%
trichloroacetic acid. After centrifugation, pellets were hydrolysed in
0.5 ml of 10 N HCl for 16 h at 110\selectlanguage{ngerman}°C. Each sample was incubated for 20
min at room temperature with 0.5 ml of 1.4\% w/v chloramine T solution,
and then incubated at 65°C for 10 min with 0.5 ml of Ehrlich's reagent
(1 M DMBA, 70\% v/v isopropanol and 30\% v/v perchloric acid). The
absorbance of each sample was then measured at 550 nm to determine the
amount of hydroxyproline present.

\textbf{Statistical analysis}

All values are expressed as mean ± S.E.M. One-way ANOVA, followed by
Dunnett's test, or Student's unpaired \emph{t} -test were used to
evaluate differences between three or more groups or between two groups,
respectively. Mac statistical analysis Ver.3.0 software (Esumi Co.,
Ltd., Tokyo, Japan) was used for the statistical analyses. Differences
were considered to be significant when \emph{P} \textless{} 0.05.

\textbf{RESULTS}

\textbf{Preferential suppression of fibroblast activity by eperisone}

A library of drugs already in clinical use was screened to identify
drugs that are not toxic to alveolar epithelial cells but are
preferentially toxic to lung fibroblasts. Specifically, LL29 or A549
cells were treated with each drug, and 24 h later, the percentages of
viable cells were determined using the methylthiazole tetrazolium
reagent. Among the drugs that showed lower IC50 values in LL29 cells
than in A549 cells, idebenone and eperisone were selected based on the
difference in IC50 values between the two cell types, their clinical
safety, and other pharmacological activities. As described above, we
previously reported the preferential suppression of fibroblast activity
by idebenone and its efficacy against BLM-induced pulmonary fibrosis
(Sugizaki et al., 2019). Therefore, in this study, we focused on
eperisone, which is used in clinical practice as a central muscle
relaxant (Iwase, Mano, Saito, \& Ishida, 1992), and examined its
efficacy against IPF using \emph{in vitro} and \emph{in vivo} systems.

As shown in Figure 1A, eperisone treatment (25--200 \selectlanguage{greek}µ\selectlanguage{english}M) decreased the
percentage of viable LL29 cells in a dose-dependent manner. In contrast,
the percentage of viable A549 cells treated with 200 \selectlanguage{greek}µ\selectlanguage{english}M of eperisone was
88.5 \selectlanguage{ngerman}± 3.0\% (mean ± SEM, \emph{n}= 4), revealing almost no decrease in
viable A549 cells after eperisone treatment. We next examined
eperisone-induced cytotoxicity in LL29 cells using CellTox\selectlanguage{english} Green Dye,
which can detect cell membrane disruption. As shown in Figure 1B, LL29
cells treated with eperisone exhibited cytotoxic effects in a time- and
concentration-dependent manner. Furthermore, we compared the effect of
eperisone on TGF-\selectlanguage{greek}β\selectlanguage{english}1--induced activation of lung fibroblasts. LL29 cells
were pre-treated with eperisone (10--30 \selectlanguage{greek}µ\selectlanguage{english}M), followed by the addition of
TGF-\selectlanguage{greek}β\selectlanguage{english}1 (5 \selectlanguage{greek}µ\selectlanguage{english}M), and the expression of fibrosis-related factors was
analyzed 72 h later by real-time RT-PCR. As shown in Figure 1C, TGF-\selectlanguage{greek}β\selectlanguage{english}1
increased the mRNA expression of Collagen 1a1 \emph{(COL1A1)} , \selectlanguage{greek}α-\selectlanguage{english}SMA
\emph{(ACTA2)} , connective tissue growth factor (\emph{CTGF} ),
vascular endothelial growth factor (\emph{VEGF} ), basic fibroblast
growth factor (\emph{BFGF} ), and platelet derived growth factor
(\emph{PDGF-A} ) in LL29 cells, but this increase was suppressed by
pre-treatment with eperisone. These results suggest that eperisone
preferentially suppressed lung fibroblast activity \emph{in vitro} .

\textbf{Effects of other drugs on lung} \textbf{fibroblast viability}

As described in the introduction, pirfenidone and nintedanib have been
used as anti-fibrotic agents in clinical practice to treat IPF patients.
Thus, to investigate the characteristic effect of eperisone on lung
fibroblasts, we measured the percentages of viable LL29 and A549 cells
after treatment with these existing drugs. After pirfenidone treatment
(up to 2 mM), almost no decrease was observed in the percentage of
viable cells of both cell types. In contrast, nintedanib decreased the
percentage of viable cells of both cell types, but there was no
difference in the degree of decrease between the cell types (Figure 2A).

Eperisone is a central muscle relaxant that has been used in clinical
practice to improve muscle tone in patients with lumbago and spastic
paralysis caused by cerebrovascular disease. Thus, we determined whether
other central muscle relaxants exert preferential effects on
fibroblasts. Among the six drugs examined, tolperisone, inaperisone, and
lanperisone preferentially reduced the viability of LL29 cells, similar
to eperisone. However, tizanidine, methocarbamol, and baclofen, at
concentrations up to 2 mM, did not reduce the viability of either cell
type (Figure 2B). As will be discussed in detail later, because
preferential suppression of fibroblasts was not observed for some
central muscle relaxants, we speculate that eperisone exerts its
preferential effects by a molecular mechanism other than its muscle
relaxant effect.

\textbf{Effect of eperisone on BLM-induced pulmonary fibrosis}

Pulmonary fibrosis was induced by intratracheal administration of BLM to
male ICR mice. Specifically, 10 days after BLM administration, mice were
divided into three groups based on the rate of change in body weight
(excluding the vehicle group), and the effect of oral eperisone
administration on lung fibrosis was examined. At 20 days after BLM
administration, lung tissue sections were prepared and stained for
collagen using Masson's trichrome stain. Collagen deposition in the
lungs was observed in a BLM administration-dependent manner. In
contrast, oral eperisone administration suppressed the BLM-dependent
collagen deposition in a dose-dependent manner (Figure 3A and 3B). Next,
we performed quantitative analysis of hydroxyproline, a
collagen-specific amino acid, in lung tissue. As shown in Figure 3C, BLM
treatment significantly increased the amount of hydroxyproline in lung
tissue, while eperisone treatment suppressed this increase. When
considering the clinical application of eperisone for the treatment of
lung fibrosis, it is important to improve respiratory function as well
as histological and biochemical indices. Moreover, our previous analysis
showed that lung elastance is increased and FVC is decreased in
BLM-induced pulmonary fibrosis (Sugizaki et al., 2019; K. I. Tanaka et
al., 2017). Thus, we measured the respiratory function of mice using a
computer-controlled ventilator and negative pressure reservoir. As shown
in Figure 3D, BLM treatment increased the total elastance (elastance of
the entire lung including the bronchi, bronchioles, and alveoli) and
tissue elastance (elastance of the alveoli) and decreased the FVC. In
contrast, eperisone significantly improved the deterioration of
respiratory function induced by BLM administration. These results
indicate that eperisone has an ameliorating effect on BLM-dependent
pulmonary fibrosis.

Fibroblasts are known to differentiate into myofibroblasts upon
activation and play an important role in the development and
exacerbation of lung fibrosis (Hinz et al., 2007; Scotton \& Chambers,
2007). Thus, we performed an immunohistochemical analysis of \selectlanguage{greek}α-\selectlanguage{english}SMA, a
myofibroblast marker. As shown in Figure 4A, BLM treatment increased the
number of \selectlanguage{greek}α-\selectlanguage{english}SMA-positive cells in the lung, i.e., myofibroblasts
increased in a BLM-dependent manner. In contrast, administration of 50
mg/kg eperisone decreased the BLM-dependent increase in \selectlanguage{greek}α-\selectlanguage{english}SMA-positive
cells in the lung to the same level as observed in the vehicle group.
These results suggest that eperisone inhibits fibroblast activation not
only \emph{in vitro} but also \emph{in vivo} and that it suppresses
BLM-dependent pulmonary fibrosis through its inhibitory effect on
fibroblast activation.

\textbf{Effects of other drugs on BLM-induced pulmonary fibrosis}

Using the same experimental approach as in Figure 3, we examined the
effect of pirfenidone and nintedanib on BLM-dependent pulmonary
fibrosis. The dose of pirfenidone (200 mg/kg) was 20 times the initial
clinical dose (600 mg/day) used in Japan (Song et al., 2020), and the
same dose of nintedanib (30 mg/kg) that had shown triple kinase
inhibition in previous animal studies was used (Wollin, Maillet,
Quesniaux, Holweg, \& Ryffel, 2014). As shown in Supplementary Figure
S1A--S1C, BLM-dependent collagen deposition and increased hydroxyproline
levels in the lung were not suppressed by oral administration of
pirfenidone or nintedanib. Nintedanib or pirfenidone administration
tended to improve the BLM-dependent deterioration of respiratory
function, but the effect was not statistically significant
(Supplementary Figure S1D).

We next analyzed the effect of tolperisone, a structural analogue of
eperisone with central muscle relaxant properties (Tekes, 2014), on
BLM-dependent pulmonary fibrosis. As shown in Supplementary Figure
S2A--S2C, oral administration of tolperisone suppressed BLM-dependent
collagen deposition and increased hydroxyproline levels in the lung
similar to eperisone administration. In addition, tolperisone
significantly improved the BLM-dependent increase in total elastance and
tissue elastance and the decrease in FVC (Supplementary Figure S2D).
These results indicate that preferential suppression of fibroblast
activity is important to prevent BLM-dependent exacerbation of lung
fibrosis.

\textbf{Safety analysis of eperisone administration}

In clinical practice, existing IPF treatments, such as pirfenidone and
nintedanib, have been reported to induce adverse effects such as
increasing markers of liver damage in the plasma and gastrointestinal
disorders (Noble et al., 2011; Richeldi et al., 2014). Therefore, we
conducted a comprehensive analysis of markers for pancreatic, hepatic,
and renal damage in plasma. The dose of eperisone was five times higher
than the dose that showed efficacy for BLM-dependent pulmonary fibrosis.
As shown in Table 1, administration of BLM or BLM plus eperisone (250
mg/kg) did not significantly alter 12 plasma markers for pancreatic,
hepatic, and renal damage. In addition, no mouse exhibited diarrhea or
hemorrhagic stool in either group (Table 2). Furthermore, we also
examined gastric and colonic mucosal injury using hematoxylin and eosin
staining. As shown in Figure 5 and Table 2, the condition of the gastric
and colonic mucosa in mice treated with BLM or BLM plus eperisone (250
mg/kg) was unchanged compared with that in vehicle-treated mice, and no
gastric and colonic mucosal injury was observed. These results suggest
that eperisone may be able to suppress pulmonary fibrosis without
inducing adverse effects.

\textbf{DISCUSSION}

In this study, we found that eperisone, a central muscle relaxant,
preferentially reduces the percentage of viable fibroblasts, an effect
not produced by the existing drugs pirfenidone and nintedanib. Moreover,
eperisone also inhibited fibroblast activation \emph{in vivo} and
markedly reduced BLM-dependent exacerbation of pulmonary fibrosis.
Furthermore, no adverse effects were observed, even when eperisone was
administered to mice at a dose five times higher than the dose at which
it inhibited BLM-induced pulmonary fibrosis. To the best of our
knowledge, this is the first study of the effects of eperisone on
fibroblasts and its therapeutic effects on a BLM-induced pulmonary
fibrosis model. Eperisone is used clinically to improve muscle tone in
patients with lumbago and spastic paralysis caused by cerebrovascular
disease, and the maximum daily dose used in Japan is 150 mg (orally).
Thus, we calculated the human equivalent dose (HED) using the dose used
in animals (15 or 50 mg/kg), animal weight (0.04 kg), and human weight
(60 kg) according to a previous report (Nair \& Jacob, 2016) and found
that the HED was 1.3 or 4.5 mg/kg. Therefore, a person weighing 60 kg
would require a dosage of 78--270 mg per day. Thus, eperisone at its
current clinical dose (150 mg/day) is expected to be effective against
IPF.

To investigate whether central muscle relaxation is involved in the
preferential effects of eperisone on fibroblasts, we examined the
percentage of viable LL29 or A549 cells when other central muscle
relaxants were administered. As shown in Figure 3, tolperisone,
inaperisone, or lanperisone, but not tizanidine, methocarbamol, or
baclofen, preferentially reduced the viability of LL29 cells. Thus, we
speculate that eperisone exerts its preferential suppression of
fibroblasts by a molecular mechanism other than its muscle relaxant
effect. In terms of chemical structure, the drugs that showed
fibroblast-preferential effects had higher ClogP values, a lipophilic
parameter related to membrane permeability (Supplementary Figure S3).
Therefore, a high ClogP value may be necessary for a drug to exert a
preferential effect on fibroblasts. In addition, it is interesting to
note that the drugs that preferentially reduced the viable percentage of
fibroblasts contain isobutyrophenone bound to the nitrogen atom of the
heterocyclic ring in the chemical structure. Chemical modification based
on this basic structure may lead to the discovery of drugs that
preferentially act on fibroblasts.

Nevertheless, the molecular mechanism by which eperisone preferentially
reduced the viability of lung fibroblasts could not be elucidated in
this study. A recent study suggested that developmentally regulated
brain protein (Drebrin), which binds to and increases the stability of
actin filaments in neurons, is mainly expressed in myofibroblasts of
mouse hearts after myocardial infarction or mouse lungs after BLM
administration and promotes the expression of fibrosis-related genes,
such as \selectlanguage{greek}\emph{α-SMA}\selectlanguage{english} and \emph{Col1A1} (Hironaka et al., 2020). Another
group demonstrated that radiation-induced DNA damage is reduced in IPF
fibroblasts and correlates with activation of the transcription factor
forkhead box M1 (FoxM1) and the subsequent upregulation of the DNA
repair proteins RAD51 and BRCA2 (Im, Lawrence, Seelig, \& Nho, 2018).
Moreover, syndecan-2 is reported to attenuate radiation-induced
pulmonary fibrosis in mice and inhibit TGF-\selectlanguage{greek}β\selectlanguage{english}1-induced
fibroblast-myofibroblast differentiation, migration, and proliferation
by down-regulating phosphoinositide 3-kinase/serine/threonine
kinase/Rho-associated coiled-coil kinase signaling and blocking serum
response factor binding to the \selectlanguage{greek}α-\selectlanguage{english}SMA promoter via CD148 (Tsoyi et al.,
2017). Furthermore, microRNA-101 has been reported to inhibit WNT5a (Wnt
ligand)-dependent lung fibroblast proliferation by inhibiting NFATc2
signaling and TGF-\selectlanguage{greek}β\selectlanguage{english}1-dependent lung fibroblast activation by inhibiting
SMAD2/3 signaling (Huang et al., 2017). Taken together, these reports
suggest that the molecular mechanisms by which eperisone preferentially
reduces the percentage of viable lung fibroblasts may involve previously
reported factors that regulate fibroblast activation.

Although pirfenidone and nintedanib are currently used in clinical
practice to treat IPF, in some cases, these drugs have not shown
efficacy and have been reported to induce adverse effects such as
elevation of liver damage markers, diarrhea, and indigestion (Noble et
al., 2011; Richeldi et al., 2014). Thus, in this study, we conducted a
''drug-repositioning strategy'' to identify safer and more effective
drugs for IPF treatment. The \emph{in vitro} studies shown in Figures 1
and 2 revealed that eperisone, but not pirfenidone or nintedanib,
exhibited a fibroblast-preferential reduction of viable cells. Moreover,
the\emph{in vivo} studies shown in Figure 3 and Supplementary Figure S1
indicated that eperisone, but not pirfenidone or nintedanib, inhibited
the exacerbation of BLM-induced pulmonary fibrosis. In addition,
eperisone did not induce adverse effects such as hepatotoxicity marker
elevation or gastrointestinal disorders. Therefore, we suggest that
eperisone may be a safer and more effective treatment for IPF than
pirfenidone or nintedanib.

After screening drugs that selectively induce fibroblast cell death, we
selected eperisone and showed its efficacy in animal models of IPF,
which is caused by fibroblast activation. As mentioned above, eperisone
has never been reported to preferentially induce cell death in
fibroblasts or effectively treat fibrosis models. However, fibrosis is
also induced in organs other than the lungs, such as the liver, heart,
and kidneys (Weiskirchen, Weiskirchen, \& Tacke, 2019). For example, in
the liver, hepatic stellate cells are activated by stimuli such as
TGF-\selectlanguage{greek}β\selectlanguage{english}1 and transdifferentiate into myofibroblasts, which promote the
production of extracellular matrix components such as collagen and
induce liver fibrosis in diseases such as nonalcoholic steatohepatitis
(Heyens, Busschots, Koek, Robaeys, \& Francque, 2021). In the kidney,
resident fibroblasts, pericytes, bone marrow-derived cells, and
endothelial cells transdifferentiate into myofibroblasts and induce
kidney fibrosis (Yuan, Tan, \& Liu, 2019). Thus, activated
myofibroblasts that transdifferentiate from fibroblasts play a role in
promoting fibrosis in organs other than the lungs. Therefore, eperisone,
which can preferentially inhibit fibroblast activity, may be effective
not only in lung fibrosis models but also in fibrosis models of other
organs; thus, the results of this study have promising applications for
future research.

\textbf{ACKNOWLEDGEMENTS}

This research was supported in parts by grants from The Mochida Memorial
Foundation for Medical and Pharmaceutical Research, Suzuken Memorial
Foundation, and a Grant-in-Aid for Scientific Research from Japan
Society for the Promotion of Science (JSPS) (KAKENHI KIBAN (C)
18K06691). We thank Lisa Kreiner, PhD, from Edanz
(\emph{https://www.jp.edanz.com/ac}) for editing a draft of this
manuscript.

\textbf{CONFLICT OF INTEREST}

Ken-ichiro Tanaka, Mikako Shimoda, Maki Ikeda, Toshifumi Sugizaki, Ayaka
Takafuji, Masahiro Kawahara, and Naoki Yamakawa do not have a financial
relationship with a commercial entity that has an interest in the
subject of this manuscript. Tohru Mizushima reports receiving personal
fees from LTT Bio-Pharma Co., Ltd. during the conduct of the study.

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\textbf{Figure legends}

\textbf{Table 1. Effect of eperisone administration on biochemical
markers in the blood.}

Mice were treated with bleomycin (BLM, 1 mg/kg) or vehicle once only on
day 0. The mice were then orally administered 250 mg/kg of eperisone
(Epe) once at day 10. After 24 h, whole blood was collected from the
mice. Analysis of the blood samples was performed by TRANS GENIC INC.
Values represent the mean \selectlanguage{ngerman}± SEM *\emph{*P \textless{} 0.01} ; \emph{*P
\textless{} 0.05} .

\textbf{Table 2. Effects of eperisone administration on gastric and
colonic mucosa}

Mice were treated with bleomycin (BLM, 1 mg/kg) or vehicle once only on
day 0. The mice were then orally administered 250 mg/kg of eperisone
(Epe) once at day 10. After 24 h, the fecal condition (diarrhea or
hemorrhagic stool) of the mice was visually examined. Gastric mucosal
injury and colonic mucosal injury were analyzed based on the hematotoxin
and eosin staining images shown in Figure 5.

\textbf{Figure 1. Preferential suppression of fibroblast activity by
eperisone.}

LL29 or A549 cells were incubated with the indicated concentrations (\selectlanguage{greek}µ\selectlanguage{english}M)
of eperisone for 24 h. The percentage of viable cells was determined
using a CellTiter-Glo\selectlanguage{ngerman}® 2.0 assay (A). LL29 cells were incubated with the
indicated concentrations (\selectlanguage{greek}µ\selectlanguage{english}M) of eperisone for 18 h. The cytotoxicity
was measured every hour using CellTox Green Dye and a microplate reader
(excitation: 485 nm, emission: 530 nm) (B). LL29 cells were incubated
with transforming growth factor (TGF)-\selectlanguage{greek}β\selectlanguage{english}1 (5 ng/ml) for 72 h in the
presence of the indicated concentrations of eperisone. Total RNA was
extracted and subjected to real-time RT-PCR using a specific primer set
for each gene. The values were normalized to \emph{Gapdh} gene
expression and expressed relative to the control sample (C). Values
represent the mean \selectlanguage{ngerman}± SEM *\emph{*} \emph{P \textless{} 0.01} ; \emph{*P
\textless{} 0.05} ; NS, not significant.

\textbf{Figure 2. Effect of other drugs on the percentage of viable LL29
and A549 cells.}

LL29 or A549 cells were incubated with the indicated concentrations of
pirfenidone, nintedanib (A), tolperisone, inaperisone, lanperisone,
tizanidine, methocarbamol, or baclofen (B) for 24 h. The percentage of
viable cells was determined using a CellTiter-Glo® 2.0 assay. Values
represent the mean ± SEM *\emph{*P \textless{} 0.01} ; \emph{*P
\textless{} 0.05.}

\textbf{Figure 3. Effect of eperisone on pre-developed pulmonary
fibrosis.}

Mice were treated with bleomycin (BLM, 1 mg/kg) or vehicle once only on
day 0. The mice were then orally administered the indicated dose of
eperisone (Epe) once daily for 9 days (from day 10 to day 18). Pulmonary
tissue sections were prepared on day 20 and subjected to a
histopathological examination (Masson's trichrome staining; scale bar =
500 \selectlanguage{greek}µ\selectlanguage{english}m) (A). The collagen-positive area was determined based on Masson's
trichrome staining images (B). The pulmonary hydroxyproline level was
determined on day 20 (C). The total respiratory system elastance, tissue
elastance, and forced vital capacity (FVC) were measured on day 20 (D).
Values represent the mean \selectlanguage{ngerman}± SEM *\emph{*P \textless{} 0.01} ; \emph{*P
\textless{} 0.05} ; NS, not significant.

\textbf{Figure 4. Effect of eperisone on bleomycin-induced increases in
myofibroblasts.}

Mice were treated with bleomycin (BLM, 1 mg/kg) or vehicle once only on
day 0. The mice were then orally administered the indicated dose of
eperisone (Epe) once daily for 9 days (from day 10 to day 18). Pulmonary
tissue sections were prepared on day 20 and subjected to
immunohistochemical analysis with an antibody against \selectlanguage{greek}α-\selectlanguage{english}smooth muscle
actin (SMA) (scale bar = 100 \selectlanguage{greek}µ\selectlanguage{english}m) (A). The \selectlanguage{greek}α-\selectlanguage{english}SMA-positive area was
determined using ImageJ software (B). Values represent the mean \selectlanguage{ngerman}± SEM
*\emph{*P \textless{} 0.01} ; \emph{*P \textless{} 0.05.}

\textbf{Figure 5. Effects of eperisone administration on gastric and
colonic mucosa.}

Mice were treated with bleomycin (BLM, 1 mg/kg) or vehicle once only on
day 0. The mice were then orally administered 250 mg/kg of eperisone
(Epe) once at day 10. After 24 h, the stomach and colon were collected
from the mice. Gastric (A) and colonic (B) tissue sections were prepared
and subjected to histopathological examination (hematotoxin and eosin
staining; scale bar = 200 \selectlanguage{greek}µ\selectlanguage{english}m).\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/Fig1/Fig1}

\end{center}
\end{figure}\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/Fig2/Fig2}

\end{center}
\end{figure}\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/Fig3/Fig3}

\end{center}
\end{figure}\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/Fig4/Fig4}

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\includegraphics[width=0.70\columnwidth]{figures/Fig5/Fig5}

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\includegraphics[width=0.70\columnwidth]{figures/Table1/Table1}

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\includegraphics[width=0.70\columnwidth]{figures/Table2/Table2}

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