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 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 α-SMA and 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-β1-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 α-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-β1-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 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, thein 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-β1 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.