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.