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 in vitro and in vivo systems.
As shown in Figure 1A, eperisone treatment (25–200 µM) decreased the percentage of viable LL29 cells in a dose-dependent manner. In contrast, the percentage of viable A549 cells treated with 200 µM of eperisone was 88.5 ± 3.0% (mean ± SEM, n= 4), revealing almost no decrease in viable A549 cells after eperisone treatment. We next examined eperisone-induced cytotoxicity in LL29 cells using CellTox™ 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-β1–induced activation of lung fibroblasts. LL29 cells were pre-treated with eperisone (10–30 µM), followed by the addition of TGF-β1 (5 µM), and the expression of fibrosis-related factors was analyzed 72 h later by real-time RT-PCR. As shown in Figure 1C, TGF-β1 increased the mRNA expression of Collagen 1a1 (COL1A1) , α-SMA (ACTA2) , connective tissue growth factor (CTGF ), vascular endothelial growth factor (VEGF ), basic fibroblast growth factor (BFGF ), and platelet derived growth factor (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 in vitro .
Effects of other drugs on lung 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.