P2X7 receptor

Preferential suppression of fibroblast activity by Eperisone [1]
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. Therefore, in this study, we focused on eperisone, which is used in clinical practice as a central muscle relaxant, and examined its efficacy against IPF using in vitro and in vivo systems.
As shown in Fig. 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. In addition, eperisone was also found to reduce the number of viable cells in other fibroblasts (HFL-1 and IMR-90 cells) in a dose-dependent manner (Supplementary Fig. S1A). Furthermore, eperisone showed little toxicity to RL-34 cells (rat liver-derived normal epithelial cells) and preferentially decreased the number of viable cells in RI-T cells (rat hepatic stellate cells), which differentiate into myofibroblasts (Supplementary Fig. S1B). We next examined eperisone-induced cytotoxicity in LL29 cells using CellTox™ Green Dye, which can detect cell membrane disruption. As shown in Fig. 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 Fig. 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.

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Effects of other drugs on lung fibroblast viability [1]
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 (Fig. 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 (Fig. 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.

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Eperisone Hydrochloride was launched in Japan in 1983 and has been used to improve muscle tone and treat spastic paralysis. However, its biochemical mechanism of action is unknown. SB Drug Discovery was used to evaluate purinergic P2X (P2X) receptor antagonism using fluorescence. In this study, we discovered that its target protein is the P2X7 receptor. Also, P2X receptor subtype selectivity was high. This finding demonstrates the (Eperisone-P2X7-pain linkage), the validity of P2X7 as a drug target, and the possibility of drug repositioning of Eperisone Hydrochloride[5].

Effect of Eperisone on BLM-induced pulmonary fibrosis [1]
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 (Fig. 3A, B). Next, we performed quantitative analysis of hydroxyproline, a collagen-specific amino acid, in lung tissue. As shown in Fig. 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. Thus, we measured the respiratory function of mice using a computer-controlled ventilator and negative pressure reservoir. As shown in Fig. 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.

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Purpose: Eperisone is an oral muscle relaxant used in musculoskeletal disorders causing muscle spasm and pain. For more effective pain control, eperisone is usually prescribed together with nonsteroidal anti-inflammatory drugs (NSAIDs). As such, eperisone may have been overlooked as the cause of anaphylaxis compared with NSAIDs. This study aimed to analyze the adverse drug reaction (ADR) reported in Korea and suggest an appropriate diagnostic approach for eperisone-induced anaphylaxis. Methods: We reviewed Eperisonee-related pharmacovigilance data (Korea Institute of Drug Safety-Korea Adverse Event Reporting System [KIDS-KAERS]) reported in Korea from 2010 to 2015. ADRs with causal relationship were selected. Clinical manifestations, severity, outcomes, and re-exposure information were analyzed. For further investigation, 7-year ADR data reported in a single center were also reviewed. Oral provocation test (OPT), skin prick test (SPT) and basophil activation test (BAT) were performed in this center. Results: During the study period, 207 patients had adverse reactions to Eperisone. The most common ADRs were cutaneous hypersensitive reactions (30.4%) such as urticaria, itchiness or angioedema. Fifth common reported ADR was anaphylaxis. There were 35 patients with anaphylaxis, comprising 16.9% of the eperisone-related ADRs. In the single center study, there were 11 patients with eperisone-induced anaphylaxis. All the patients underwent OPT and all the provoked patients showed a positive reaction. Four of the 11 patients with anaphylaxis also underwent SPT and BAT, which were all negative. Conclusions: Incidence of Eperisone-induced anaphylaxis calculated from the KIDS-KAERS database was 0.001%. Eperisone can cause hypersensitive reactions, including anaphylaxis, possibly by inducing non-immunoglobulin E-mediated immediate hypersensitivity.

Protocol of P2X panel screening: Cells (132N1 astrocytoma cells for the P2X1 receptor and HEK293 cells for other P2X receptors) stably expressing P2X receptors were seeded in black, clear-bottomed 96-well plates at a density of 50000 cells per well and incubated overnight at 37 °C. The next day, medium was removed from the cell plates and 25 µL of the assay buffer (1.11 mM CaCl2, 0.43 mM MgCl.6H2O, 0.36 mM MgSO4.7H2O, 4.98 mM KCl, 0.39 mM KH2PO4, 122 mM NaCl, 0.3 mM Na2HPO4, 4.86 mM D-glucose, 17.7 mM N-(2-hydroxyethyl) piperazine-N′-2-ethanesulfonic acid (HEPES), pH 7.4) was added. A calcium dye solution (10 µL) was added to the wells and incubated at 37 °C for 1 h. Test compounds were added (5 µL) and incubated for 10 min at room temperature. The plates were then placed in FLIPR, and fluorescence was monitored every 1.52 s. After 20 s, 10 µL of the agonist at approximately EC80 concentration was added and the fluorescence was monitored for 5 min at an ex/em of 488 nm/510–570 nm. The IC50 values of the test compounds were determined using the GraphPad Prism software. Protocol of YO-PRO-1 uptake assay: THP-1 cells were seeded onto 100 mm dish at a density of 10000000 cells per dish and treated with 500 nmol/L phorbol 12-myristate 13-acetate for 3 h. Cells were harvested and washed with phosphate buffered saline (PBS) by centrifugation and the cells were re-suspended in medium (10% fetal bovine serum (FBS)/RPMI1640). The cells were seeded in Black-wall 96 well plate at a density of 80000 cells/0.2 mL/well and incubated over night at 37 °C. After then, lipopolysaccharide (LPS) (1 µg/mL) was added in wells and the cells were treated for 6 h (Priming). The cells were treated with test compounds, Yo-PRO-1 (2 µmol/L) was added in wells and incubated for 15 min at 37 °C. Finally, BzATP (300 micromol/L, an agonist of P2X7 receptor) was added in wells and fluorescence monitored every 1 min for 60 min at ex/em: 485 nm/535 nm. Maximum slope of fluorescence for 10 min was measured [5].

Treatment of mice with BLM, Eperisone, and other reagents [1]
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.

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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 ± SEM.[1]

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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. The analysis of fecal condition was conducted by an investigator blinded to the study protocol. Gastric mucosal injury and colonic mucosal injury were analyzed based on the hematotoxin and eosin staining images shown in Fig. 5.[1]

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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 µm).[1]

Safety analysis of ipilisone administration [1] In clinical practice, it has been reported that existing drugs for the treatment of idiopathic pulmonary fibrosis (IPF), such as pirfenidone and nintedanib, can cause adverse reactions, such as elevated plasma markers of liver injury and gastrointestinal disorders. Therefore, we conducted a comprehensive analysis of plasma markers of pancreatic, liver and kidney injury. The dose of ipilisone was five times the effective dose for bleomycin (BLM)-dependent pulmonary fibrosis. As shown in Table 1, neither bleomycin alone nor bleomycin in combination with ipilisone (250 mg/kg, single dose on day 10) significantly altered 12 plasma markers of pancreatic, liver and kidney injury. In addition, after 9 consecutive days (days 10 to 18) of ipilisone (50 mg/kg), no significant changes were observed in four plasma markers indicating liver and kidney injury (Supplementary Table S1). Furthermore, no diarrhea or bloody stools were observed in either group of mice (Table 2). In addition, we used hematoxylin-eosin staining to detect gastric and colonic mucosal damage. As shown in Figure 5 and Table 2, compared with the carrier control group, the gastric and colonic mucosal conditions of mice in the bleomycin (BLM) group and the bleomycin combined with ipilisone (250 mg/kg) group were unchanged, and no gastric and colonic mucosal damage was observed. These results suggest that ipilisone may be able to inhibit pulmonary fibrosis without causing adverse reactions.

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Ipilisone hydrochloride (4'-ethyl-2-methyl-3-piperidinylphenylacetone hydrochloride) is an antispasmodic drug used to treat diseases characterized by muscle stiffness and pain. This study aimed to investigate the efficacy of ipilisone in treating patients with acute low back pain and spinal muscle spasms. This study used a randomized, double-blind (double-dummy) design and included 160 patients with low back pain who had no history of major spinal diseases. Patients were randomly assigned to receive either epirlisone 100 mg orally three times daily (tid) or colchicine 8 mg orally twice daily (bid) for 12 consecutive days. Analgesic activity was assessed using spontaneous pain (VAS score) and motor/pressure pain (4-digit score), while muscle relaxant activity was assessed using hand-to-ground distance and the Lasseg test. All measurements were taken on enrollment and on days 3, 7, and 12 post-treatment. The analgesic and muscle relaxant effects of both drugs were comparable. Both treatments significantly reduced spontaneous pain and motor/pressure pain. Furthermore, patients treated with both epirlisone and colchicine showed clinically significant muscle relaxation, as evidenced by a gradual decrease in hand-to-ground distance and an increase in joint range of motion (Lasseg test). Only 5% of patients in the epirlisone group experienced mild gastrointestinal side effects, compared to 21.25% in the colchicine group. In addition, diarrhea occurred in patients treated with colchicine, with some cases being moderate. In conclusion, epirlisone is a more effective and safer alternative to other muscle relaxants for the treatment of low back pain. [2] Epirlisone is an analgesic central muscle relaxant that has been used to treat low back pain (LBP). This systematic review aimed to evaluate the efficacy and safety of epirlisone in patients with low back pain. This systematic review followed the Cochrane Back and Neck (CBN) group and the Preferred Reporting Items (PRISMA) guidelines for systematic reviews and meta-analyses. Risk of bias was assessed using the CBN group and the Moga tool. A total of 7 studies (5 randomized controlled trials [RCTs] and 2 uncontrolled studies) involving 801 participants were included. Epirlisone intervention may be effective in patients with acute low back pain with fewer adverse reactions (relative risk, 0.25; 95% confidence interval, 0.15–0.41; p<0.0001). Epirlisone also improves paravertebral blood flow and has similar efficacy to tizanidine in patients with chronic low back pain. The studies included in this review had small sample sizes and short follow-up periods, which are insufficient to support the use of ipilisone for the treatment of low back pain. However, we recommend conducting well-designed, high-quality, large-sample, and long-follow-up randomized controlled trials to confirm the clinical benefit of ipilisone in the treatment of acute or chronic low back pain. [3]

[1]. Therapeutic effects of eperisone on pulmonary fibrosis via preferential suppression of fibroblast activity. Cell Death Discov. 2022 Feb 8;8(1):52.
[2]. Efficacy and safety of eperisone in patients with low back pain: a double blind randomized study. Eur Rev Med Pharmacol Sci. 2008 Jul-Aug;12(4):229-35.
[3]. Clinical efficacy and safety of eperisone for low back pain: A systematic literature review. Pharmacol Rep. 2016 Oct;68(5):903-12.
[4]. Eperisone-Induced Anaphylaxis: Pharmacovigilance Data and Results of Allergy Testing. Allergy Asthma Immunol Res. 2019;11(2):231-240.
[5]. Eperisone Hydrochloride, a Muscle Relaxant, Is a Potent P2X7 Receptor Antagonist. Chem Pharm Bull (Tokyo). 2024;72(3):345-348.

Eperisone hydrochloride is an aromatic ketone compound. Although the exact pathogenesis of idiopathic pulmonary fibrosis (IPF) remains unclear, the transdifferentiation of fibroblasts to myofibroblasts triggered by alveolar epithelial cell damage and the generation of extracellular matrix components such as collagen are important mechanisms in the development and progression of IPF. In the lungs of IPF patients, the apoptosis rate of fibroblasts is lower than that of alveolar epithelial cells, and this process is closely related to the pathogenesis of IPF. We used a compound library containing approved drugs to screen for drugs that preferentially reduce the viability of LL29 cells (lung fibroblasts from IPF patients) rather than A549 cells (human alveolar epithelial cell line). After screening, we selected Eperisone, a clinically used centrally acting muscle relaxant. Eperisone has low toxicity to A549 cells and preferentially reduces the survival rate of LL29 cells, while pirfenidone and nintedanib do not have this effect. Ipirisone also significantly inhibited the transforming growth factor-β1-dependent transdifferentiation of LL29 cells into myofibroblasts. In ICR mice, ipirisone inhibited bleomycin (BLM)-induced pulmonary fibrosis, respiratory dysfunction, and fibroblast activation. In contrast, under the same experimental conditions, pirfenidone and nintedanib were less effective than ipirisone in inhibiting BLM-induced pulmonary fibrosis. Finally, we found that ipirisone did not cause adverse liver and gastrointestinal reactions in the BLM-induced pulmonary fibrosis model. Based on these results, we believe that ipirisone may be safer than existing therapies and may be more beneficial for patients with idiopathic pulmonary fibrosis (IPF). [1] Although pirfenidone and nintedanib are currently used clinically to treat idiopathic pulmonary fibrosis (IPF), these drugs are not effective in some cases and have been reported to cause adverse reactions such as elevated liver injury markers, diarrhea, and indigestion. Therefore, this study adopted a "drug repositioning strategy" to find safer and more effective drugs for the treatment of IPF. The in vitro studies shown in Figures 1 and 2 demonstrate that ipilisone (rather than pirfenidone or nintedanib) preferentially reduces fibroblast survival. Furthermore, the in vivo studies shown in Figures 3 and Supplementary Figure S1 demonstrate that ipilisone (rather than pirfenidone or nintedanib) inhibits bleomycin (BLM)-induced pulmonary fibrosis exacerbation. Moreover, ipilisone does not cause adverse reactions such as elevated hepatotoxicity markers or gastrointestinal disorders. Therefore, we believe that ipilisone may be safer and more effective than pirfenidone or nintedanib, making it a better option for treating idiopathic pulmonary fibrosis (IPF). After screening for drugs that selectively induce fibroblast death, we selected ipilisone and confirmed its efficacy in an animal model of IPF induced by fibroblast activation. As mentioned above, there has never been a previous report indicating that ipilisone preferentially induces fibroblast death or effectively treats fibrosis models. However, fibrosis can also occur in other organs besides the lungs, such as the liver, heart, and kidneys. For example, in the liver, hepatic stellate cells are activated by stimulation such as TGF-β1 and transdifferentiate into myofibroblasts, which promote the production of extracellular matrix components such as collagen, thereby inducing liver fibrosis in diseases such as non-alcoholic steatohepatitis. In the kidney, resident fibroblasts, pericytes, bone marrow-derived cells and endothelial cells transdifferentiate into myofibroblasts and induce renal fibrosis. Therefore, activated myofibroblasts derived from fibroblasts also play a role in fibrosis in other organs besides the lung. Therefore, ipilisone can preferentially inhibit the activity of fibroblasts and may be effective not only in pulmonary fibrosis models but also in fibrosis models of other organs; therefore, the results of this study have broad application prospects for future research. [1]

C₁₇H₂₆CLNO

295.85

295.17

C, 69.02; H, 8.86; Cl, 11.98; N, 4.73; O, 5.41

56839-43-1

Eperisone-d10 hydrochloride;1246819-46-4;Eperisone;64840-90-0

123698

White to off-white solid powder

0.994g/cm3

386.8ºC at 760mmHg

168-174ºC

137.4ºC

4.293

1

2

5

20

275

0

CCC1=CC=C(C=C1)C(=O)C(C)CN2CCCCC2.Cl

GTAXGNCCEYZRII-UHFFFAOYSA-N

InChI=1S/C17H25NO.ClH/c1-3-15-7-9-16(10-8-15)17(19)14(2)13-18-11-5-4-6-12-18;/h7-10,14H,3-6,11-13H2,1-2H3;1H

1-(4-ethylphenyl)-2-methyl-3-piperidin-1-ylpropan-1-one;hydrochloride

Eperisone hydrochloride; 56839-43-1; eperisone; 64840-90-0; Eperisone [INN]; Eperisona; (+-)-Eperisone; Eperisonum; Eperisonum [INN-Latin]; Eperisona [INN-Spanish]; Eperisone HCl; Myonal; Mional; E-646; UNII-U38O8U7P6X; Epenard (TN);

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: Please store this product in a sealed and protected environment, avoid exposure to moisture.

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

H2O : ~100 mg/mL (~338.01 mM)
DMSO : ~31.25 mg/mL (~105.63 mM)

Solubility in Formulation 1: ≥ 2.08 mg/mL (7.03 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: ≥ 2.08 mg/mL (7.03 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

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Solubility in Formulation 3: ≥ 2.08 mg/mL (7.03 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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Solubility in Formulation 4: 16.67 mg/mL (56.35 mM) in PBS (add these co-solvents sequentially from left to right, and one by one), clear solution; with ultrasonication.

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 (Please use freshly prepared in vivo formulations for optimal results.)