Regular Article
Eperisone Hydrochloride, a Muscle Relaxant, Is a Potent P2X7 Receptor Antagonist
2024 年 72 巻 3 号 p. 345-348
詳細
2024 年 72 巻 3 号 p. 345-348
Eperisone Hydrochloride was launched in Japan in 1983 and has been used to improve muscle tone and treat spastic paralysis (Originator: Eisai Co., Ltd.). 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.
Chemical Biology, proposed by Stuart L. Schreiber in the late 1980s, is a field of study that uses chemical compounds to elucidate biological phenomena.1) It is a very interesting method with an element of solving mysteries.
There have been reports of a relationship between Eperisone and pain (Eperisone-pain linkage).
Cabitza and Randelli reported the efficacy of Eperisone in patients with acute low back pain and spasticity of the spinal muscles.2) Beltrame et al. reported that the administration of Eperisone resulted in a prompt reduction of both spontaneous and provoked pain.3)
Microglial purinergic receptors (P2Xs) contribute to the pathogenesis of neuropathic pain (P2Xs-pain linkage).4)
Eperisone also has the structural characteristics of P2X receptor antagonists (Table 1). P2X receptor antagonists commonly have (1) a carbonyl/sulfonyl oxygen atom, (2) multiple rings, and (3) a low-molecular weight. Eperisone is also a small molecule with a carbonyl oxygen atom and multiple rings. In addition, since Eperisone is pharmacologically pain-relieving, we hypothesized the P2X receptor as the biochemical mechanism of action (MoA).
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These two linkages and the structural characteristics inspired us to investigate Eperisone in the P2X panel screening.
The effect of Eperisone Hydrochloride (Fig. 1) on the P2X receptor subtypes was examined using a fluorescence assay. MoA of the muscle relaxant Eperisone Hydrochloride has been unknown for more than 40 years; however, we found that Eperisone antagonizes the P2X7 receptor (Table 2). It has also been found to be highly selective towards P2X receptor subtypes. Eperisone Hydrochloride antagonizes the human-mouse P2X7 receptor. In contrast, Eperisone Hydrochloride also showed weak antagonism for human P2X3 but was 569-fold more selective for human P2X7. Gefapixant, a P2X3 receptor antagonist, causes dysgeusia as an adverse effect.5) However, because Eperisone has a high selectivity (569-fold) for P2X3, no taste disorders have been reported.6)
| Target | Eperisone hydrochloride (IC50, µmol/L) | Positive control substance (IC50, µmol/L) | Eperisone hydrochloride selectivity (human) P2X (1–5)/P2X7 |
|---|---|---|---|
| Human P2X1 | >10 | 4.536 (PPNDS) | >794 |
| Human P2X2 | >10 | 0.5904 (PPADS) | >794 |
| Human P2X3 | 7.158 | 0.6441 (RO-3) | 569 |
| Human P2X4 | >10 | 2.309 (BX430) | >794 |
| Human P2X5 | >10 | 312.6 (PPADS) | >794 |
| Human P2X7 | 0.01259 | 0.2701 (A-438079) | 1 |
| Mouse P2X7 | 0.04784 | 0.09006 (CN-62) | — |
Evaluated by double measurement.
“Eperisone Hydrochloride Data (Table 2)” Human P2X7: IC50 = 12.6 nmol/L, Selectivity for P2X (1–5) receptors: >569 fold.
We next performed YO-PRO-1 uptake assay known as another standard method for evaluation of P2X7 receptor antagonists. IC50s of Eperisone Hydrochloride and A-804598 (an antagonist of P2X7 receptor) were 15 and 6.9 nmol/L, respectively (a representative result of repeated 2 assays).
Eperisone Hydrochloride was launched in 1983, and the P2X7 receptor was cloned in 1996.7,8) The antagonist Eperisone was discovered more than 13 years before receptor cloning. This is interesting because it demonstrates the usefulness of phenotypic screening.
Eperisone is believed to act on the γ-motoneuron system in the spinal cord to inhibit the afferent firing of muscle spindles and to relax muscle tone by suppressing the polysynaptic reflex in the spinal cord.6) On the other hand, microglia are known to be activated in the dorsal horn of the spinal cord during the onset of pain,4) microglia are known to be one of the cells expressing P2X receptors.8)
These two linkages, (Eperisone-pain linkage) and (P2Xs-pain linkage), and the structural characteristics led to the Eperisone-P2X linkage hypothesis. In this study, we confirmed the compound-target-indication linkage by elucidating the MoA of the existing drug, Eperisone Hydrochloride.
The relationship between P2X7 and pain has been widely reported (P2X7-pain linkage). Dell’Antonio et al. used the irreversible inhibitor of the P2X7 receptor, designated oxidized ATP (oATP), to test its possible antinociceptive activity in arthritic rats.9) Chessell et al. demonstrated that in mice lacking this receptor, inflammatory (in an adjuvant-induced model) and neuropathic (in a partial nerve ligation model) hypersensitivity was completely absent to both mechanical and thermal stimuli, whilst normal nociceptive processing was preserved.10) Honore et al. demonstrated that selective blockade of P2X7 receptors in vivo produced significant antinociception in animal models of neuropathic and inflammatory pain.11) King reviewed the role of P2X7 receptors in the onset and persistence of chronic pain in animal models.12) Ristić and Ellrich reported that P2X7 receptor blockade reverses purinergic facilitation of neck muscle nociception in mice.13)
Our findings revealed that the P2X7 receptor is a druggable target. We also showed the possibility of using a P2X7 receptor antagonist for the indications of Eperisone (improvement of muscle tone and spastic paralysis) and Eperisone for the indications currently under development (neuropathic pain, etc.).
Based on the data of three linkages, (Eperisone-pain linkage), (P2X7-pain linkage) (Eperisone-P2X7 linkage), the analgesic effects of Eperisone were presumed to be mediated by the P2X7 receptor (Eperisone-P2X7-pain linkage) (Fig. 2).
However, no reports are indicating that the inhibition of P2X7 causes skeletal muscle relaxation or spastic paralysis. Eperisone may be mediated by P2X7 to improve muscle tone and spastic paralysis. However, further studies are required.
LY3857210 (AK1780), a P2X7 receptor antagonist of ASAHI KASEI-RaQualia–Lilly, is currently in the clinical stage of treatment for chronic lower back pain, pain due to knee osteoarthritis, and diabetic peripheral neuropathic pain.
As the MoA is now known, drug repositioning of Eperisone may expand the indications for neuropathic pain and other indications. The discovery of P2X7 as a target for existing drugs and the fact that the P2X7 receptor is a druggable target with low-class effects (3.8% incidence of adverse effects for Eperisone Hydrochloride6)) are expected to encourage the development of P2X7 receptor antagonists. It would also be interesting to elucidate the pharmacological effects of P2X7 based on the chemical biology of Eperisone.
Tolperisone Hydrochloride (Fig. 3) is a central muscle relaxant discovered before Eperisone (1958).14) It is presumed that Tolperisone is also likely to act on P2X7.
We elucidated one of the MoAs of Eperisone, which has been used as an unknown for over 40 years. We found that Eperisone selectively antagonized the P2X7 receptor. This finding indicates that P2X7 is a druggable target. We hope that this finding will contribute to the elucidation of the pharmacological effects and safety of P2X7 receptor antagonists and the development of new drugs for the treatment of many patients.
There have been no reports on the interaction between Eperisone and P2X receptors. It has been reported that the ion channels may be part of the MoA of Eperisone in a neuron of Achatina fulica.15) P2X receptors are largely involved in pain, as described above; therefore, the antagonism of Eperisone against P2X receptors was investigated.
Panel screening for P2X receptor antagonism was outsourced, and P2X receptor subtype selectivity was confirmed.
Materials and MethodsEperisone Hydrochloride was purchased from FUJIFILM Wako Pure Chemical Corporation (Osaka, Japan). The content of the standard was 98% (titrated).
SB Drug Discovery Screening Services (www.sbdrugdiscovery.com, Glasgow, Lanarkshire, U.K.) were used to evaluate receptor antagonism of human P2X1, human P2X2, human P2X3, human P2X4, human P2X5, human P2X7, and mouse P2X7 using fluorescence. Eperisone Hydrochloride was used at concentrations of 10, 100, 1000, and 10000 nmol/L in duplicate.
ProtocolProtocol 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 (a human monocytic cell line, obtained from American Tissue Culture Collection, Manassas, VA, U.S.A.) 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. The IC50 values of the test compound were determined using GraphPad Prism software.
Search EnginesPub Med, Google Scholar, Google, and Cortellis Drug Discovery Intelligence were used.
We dedicate our works to the late Emeritus Professor Chikara Kaneko of Tohoku University, who taught us the fun of chemistry, frontier orbital theory, tennis, and others. We sincerely thank Professor Nobuya Katagiri, who made us interested in the experimentation. We would like to thank Shuzo Watanabe, Koji Bandoh, Yasuhiro Tsukimi, Katsuyuki Keino, Shuji Ohta, Shuichiro Sato, Jun-ya Kato, Satoshi Maeda, Tomoaki Watanabe, Masayuki Yoshizawa, Miho Yoshizawa, Atsushi Sinbo, Youichi Nakano, Hideo Kobayashi, Hiroyuki Suzuki, Kouki Morita, Takeo Shibata, Nobuyoshi Minami, Kouichi Hasumi, special members of Aska Pharmaceutical Co., Ltd., who supported us patiently.
The authors declare no conflict of interest.
