P2X7 receptor [5]

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]

123698 rat LD50 oral 1002 mg/kg SENSE ORGANS AND SPECIAL SENSES: LACRIMATION: EYE; AUTONOMIC NERVOUS SYSTEM: SMOOTH MUSCLE RELAXANT (MECHANISM UNDEFINED, SPASMOLYTIC); BEHAVIORAL: CONVULSIONS OR EFFECT ON SEIZURE THRESHOLD Yakuri to Chiryo. Pharmacology and Therapeutics., 19(1743), 1991
123698 rat LD50 subcutaneous 490 mg/kg BEHAVIORAL: EXCITEMENT; BEHAVIORAL: ATAXIA; LUNGS, THORAX, OR RESPIRATION: RESPIRATORY DEPRESSION Oyo Yakuri. Pharmacometrics., 21(939), 1981
123698 rat LD50 intravenous 51 mg/kg BEHAVIORAL: EXCITEMENT; BEHAVIORAL: ATAXIA; LUNGS, THORAX, OR RESPIRATION: RESPIRATORY DEPRESSION Oyo Yakuri. Pharmacometrics., 21(939), 1981
123698 rat LD50 intramuscular 400 mg/kg BEHAVIORAL: EXCITEMENT; BEHAVIORAL: ATAXIA; LUNGS, THORAX, OR RESPIRATION: RESPIRATORY DEPRESSION Oyo Yakuri. Pharmacometrics., 21(939), 1981
123698 mouse LD50 oral 324 mg/kg Yakugaku Zasshi. Journal of Pharmacy., 107(705), 1987 [PMID:3437397]

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Safety analysis of Eperisone administration [1]
In clinical practice, existing IPF treatments, such as pirfenidone and nintedanib, have been reported to induce adverse effects such as increasing markers of liver damage in the plasma and gastrointestinal disorders. Therefore, we conducted a comprehensive analysis of markers for pancreatic, hepatic, and renal damage in plasma. The dose of eperisone was five times higher than the dose that showed efficacy for BLM-dependent pulmonary fibrosis. As shown in Table 1, administration of BLM or BLM plus Eperisone (250 mg/kg, once at day 10) did not significantly alter 12 plasma markers for pancreatic, hepatic, and renal damage. Moreover, administration of eperisone (50 mg/kg) for 9 consecutive days (from day 10 to day 18) did not cause any significant changes in the four plasma markers indicating hepatic and renal damage (Supplementary Table S1). In addition, no mouse exhibited diarrhea or hemorrhagic stool in either group (Table 2). Furthermore, we also examined gastric and colonic mucosal injury using hematoxylin and eosin staining. As shown in Fig. 5 and Table 2, the condition of the gastric and colonic mucosa in mice treated with BLM or BLM plus eperisone (250 mg/kg) was unchanged compared with that in vehicle-treated mice, and no gastric and colonic mucosal injury was observed. These results suggest that eperisone may be able to suppress pulmonary fibrosis without inducing adverse effects.

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Eperisone hydrochloride (4'-ethyl-2-methyl-3-piperidinopropiophenone hydrochloride) is an antispastic agent used for treatment of diseases characterized by muscle stiffness and pain. The aim of this research was to investigate the efficacy of eperisone in patients with acute low back pain and spasticity of spinal muscles. The study design was a randomized, double-blind (double-dummy) study in 160 patients with low back pain and no Rx finding of major spinal diseases, randomly assigned to a treatment with oral eperisone 100 mg three times daily (t.i.d.) or thiocolchicoside 8 mg twice daily (b.i.d.) for 12 consecutive days. Analgesic activity was evaluated by scoring "spontaneous pain" (VAS) and pain on movement and pression (4-digit scale), while muscle relaxant activity of the medication was evaluated by means of the "hand-to-floor" distance and the Lasegue's manoeuvre. All the measures were done at the inclusion day and after 3, 7 and 12 days of treatment. The two medications had comparable analgesic and muscle relaxant efficacy. Sponta-neous pain and pain on movement/pressure were significantly reduced by both treatments. Moreover, both eperisone- and thiocolchicoside-treated patients showed a clinically evident muscle relaxation as proved by a progressive reduction in the "hand-to-floor" distance and increase in the articular excursion (Lasegue's manoeuvre). Only 5% of eperisone-treated patients showed minor gastrointestinal side effects, while the incidence of side effects in the thiocolchicoside group was 21.25%. Moreover, in the thiocolchicoside-treated patients also diarrhoea was present, which reached a moderate intensity in some cases. In conclusions, eperisone represents a valuable and safer alternative to other muscle relaxant agents for treatment of low back pain. [2]

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Eperisone, an analgesic and centrally acting muscle relaxant has been in use for the treatment of low back pain (LBP). The present systematic review evaluates the efficacy and safety of eperisone in patients with LBP. Cochrane Back and Neck (CBN) Group and Preferred Reporting Items for Systematic reviews and Meta-Analyses (PRISMA) guidelines were adopted to perform this systematic review. For risk of bias assessment CBN Group and Moga tools were used. Seven (5 randomized controlled trials [RCTs] and 2 uncontrolled studies) studies involving 801 participants were included. Eperisone intervention may be effective in acute LBP patients with less adverse effects (relative risk, 0.25; 95% confidence interval, 0.15-0.41; p<0.0001). Eperisone also improved paraspinal blood flow and was found to have efficacy similar to tizanidine in chronic LBP patients. The included studies in this review are of smaller sample size and short duration to support eperisone use in LBP. However, we recommend well-designed RCTs of high quality with larger sample size and longer follow-up to confirm the clinical benefits of eperisone in the treatment of acute or chronic LBP. [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.

1-(4-ethylphenyl)-2-methyl-3-(piperidin-1-yl)propan-1-one is an aromatic ketone that is N-propylpiperidine in which a hydrogen at positon 2 of the propyl group is replaced by a p-ethylbenzoyl group. It is a member of piperidines and an aromatic ketone.
Eperisone is an antispasmodic drug which relaxes both skeletal muscles and vascular smooth muscles, and demonstrates a variety of effects such as reduction of myotonia, improvement of circulation, and suppression of the pain reflex. It is not approved for use in the United States, but is available in other countries like India, South Korea, and Bangladesh.
See also: Esorubicin (annotation moved to).

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Although the exact pathogenesis of idiopathic pulmonary fibrosis (IPF) is still unknown, the transdifferentiation of fibroblasts into myofibroblasts and the production of extracellular matrix components such as collagen, triggered by alveolar epithelial cell injury, are important mechanisms of IPF development. In the lungs of IPF patients, apoptosis is less likely to be induced in fibroblasts than in alveolar epithelial cells, and this process is involved in the pathogenesis of IPF. We used a library containing approved drugs to screen for drugs that preferentially reduce cell viability in LL29 cells (lung fibroblasts from an IPF patient) compared with A549 cells (human alveolar epithelial cell line). After screening, we selected eperisone, a central muscle relaxant used in clinical practice. Eperisone showed little toxicity in A549 cells and preferentially reduced the percentage of viable LL29 cells, while pirfenidone and nintedanib did not have this effect. Eperisone also significantly inhibited transforming growth factor-β1-dependent transdifferentiation of LL29 cells into myofibroblasts. In an in vivo study using ICR mice, eperisone inhibited bleomycin (BLM)-induced pulmonary fibrosis, respiratory dysfunction, and fibroblast activation. In contrast, pirfenidone and nintedanib were less effective than eperisone in inhibiting BLM-induced pulmonary fibrosis under this experimental condition. Finally, we showed that eperisone did not induce adverse effects in the liver and gastrointestinal tract in the BLM-induced pulmonary fibrosis model. Considering these results, we propose that eperisone may be safer and more therapeutically beneficial for IPF patients than current therapies. [1]

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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. 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 Figs. 1 and 2 revealed that eperisone, but not pirfenidone or nintedanib, exhibited a fibroblast-preferential reduction of viable cells. Moreover, the in vivo studies shown in Fig. 3 and Supplementary Fig. 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. 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. In the kidney, resident fibroblasts, pericytes, bone marrow-derived cells, and endothelial cells transdifferentiate into myofibroblasts and induce kidney fibrosis. 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.[1]

C₁₇H₂₅NO

259.39

259.194

C, 78.72; H, 9.71; N, 5.40; O, 6.17

64840-90-0

Eperisone hydrochloride;56839-43-1

3236

Typically exists as solid at room temperature

0.994 g/cm3

386.8ºC at 760 mmHg

170-172ºC

137.4ºC

3.491

0

2

5

19

275

0

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

SQUNAWUMZGQQJD-UHFFFAOYSA-N

InChI=1S/C17H25NO/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

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

eperisone; 64840-90-0; Eperisone [INN]; Eperisona; (+-)-Eperisone; Eperisonum; Eperisonum [INN-Latin]; Eperisona [INN-Spanish];

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

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

May dissolve in DMSO (in most cases), if not, try other solvents such as H2O, Ethanol, or DMF with a minute amount of products to avoid loss of samples

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

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Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*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.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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