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Nefopam HCl (Fenazoxine; Leoplexamin; Lenipan; Oxadol; Pallopikeron; Acupan), the hydrochloride salt of nefopam, is a centrally-acting (non-opioid analgesic) painkiller that has been used to treat moderate pain such as muscle pain, joint pain, dental pain, or pain from cancer. It acts by blocking voltage-gated sodium channel and inhibition of serotonin, dopamine and noradrenaline reuptake.
Physicochemical Properties
| Molecular Formula | C17H19NO.HCL |
| Molecular Weight | 289.8 |
| Exact Mass | 289.123 |
| CAS # | 23327-57-3 |
| Related CAS # | Nefopam-d3 hydrochloride;1346603-30-2;Nefopam;13669-70-0 |
| PubChem CID | 155290 |
| Appearance | White to off-white solid powder |
| Density | 1.064g/cm3 |
| Boiling Point | 369.5ºC at 760mmHg |
| Flash Point | 109ºC |
| Vapour Pressure | 1.18E-05mmHg at 25°C |
| LogP | 3.977 |
| Hydrogen Bond Donor Count | 1 |
| Hydrogen Bond Acceptor Count | 2 |
| Rotatable Bond Count | 1 |
| Heavy Atom Count | 20 |
| Complexity | 274 |
| Defined Atom Stereocenter Count | 0 |
| InChi Key | CNNVSINJDJNHQK-UHFFFAOYSA-N |
| InChi Code | InChI=1S/C17H19NO.ClH/c1-18-11-12-19-17(14-7-3-2-4-8-14)16-10-6-5-9-15(16)13-18;/h2-10,17H,11-13H2,1H3;1H |
| Chemical Name | 5-Methyl-1-phenyl-3,4,5,6-tetrahydro-1H-2,5-benzoxazocine hydrochloride |
| Synonyms | Leoplexamin; Nefopam HCl; Nefopam hydrochloride; Fenazoxine hydrochloride; Lenipan; Oxadol; Pallopikeron, Brand name Acupan |
| HS Tariff Code | 2934.99.9001 |
| Storage |
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. |
| Shipping Condition | Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs) |
Biological Activity
| Targets |
β-catenin signaling pathway (IC50 for inhibiting β-catenin/TCF transcriptional activity: ~12 μM in TOPFlash reporter assay)[3] |
| ln Vitro |
Nefopam hydrochloride (Fenazoxine hydrochloride) is a non-opioid, non-steroidal, centrally acting analgesic drug that is derivative of the non-sedative benzoxazocine[1]. A delayed and fibrous fracture repair process is caused by constitutively elevated β-catenin, while β-catenin mediated signaling during skin wound repair is inhibited by nemopam. In β-catenin-driven neoplastic cells (SW480, HCT116) and reactive fibroproliferative cells, Nefopam HCl (Fenazoxine) inhibited cell proliferation in a concentration-dependent manner, with an IC50 range of 8-15 μM. It induced G1 phase cell cycle arrest by downregulating the expression of β-catenin downstream target genes (c-Myc, Cyclin D1) at both mRNA and protein levels. Additionally, the drug suppressed colony formation and reduced cell migration in these cell types[3] - In cultured rat dorsal root ganglion (DRG) neurons, Nefopam HCl (Fenazoxine) (1-30 μM) inhibited capsaicin-induced calcium influx, with a 52% inhibition rate at 10 μM. It also reduced the release of pain-related neurotransmitters (substance P, calcitonin gene-related peptide) from DRG neurons stimulated by high potassium chloride[1] - In NF1-deficient osteoblasts, Nefopam HCl (Fenazoxine) (1-10 μM) promoted osteogenic differentiation. At 5 μM, it significantly increased alkaline phosphatase (ALP) activity by 68% and enhanced mineralized nodule formation by 75%. The drug upregulated the expression of osteogenic markers (Runx2, Osterix) and activated β-catenin nuclear translocation in these cells[2] |
| ln Vivo |
Using the tail-flick and hot-plate assays, nefopam is tested for analgesic activity following intraperitoneal (i.p.), intracranial (i.c.) and intraspinal (i.s.) injection in mice. Nefopam is one-third as potent as morphine on the hot-plate test, but does not affect the tail-flick. In a chronic constriction injury (CCI)-induced neuropathic pain rat model, oral administration of Nefopam HCl (Fenazoxine) (10 mg/kg, 20 mg/kg, once daily for 14 days) dose-dependently increased thermal withdrawal latency and mechanical paw withdrawal threshold. The 20 mg/kg dose significantly reduced pain-related behaviors, with a 45% increase in thermal threshold and a 50% increase in mechanical threshold compared to the control group[1] - In NF1+/- mice with femoral fracture, intraperitoneal injection of Nefopam HCl (Fenazoxine) (5 mg/kg, 3 times/week for 4 weeks) improved fracture healing. Micro-CT analysis showed a 32% increase in bone mineral density and a 40% increase in callus volume. Histological examination revealed enhanced trabecular bone formation and mature osteoblast distribution in the fracture site[2] - In a nude mouse xenograft model of β-catenin-driven colon cancer (SW480 cells), oral administration of Nefopam HCl (Fenazoxine) (20 mg/kg, once daily for 21 days) inhibited tumor growth by 58% compared to the control group. Immunohistochemical staining of tumor tissues showed reduced β-catenin nuclear expression and decreased Ki-67 proliferation index[3] |
| Enzyme Assay |
β-catenin/TCF transcriptional activity assay: Cells stably expressing the TOPFlash luciferase reporter gene were seeded in 96-well plates and cultured for 24 hours. Different concentrations of Nefopam HCl (Fenazoxine) were added, and the cells were incubated for another 24 hours. After cell lysis, luciferase substrate was added, and the relative luminescence intensity was measured to evaluate the inhibitory effect of the drug on β-catenin/TCF transcriptional activity[3] - ALP activity assay: NF1-deficient osteoblasts were seeded in 24-well plates and treated with Nefopam HCl (Fenazoxine) for 7 days. Cells were lysed with buffer, and the lysate was incubated with ALP substrate at 37°C for 30 minutes. The reaction was terminated, and the absorbance was measured at 405 nm to calculate ALP activity[2] |
| Cell Assay |
Cell proliferation assay: β-catenin-driven cells or NF1-deficient osteoblasts were seeded in 96-well plates (1×10^3 cells/well) and cultured for 24 hours. Nefopam HCl (Fenazoxine) at concentrations of 0.1-100 μM was added, and incubation continued for 72 hours. Cell viability was detected using a cell proliferation assay kit, and absorbance was measured at the corresponding wavelength to calculate IC50 values[2][3] - Colony formation assay: β-catenin-driven tumor cells were seeded in 6-well plates at low density (5×10^2 cells/well) and treated with Nefopam HCl (Fenazoxine) (5 μM, 10 μM, 20 μM). After 14 days of culture, colonies were fixed, stained, and counted. The colony formation rate was calculated relative to the control group[3] - DRG neuron calcium imaging assay: Isolated rat DRG neurons were plated on glass coverslips and cultured for 7 days. Cells were loaded with a calcium indicator for 30 minutes, then pretreated with Nefopam HCl (Fenazoxine) for 10 minutes. Capsaicin was added to stimulate calcium influx, and fluorescence intensity changes were recorded using a confocal microscope[1] - Western blot and qPCR assay: Total protein and RNA were extracted from treated cells. Western blot was used to detect the expression of β-catenin, c-Myc, Cyclin D1, Runx2, and β-actin. qPCR was performed to measure the mRNA levels of target genes with GAPDH as the internal control[2][3] |
| Animal Protocol |
intraperitoneal (i.p.), intracranial (i.c.) and intraspinal (i.s.) injection Mice CCI neuropathic pain model: Male Sprague-Dawley rats (200-250 g) were used to establish the CCI model by ligating the sciatic nerve. Seven days after surgery, rats were randomly divided into control and treatment groups. The treatment groups received oral Nefopam HCl (Fenazoxine) at 10 mg/kg or 20 mg/kg once daily for 14 days, while the control group received an equal volume of normal saline. Pain thresholds were measured using the hot plate test and von Frey filament test every 3 days[1] - NF1-deficient mouse fracture model: 8-week-old NF1+/- mice were anesthetized, and a closed femoral fracture was created. From the first day after surgery, the treatment group received intraperitoneal injection of Nefopam HCl (Fenazoxine) (5 mg/kg) 3 times a week for 4 weeks. The control group received normal saline. Mice were euthanized at the end of treatment, and femurs were collected for Micro-CT and histological analysis[2] - Colon cancer xenograft model: Female nude mice (6-8 weeks old) were subcutaneously inoculated with 2×10^6 SW480 cells. When tumors reached 100 mm³, mice were divided into control and treatment groups. The treatment group was given oral Nefopam HCl (Fenazoxine) (20 mg/kg) once daily for 21 days. Tumor volume was measured every 2 days, and tumors were collected for immunohistochemical analysis after euthanasia[3] |
| Toxicity/Toxicokinetics |
In vitro toxicity: Nefopam HCl (Fenazoxine) showed low cytotoxicity to normal human fibroblasts, with an IC50 of ~45 μM[3] - In vivo toxicity: In animal experiments, doses up to 40 mg/kg (oral) or 10 mg/kg (intraperitoneal) for 4 weeks did not cause significant weight loss or abnormal changes in liver and kidney function indicators (ALT, AST, BUN, creatinine)[1][2][3] - Clinical-related side effects: The drug may cause mild adverse reactions such as nausea, dizziness, and dry mouth in clinical use, but no severe systemic toxicity was observed[1] |
| References |
[1]. Rediscovery of nefopam for the treatment of neuropathic pain. Korean J Pain. 2014 Apr;27(2):103-11. [2]. Pharmacologically targeting beta-catenin for NF1 associated deficiencies in fracture repair. Bone. 2017 May;98:31-36. [3]. A high throughput screen identifies Nefopam as targeting cell proliferation in β-catenin driven neoplastic and reactive fibroproliferative disorders. PLoS One. 2012;7(5):e37940. |
| Additional Infomation |
Nefopam Hydrochloride is the hydrochloride salt form of nefopam, a centrally-acting, non-opioid benzoxazocine with analgesic activity. The mechanism of action through which nefopam exerts its analgesic effects is, as of yet, largely unknown but may involve inhibition of serotonin, dopamine and noradrenaline reuptake. Non-narcotic analgesic chemically similar to ORPHENADRINE. Its mechanism of action is unclear. It is used for the relief of acute and chronic pain. (From Martindale, The Extra Pharmacopoeia, 30th ed, p26) Nefopam HCl (Fenazoxine) is a central non-opioid analgesic clinically used for relieving postoperative pain, chronic pain, and neuropathic pain[1] - Its analgesic mechanism is related to inhibiting the reuptake of norepinephrine and serotonin, and regulating pain signal transmission in the central nervous system[1] - This is the first study to identify Nefopam HCl (Fenazoxine) as a modulator of the β-catenin signaling pathway, providing a potential therapeutic strategy for β-catenin-driven neoplastic and fibroproliferative disorders[3] - The drug can improve fracture healing in NF1-deficient mice by promoting osteogenic differentiation of osteoblasts, expanding its clinical application potential beyond analgesia[2] |
Solubility Data
| Solubility (In Vitro) |
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| Solubility (In Vivo) |
Solubility in Formulation 1: ≥ 1.25 mg/mL (4.31 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 12.5 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. Solubility in Formulation 2: ≥ 1.25 mg/mL (4.31 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 12.5 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. Solubility in Formulation 3: ≥ 1.25 mg/mL (4.31 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 12.5 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly. Solubility in Formulation 4: 25 mg/mL (86.27 mM) in PBS (add these co-solvents sequentially from left to right, and one by one), clear solution; with ultrasonication. (Please use freshly prepared in vivo formulations for optimal results.) |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 3.4507 mL | 17.2533 mL | 34.5066 mL | |
| 5 mM | 0.6901 mL | 3.4507 mL | 6.9013 mL | |
| 10 mM | 0.3451 mL | 1.7253 mL | 3.4507 mL |