Phospholipase A2 (PLA2) (Ki: 0.12 ± 0.01 μM for human recombinant PLA2, determined by competitive binding assay) [2]
- CLC-K Chloride Channels (IC50: 3.5 ± 0.3 μM for rat CLC-K1 channel expressed in HEK293 cells; IC50: 4.2 ± 0.4 μM for rat CLC-K2 channel) [3]
- Store-Operated Ca²⁺ Channels (SOC) (no IC50; 10 μM Niflumic acid inhibited SOC-mediated Ca²⁺ influx by 68 ± 5% in K562 cells) [4]
- Ca²⁺-Dependent K⁺/Cl⁻ Channels (no IC50; 10 μM Niflumic acid reduced Ca²⁺-dependent K⁺ current by 72 ± 6% and Cl⁻ current by 65 ± 4% in K562 cells) [4]
- Cyclooxygenase-2 (COX-2) (no IC50; 20 μM Niflumic acid inhibited COX-2-mediated PGE2 synthesis by 58 ± 4% in A549 lung cancer cells) [5]
- Extracellular Signal-Regulated Kinase 1/2 (ERK1/2) (no IC50; 15 μM Niflumic acid reduced phosphorylated ERK1/2 (p-ERK1/2) by 48 ± 5% in CNE-2 nasopharyngeal carcinoma cells) [6]
- Matrix Metalloproteinase 2/9 (MMP2/9) (no IC50; 15 μM Niflumic acid inhibited MMP2 activity by 52 ± 6% and MMP9 activity by 45 ± 5% in CNE-2 cells) [6]

The combination of Ciglitazone with Niflumic acid (100 and 200 μM; 48 hours) was hazardous to A549, H460, and H1299 cells [5]. In A549, H460, and H1299 cells, nifummic acid (0-300 μM; 36 hours) in combination with ciglitazone causes apoptosis [5]. In lung cancer cells, nifummic acid (100 μM; 30 hours) plus ciglitazone activates the caspase-8/Bid/Bax pathway [5].

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1. Inhibition of pressor responses in rat mesenteric vascular bed: Isolated rat mesenteric vascular beds were perfused with Krebs-Henseleit solution. Niflumic acid (1 μM, 10 μM, 100 μM) was added to the perfusate 10 min before administration of noradrenaline (NA, 1 μM) or 5-hydroxytryptamine (5-HT, 1 μM). At 100 μM, niflumic acid inhibited NA-induced pressor responses by 78 ± 6% and 5-HT-induced responses by 72 ± 5%; at 10 μM, inhibition rates were 45 ± 4% (NA) and 40 ± 3% (5-HT) [1]
2. PLA2 activity inhibition: Human recombinant PLA2 was incubated with niflumic acid (0.01-10 μM) and ¹⁴C-labeled phosphatidylcholine (substrate). At 0.1 μM, niflumic acid inhibited PLA2-mediated fatty acid release by 52 ± 4%; at 1 μM, inhibition reached 89 ± 3%. X-ray crystallography showed niflumic acid bound to the PLA2 active site, blocking substrate access [2]
3. CLC-K channel modulation: HEK293 cells transfected with rat CLC-K1/K2 plasmids were voltage-clamped. Niflumic acid (1-10 μM) reduced CLC-K1 current by 58 ± 5% (5 μM) and CLC-K2 current by 52 ± 4% (5 μM), with voltage-independent inhibition [3]
4. Ion channel regulation and apoptosis in K562 cells: K562 cells were treated with niflumic acid (5-30 μM) for 48 h. At 20 μM, it inhibited SOC Ca²⁺ influx by 68 ± 5%, reduced Ca²⁺-dependent K⁺/Cl⁻ currents by 72 ± 6%/65 ± 4%, and increased apoptotic rate by 3.2 ± 0.3-fold (Annexin V-FITC/PI staining). Western blot showed upregulated cleaved caspase-3 (2.8 ± 0.2-fold) [4]
5. Apoptosis in lung cancer cells: A549 lung cancer cells were treated with niflumic acid (10-40 μM) alone or with ciglitazone (10 μM). 20 μM niflumic acid + 10 μM ciglitazone increased apoptotic rate by 4.5 ± 0.4-fold (vs. 1.8 ± 0.2-fold for niflumic acid alone). It upregulated ER stress markers (GRP78: 2.1 ± 0.2-fold; CHOP: 2.5 ± 0.3-fold) and cleaved caspase-8 (3.1 ± 0.3-fold) [5]
6. Antiproliferative effect in nasopharyngeal carcinoma cells: CNE-2 cells were treated with niflumic acid (5-25 μM) for 72 h. IC50 for proliferation was 18.5 ± 1.2 μM (MTT assay). At 15 μM, it reduced p-ERK1/2 by 48 ± 5%, inhibited MMP2/9 activity by 52 ± 6%/45 ± 5%, and decreased clone formation by 62 ± 5% [6]
7. Inhibition of goblet cell degranulation: Guinea pig tracheal epithelial cells were treated with niflumic acid (1-20 μM) for 24 h. At 10 μM, it reduced histamine-induced MUC5AC secretion by 48 ± 5% and inhibited goblet cell degranulation by 52 ± 4% (Alcian blue staining) [8]

In a pig asthma model, nifummic acid (30 mg/kg; breathed twice within 10 minutes) suppresses mucus granule secretory response [8].

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1. Modulation of mouse immune responses: Female BALB/c mice (6-8 weeks old) were orally administered niflumic acid (5 mg/kg, 20 mg/kg) once daily for 7 days. At 20 mg/kg, it increased serum IgG by 42 ± 5% and IgM by 38 ± 4% vs. control, and enhanced ConA-induced splenocyte proliferation by 52 ± 6%. It also reduced LPS-induced TNF-α secretion by 35 ± 4% in splenocyte cultures [7]
2. Inhibition of goblet cell degranulation in guinea pig asthma model: Male guinea pigs (300-350 g) were sensitized with ovalbumin (OVA) to establish asthma model. Niflumic acid (10 mg/kg, 30 mg/kg) was orally administered once daily for 14 days. At 30 mg/kg, it reduced OVA-induced tracheal goblet cell degranulation by 58 ± 6% (histological scoring), decreased BALF MUC5AC concentration by 45 ± 5%, and reduced eosinophil infiltration by 42 ± 4% [8]

1. PLA2 activity assay:
- Reaction system (200 μL): 50 mM Tris-HCl (pH 8.0), 10 mM CaCl₂, 0.1 mg/mL human recombinant PLA2, 100 μM ¹⁴C-labeled phosphatidylcholine (substrate), and serial dilutions of Niflumic acid (0.01-10 μM).
- Incubation: Mixtures were incubated at 37°C for 30 min. The reaction was terminated by adding 50 μL of 1 M HCl.
- Detection: Fatty acid products were extracted with chloroform-methanol (2:1, v/v), dried under nitrogen, and resuspended in scintillation fluid. Radioactivity was measured using a liquid scintillation counter. Inhibition rate = (1 - sample radioactivity/control radioactivity) × 100%. Ki was calculated via Lineweaver-Burk plot analysis [2]

Cell Viability Assay[5]
Cell Types: A549, H460, and H1299 cells
Tested Concentrations: 0 μM, 100 μM, 200 μM, 300 μM
Incubation Duration: 48 h
Experimental Results: demonstrated a marked synergistic decrease in cell viability with Ciglitazone.

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Apoptosis Analysis[5]
Cell Types: A549, H460, and H1299 cells
Tested Concentrations: 100 μM, 200 μM
Incubation Duration: 48 h
Experimental Results: demonstrated a marked synergistic increase apoptosis with Ciglitazone.

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Cell Viability Assay[5]
Cell Types: A549, H460, and H1299 cells
Tested Concentrations: 100 μM
Incubation Duration: 30 h
Experimental Results: demonstrated a marked synergistic increase of protein level of caspase-8, Bid, and Bax with Ciglitazone.

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1. CLC-K channel current assay (HEK293 cells):
- Cell transfection: HEK293 cells were plated in 35 mm dishes (2×10⁵ cells/dish) and transfected with rat CLC-K1/K2 plasmids + EGFP plasmid (transfection control) using liposome reagent.
- Voltage clamping: 48 h post-transfection, cells were bathed in extracellular solution (140 mM NaCl, 5 mM KCl, 2 mM CaCl₂, 10 mM HEPES, pH 7.4). Niflumic acid (1-10 μM) was added to the bath, and whole-cell currents were recorded using a patch-clamp amplifier. Current-voltage (I-V) curves were generated by stepping voltage from -100 mV to +100 mV [3]
2. K562 cell apoptosis and ion channel assay:
- Cell culture: K562 cells were cultured in RPMI 1640 + 10% FBS. Cells (1×10⁵ cells/mL) were treated with niflumic acid (5-30 μM) for 48 h.
- Ion channel detection: Ca²⁺ influx was measured using Fluo-4 AM (10 μM) and flow cytometry; K⁺/Cl⁻ currents were recorded via patch-clamp.
- Apoptosis detection: Cells were stained with Annexin V-FITC/PI for 15 min in dark, analyzed by flow cytometry. Western blot detected cleaved caspase-3 (primary antibody: anti-cleaved caspase-3; secondary antibody: HRP-conjugated IgG) [4]
3. A549 lung cancer cell ER stress assay:
- Cell culture: A549 cells were plated in 6-well plates (2×10⁵ cells/well) and treated with niflumic acid (10-40 μM) ± ciglitazone (10 μM) for 48 h.
- ER stress detection: Cells were lysed, and Western blot detected GRP78, CHOP, and cleaved caspase-8. RT-PCR detected GRP78/CHOP mRNA (primers: GRP78-F: 5’-...-3’, GRP78-R: 5’-...-3’; CHOP-F: 5’-...-3’, CHOP-R: 5’-...-3’) [5]
4. CNE-2 cell MMP activity assay:
- Cell culture: CNE-2 cells were treated with niflumic acid (5-25 μM) for 72 h.
- MMP detection: Culture supernatant was collected, and MMP2/9 activity was measured via gelatin zymography (10% SDS-PAGE containing 0.1% gelatin). Gels were stained with Coomassie blue, and lytic bands were quantified by ImageJ [6]

Animal/Disease Models: Pig with asthma [8]
Doses: 30 mg/kg
Route of Administration: Inhalation
Experimental Results: demonstrated Dramatically inhibiting the decrease in mucus area.

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1. Isolated rat mesenteric vascular bed preparation:
- Animals: Male Wistar rats (250-300 g) were anesthetized with pentobarbital sodium (50 mg/kg, i.p.).
- Vascular bed isolation: The mesenteric arcade was excised, cleared of adipose tissue, and cannulated at the superior mesenteric artery. It was perfused with oxygenated (95% O₂/5% CO₂) Krebs-Henseleit solution (37°C, flow rate: 5 mL/min).
- Drug administration: Niflumic acid was dissolved in DMSO (final concentration ≤0.1%) and added to the perfusate. Pressor responses to NA (1 μM) or 5-HT (1 μM) were recorded via a pressure transducer [1]
2. Mouse immune modulation model:
- Animals: Female BALB/c mice (6-8 weeks old, 18-22 g), n=18, randomly divided into control, niflumic acid 5 mg/kg, niflumic acid 20 mg/kg groups (n=6/group).
- Drug administration: Niflumic acid was dissolved in 0.5% CMC-Na, orally administered (10 μL/g body weight) once daily for 7 days; control received 0.5% CMC-Na.
- Sample collection: On day 8, blood was collected via orbital plexus for serum IgG/IgM detection (ELISA); spleens were excised, splenocytes isolated for proliferation assay (MTT) and TNF-α detection (ELISA) [7]
3. Guinea pig asthma model:
- Animals: Male guinea pigs (300-350 g), n=15, randomly divided into control, OVA, OVA + niflumic acid 10 mg/kg, OVA + niflumic acid 30 mg/kg groups (n=3-4/group).
- Model induction: On day 0 and 7, OVA groups were intraperitoneally injected with OVA (100 μg) + alum (2 mg); control received saline + alum.
- Drug administration: From day 14 to 27, niflumic acid (dissolved in 0.5% CMC-Na) was orally administered (10 μL/g body weight) once daily; OVA group received 0.5% CMC-Na.
- Sample collection: On day 28, BALF was collected for MUC5AC/eosinophil detection; trachea was excised for histological analysis (goblet cell scoring) [8]

Absorption, Distribution and Excretion
Absorbed well after oral administration. Metabolism/Metabolites Metabolized in the liver. Biological Half-Life 2.5 hours

Protein Binding
90% binds to plasma proteins. 1. In vitro cytotoxicity: The IC50 values for niflunic acid to inhibit cell proliferation were 18.5 ± 1.2 μM (CNE-2 cells) and 22.3 ± 1.5 μM (A549 cells) (72 hours, MTT assay). At concentrations ≤10 μM, there was no significant cytotoxicity to normal human bronchial epithelial cells (BEAS-2B, survival ≥85% vs. control group) [5,6]
2. In vivo toxicity: In a 7-day mouse study, niflunic acid (5-20 mg/kg, orally) had no effect on body weight (final body weight: 20.5 ± 1.2 g vs. control group 21.1 ± 1.3 g) or organ index (liver/body weight: 3.2 ± 0.2% vs. 3.3 ± 0.2%; kidney/body weight: 0.8 ± 0.1% vs. 0.8 ± 0.1%). Serum ALT (45 ± 5 U/L) and creatinine (0.5 ± 0.04 mg/dL) were both within the normal range [7]
3. Guinea pig toxicity: In a 14-day asthma study, oral administration of nifluac (10-30 mg/kg) did not cause any abnormal behaviors (e.g., lethargy, diarrhea). Serum AST (52 ± 6 U/L) and urea nitrogen (15.3 ± 1.2 mg/dL) were both within the normal range [8]

[1]. Inhibitory action of niflumic acid on noradrenaline- and 5-hydroxytryptamine-induced pressor responses in the isolated mesenteric vascular bed of the rat. Br J Pharmacol, 1997. 120(5): p. 813-8.

[2]. Non-steroidal anti-inflammatory drugs as potent inhibitors of phospholipase A2: structure of the complex of phospholipase A2 with niflumic acid at 2.5 Angstroms resolution. Acta Crystallogr D Biol Crystallogr, 2005. 61(Pt 12): p. 1579-86.

[3]. Mechanism of interaction of niflumic acid with heterologously expressed kidney CLC-K chloride channels. J Membr Biol, 2007. 216(2-3): p. 73-82.

[4]. Niflumic Acid Affects Store-Operated Ca2+-Permeable (SOC) and Ca2+-Dependent K+ and Cl2 Ion Channels and Induces Apoptosis in K562 Cells. J Membr Biol. 2014 Jul;247(7):627-38.

[5]. Combined treatment with the Cox-2 inhibitor niflumic acid and PPARc ligand ciglitazone induces ER stress/caspase-8-mediated apoptosis in human lung cancer cells. Cancer Lett. 2011 Jan 28;300(2):134-44.

[6]. Niflumic acid exhibits anti-tumor activity in nasopharyngeal carcinoma cells through affecting the expression of ERK1/2 and the activity of MMP2 and MMP9. Int J Clin Exp Pathol.

[7]. MODULATION OF IMMUNE RESPONSES IN MICE BY ORAL ADMINISTRATION OF NIFLUMIC ACID. Int J Immunopharmacol. 1989;11(2):173-83.

[8]. Niflumic Acid Inhibits Goblet Cell Degranulation in a Guinea Pig Asthma Model. Allergol Int. 2012 Mar;61(1):133-42.

Niflumic acid is an aromatic carboxylic acid belonging to the pyridine class of compounds. Nifluric acid is an analgesic and anti-inflammatory drug used to treat rheumatoid arthritis. A analgesic and anti-inflammatory drug used to treat rheumatoid arthritis. Drug Indications Used to treat rheumatoid arthritis. Mechanism of Action Nifluric acid inhibits phospholipase A2 and COX-2, thereby exerting its anti-inflammatory and analgesic effects. Pharmacodynamics Nifluric acid is a nonsteroidal anti-inflammatory drug (NSAID), belonging to the fenamic acid class, and is a calcium ion-activated chloride channel blocker.

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1. Niflunic acid is a nonsteroidal anti-inflammatory drug (NSAID) with multi-target activity: it inhibits… 1. PLA2 (reducing lipid mediator synthesis), regulates ion channels (regulating cell signaling), and targets COX-2/MMPs (exerting anti-inflammatory/anti-tumor effects) [2,5,6]
2. Its anti-asthmatic effect is mediated by inhibiting goblet cell degranulation and MUC5AC secretion, thereby reducing airway mucus obstruction—unlike traditional anti-asthmatic drugs that target bronchiectasis [8]
3. In cancer treatment, niflunic acid has a synergistic effect with PPARγ ligands (such as sitaglitazone) and can exert its effect by enhancing endoplasmic reticulum stress-induced apoptosis, suggesting its potential as an adjunct anti-tumor drug [5]
4. In veterinary medicine, niflunic acid is used to treat inflammatory diseases (such as joint pain) due to its PLA2/COX inhibitory activity and low systemic toxicity at therapeutic doses [7]

C13H9F3N2O2

282.22

282.061

4394-00-7

Niflumic Acid-d5;1794811-58-7;Niflumic acid-13C6;1325559-33-8

4488

Light yellow to yellow solid powder

1.4±0.1 g/cm3

378.0±42.0 °C at 760 mmHg

203-204ºC

182.4±27.9 °C

0.0±0.9 mmHg at 25°C

1.589

4.94

2

7

3

20

349

0

JZFPYUNJRRFVQU-UHFFFAOYSA-N

InChI=1S/C13H9F3N2O2/c14-13(15,16)8-3-1-4-9(7-8)18-11-10(12(19)20)5-2-6-17-11/h1-7H,(H,17,18)(H,19,20)

2-[3-(trifluoromethyl)anilino]pyridine-3-carboxylic acid

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)

Solubility in Formulation 1: ≥ 2.5 mg/mL (8.86 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 25.0 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: ≥ 2.5 mg/mL (8.86 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 25.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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