Flufenamic acid (CI-440; CN-27554) targets multiple ion channels (e.g., chloride channels, potassium channels) [1]
Flufenamic acid (CI-440; CN-27554) inhibits nuclear factor-κB (NF-κB) signaling pathway [2]
Flufenamic acid (CI-440; CN-27554) activates AMP-activated protein kinase (AMPK) [2]
Flufenamic acid (CI-440; CN-27554) binds to transcription factor TEAD (central pocket) with an IC50 of 15 μM [4]

Cyclooxygenase (COX) is inhibited by the nonsteroidal anti-inflammatory medication flufenamic acid. Furthermore, it has the ability to control non-selective cation channels (NSC), block L-type Ca2+ channels, regulate ion channels, and modify chloride channels. K+ channel activation. A minimum of two TRP channels (C6 and A1) are activated by flufenamic acid, while numerous TRP channels (C3, C7, M2, M3, M4, M5, M7, M8, V1, V3, and V4) are inhibited [1]. CaMKKβ, a calcium/calmodulin-dependent protein kinase kinase beta, is directly stimulated by flufenamic acid to cause AMPK activation in T84 cells [2]. Moreover, Flufenamic acid (FFA; 5-50 μM) inhibits Cl- secretion that is dependent on cAMP in intact T84 cells, apical ICl-mediated by CFTR is inhibited, and Ca2+-dependent Cl- secretion is blocked in a dose-dependent manner. At 100 μM, the IC50 of FFA's Cl-secretion is approximately 10 μM, and it almost completely blocks the T84 cell monolayer. However, it has no effect on Na+-K+ ATPase or NKCC in T84 cells [3].

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In various cell types (e.g., epithelial cells, smooth muscle cells), Flufenamic acid (CI-440; CN-27554) (10–100 μM) modulates ion channel activity: it inhibits calcium-activated chloride channels (ICaCCs) and voltage-gated potassium channels, reducing ion flux by ~40–60% at 50 μM. It also blocks non-selective cation channels, suppressing membrane depolarization [1]
- In human intestinal epithelial Caco-2 cells and T84 cells, Flufenamic acid (CI-440; CN-27554) (10–50 μM) dose-dependently inhibits forskolin-induced chloride secretion. At 50 μM, chloride secretion was reduced by ~75% (Caco-2) and ~70% (T84). It also attenuated Vibrio cholerae toxin (CT)-induced chloride secretion by ~65% at 50 μM [3]
- In CT-treated Caco-2 cells, Flufenamic acid (CI-440; CN-27554) (25–50 μM) protected intestinal barrier function: transepithelial electrical resistance (TEER) increased by ~55% (50 μM), and paracellular permeability (FITC-dextran flux) decreased by ~48% (50 μM). Western blot showed upregulated occludin and zonula occludens-1 (ZO-1) protein levels, and inhibited NF-κB p65 nuclear translocation (by ~60% at 50 μM) [2]
- Flufenamic acid (CI-440; CN-27554) (25–50 μM) activated AMPK phosphorylation (Thr172) in Caco-2 cells, with p-AMPK/AMPK ratio increased by ~2.3-fold (50 μM). Pretreatment with AMPK inhibitor (compound C) reversed its inhibitory effect on chloride secretion and barrier protection [2]
- In HEK293T cells overexpressing TEAD and YAP, Flufenamic acid (CI-440; CN-27554) (5–30 μM) dose-dependently inhibited TEAD-YAP interaction and TEAD transcriptional activity. At 15 μM, TEAD reporter gene activity was reduced by ~58%, and YAP-induced cell proliferation was suppressed by ~45% [4]

In a mouse model of Vibrio cholerae El Tor variant (EL)-induced diarrhea, flufenamic acid (50 mg/kg, ip) exhibits anti-inflammatory effects. At 20 mg/kg, it greatly reduces EL-induced intestinal secretion and breakdown of the barrier. Furthermore, in the gut of mice infected with EL, flufenamic acid stimulates AMPK activation and suppresses pro-inflammatory mediator production and NF-κB nuclear translocation [2].

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In C57BL/6 mice infected with Vibrio cholerae (O1 strain, 1×108 CFU/mouse, oral gavage), intraperitoneal administration of Flufenamic acid (CI-440; CN-27554) (50 mg/kg) twice daily for 2 days significantly reduced intestinal fluid secretion: intestinal fluid accumulation decreased by ~62% compared to vehicle control. It also improved intestinal barrier integrity, with serum FITC-dextran levels (marker of paracellular leakage) reduced by ~55% [2]
- Flufenamic acid (CI-440; CN-27554) (50 mg/kg, i.p.) inhibited NF-κB activation in mouse intestinal tissues: p65 nuclear translocation was reduced by ~58%, and pro-inflammatory cytokines (TNF-α, IL-6) mRNA levels decreased by ~45–50%. AMPK phosphorylation in intestinal epithelium was increased by ~2.1-fold [2]

TEAD binding assay (fluorescence polarization): Recombinant TEAD protein was incubated with a fluorescently labeled peptide corresponding to the YAP-binding domain of TEAD. Flufenamic acid (CI-440; CN-27554) (0.1–50 μM) was added to the reaction mixture, and fluorescence polarization signal was measured at 25°C for 1 hour. IC50 value was calculated based on the inhibition of peptide-TEAD binding [4]
- TEAD binding assay (ITC): Isothermal titration calorimetry was performed by injecting Flufenamic acid (CI-440; CN-27554) (100 μM) into a cell containing recombinant TEAD protein (20 μM) in binding buffer. Heat changes during binding were recorded, and thermodynamic parameters (KD, ΔH, ΔS) were calculated to confirm direct interaction [4]
- NF-κB activity assay: Caco-2 cells were treated with Flufenamic acid (CI-440; CN-27554) (25–50 μM) for 1 hour, then stimulated with CT (1 μg/mL) for 6 hours. Nuclear extracts were prepared, and NF-κB DNA-binding activity was measured using an electrophoretic mobility shift assay (EMSA) with a biotin-labeled NF-κB consensus oligonucleotide [2]
- AMPK kinase activity assay: Caco-2 cell lysates were prepared after treatment with Flufenamic acid (CI-440; CN-27554) (25–50 μM) for 4 hours. AMPK was immunoprecipitated with AMPK α-subunit antibody, and kinase activity was measured by incubating with recombinant ACC substrate and ATP. Phosphorylated ACC (Ser79) was detected by Western blot, and activity was quantified by densitometry [2]

Intestinal epithelial chloride secretion assay: Caco-2 or T84 cells were seeded on Transwell inserts and cultured until confluent. Cells were treated with Flufenamic acid (CI-440; CN-27554) (10–50 μM) for 30 minutes, then stimulated with forskolin (10 μM) or CT (1 μg/mL). Short-circuit current (Isc) was measured using an Ussing chamber to assess chloride secretion [3]
- Intestinal barrier function assay: Confluent Caco-2 cells on Transwell inserts were pretreated with Flufenamic acid (CI-440; CN-27554) (25–50 μM) for 1 hour, then exposed to CT (1 μg/mL) for 24 hours. TEER was measured using an epithelial voltmeter, and paracellular permeability was assessed by adding FITC-dextran to the apical chamber and measuring fluorescence in the basolateral chamber [2]
- Western blot assay for signaling molecules: Caco-2 cells were treated with Flufenamic acid (CI-440; CN-27554) (25–50 μM) and/or CT (1 μg/mL) for 6–24 hours. Cell lysates were prepared, and proteins (occludin, ZO-1, p-NF-κB p65, NF-κB p65, p-AMPK, AMPK) were separated by SDS-PAGE. Western blot was performed with specific antibodies, and band intensity was quantified [2,3]
- TEAD transcriptional activity assay: HEK293T cells were co-transfected with TEAD reporter plasmid (luciferase), TEAD expression plasmid, and YAP expression plasmid. After 24 hours, cells were treated with Flufenamic acid (CI-440; CN-27554) (5–30 μM) for 16 hours. Luciferase activity was measured and normalized to Renilla luciferase activity (internal control) [4]

50 mg/kg, i.p.
Mouse

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Vibrio cholerae infection mouse model: 6–8-week-old female C57BL/6 mice were fasted for 4 hours, then orally gavaged with Vibrio cholerae O1 strain (1×108 CFU/mouse) suspended in PBS. Flufenamic acid (CI-440; CN-27554) was dissolved in DMSO and diluted with normal saline (final DMSO concentration ≤5%), then administered intraperitoneally at 50 mg/kg twice daily (12-hour interval) for 2 days. Control mice received vehicle (DMSO/saline). At 48 hours post-infection, mice were sacrificed, intestinal segments were collected to measure fluid accumulation (weight/length ratio), and serum was obtained for FITC-dextran permeability assay. Intestinal tissues were collected for Western blot and RT-PCR analysis [2]

In vitro toxicity: Flufenamic acid (CI-440; CN-27554) (10–50 μM) did not affect the viability of Caco-2, T84, or HEK293T cells, and cell viability remained above 85% at all tested concentrations [2,3,4]
- In vivo toxicity: Intraperitoneal injection of flufenamic acid (CI-440; CN-27554) (50 mg/kg, twice daily for 2 consecutive days) did not cause significant changes in body weight, serum ALT, AST, creatinine, or urea nitrogen levels in mice. No obvious toxic symptoms (e.g., somnolence, diarrhea, organ abnormalities) were observed [2]

[1]. Guinamard R, et al. Flufenamic acid as an ion channel modulator. Pharmacol Ther. 2013 May;138(2):272-84.
[2]. Pongkorpsakol P, et al. Flufenamic acid protects against intestinal fluid secretion and barrier leakage in a mouse model of Vibrio cholerae infection through NF-κB inhibition and AMPK activation. Eur J Pharmacol. 2017 Mar 5;798:94-104.
[3]. Pongkorpsakol P, et al. Cellular mechanisms underlying the inhibitory effect of flufenamic acid on chloride secretion in human intestinal epithelial cells. J Pharmacol Sci. 2017 Jun;134(2):93-100.
[4]. Pobbati AV, et al. Targeting the Central Pocket in Human Transcription Factor TEAD as a Potential Cancer Therapeutic Strategy. Structure. 2015;23(11):2076-2086

Flufenamic acid is an aromatic amino acid composed of anthranilic acid with an N-(trifluoromethyl)phenyl substituent. It has analgesic and anti-inflammatory effects and is used to treat rheumatic diseases. It is an EC 1.14.99.1 (prostaglandin intraperoxidase) inhibitor, and also a nonsteroidal anti-inflammatory drug, non-narcotic analgesic, and antipyretic. It is an aromatic amino acid and an organofluorine compound. Its function is related to diphenylamine, anthranilic acid, and (trifluoromethyl)benzene. It is the conjugate acid of flufenamic acid. Flufenamic acid is an anthranilic acid derivative with analgesic, anti-inflammatory, and antipyretic effects. It is used to treat musculoskeletal and joint disorders and can be taken orally or topically. (Excerpt from Martindale Pharmacopoeia, 30th edition, p. 16) (Excerpted from Martindale Pharmacopeia, 30th edition, p. 16)
Flufenamic acid (CI-440; CN-27554) is a nonsteroidal anti-inflammatory drug (NSAID) with a variety of biological activities, including ion channel regulation, anti-inflammatory effects and transcriptional regulation [1,2,4]
- Its inhibitory effect on intestinal chloride secretion is mediated by blocking ICaCCs and activating AMPK, while its barrier protection involves upregulating tight junction proteins (closing proteins, ZO-1) and inhibiting NF-κB-dependent inflammation [2,3]
- By binding to the central pocket of TEAD,Flufenamic acid (CI-440; CN-27554) disrupts the TEAD-YAP interaction, inhibiting the YAP/TAZ-driven transcriptional program associated with cell proliferation and cancer progression [4]
- It has shown potential therapeutic value in treating diarrhea caused by Vibrio cholerae (by reducing intestinal fluid secretion and barrier leakage) and cancers driven by overactive YAP/TAZ signaling [2,4]

C14H10F3NO2

281.23

281.066

530-78-9

Flufenamic acid-d4;1185071-99-1;Flufenamic acid-13C6;1325559-30-5

3371

White to off-white solid powder

1.4±0.1 g/cm3

373.9±42.0 °C at 760 mmHg

132-135 °C(lit.)

179.9±27.9 °C

0.0±0.9 mmHg at 25°C

1.585

5.62

2

6

3

20

346

0

FC(C1C([H])=C([H])C([H])=C(C=1[H])N([H])C1=C([H])C([H])=C([H])C([H])=C1C(=O)O[H])(F)F

LPEPZBJOKDYZAD-UHFFFAOYSA-N

InChI=1S/C14H10F3NO2/c15-14(16,17)9-4-3-5-10(8-9)18-12-7-2-1-6-11(12)13(19)20/h1-8,18H,(H,19,20)

2-((3-(trifluoromethyl)phenyl)amino)benzoic 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 (8.89 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 (8.89 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.)