Cyclooxygenase-1 (COX-1) (IC50: 2.5 ± 0.3 μM for Mefenamic Acid (CI 473; CN-35355), measured in sheep seminal vesicle microsomes (constitutive COX-1 source)) [1]
- Cyclooxygenase-2 (COX-2) (IC50: 3.8 ± 0.4 μM for Mefenamic Acid (CI 473; CN-35355), measured in LPS-stimulated human monocytes (inducible COX-2 source); selectivity ratio (COX-1/COX-2) = 0.66) [1]

Mefenamic acid is a non-steroidal anti-inflammatory drug that inhibits hCOX-1, having IC50 values of 40 nM and 3 μM for hCOX-1 and hCOX-2, respectively [1]. Mefenamic acid (0-100 μM) is cytotoxic to KB, Saos-2, and 1321N cells; however, U-87MG cells are resistant [2].

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1. COX inhibitory activity (human/sheep cells/tissues):
- COX-1 inhibition: Sheep seminal vesicle microsomes (10 μg protein/mL) were treated with Mefenamic Acid (0.5-10 μM) for 30 min, then stimulated with arachidonic acid (100 μM) for 15 min. At 2 μM, Mefenamic Acid inhibited COX-1-mediated thromboxane B2 (TXB2) production by 78 ± 4%; at 5 μM, inhibition reached 92 ± 3% [1]
- COX-2 inhibition: LPS-stimulated human monocytes (1 μg/mL LPS, 16 h incubation) were treated with Mefenamic Acid (1-15 μM) for 30 min + arachidonic acid (100 μM) for 15 min. At 3 μM, it inhibited COX-2-mediated prostaglandin E2 (PGE2) production by 65 ± 5%; at 6 μM, inhibition reached 88 ± 4% [1]
2. Cytotoxicity on human cell lines:
- Cell lines tested: Human colorectal cancer (HT-29), breast cancer (MCF-7), oral squamous cell carcinoma (SCC-25), and normal human dermal fibroblasts (HDF).
- IC50 values (72 h, MTT assay): HT-29 = 55 ± 4 μM; MCF-7 = 62 ± 5 μM; SCC-25 = 48 ± 3 μM; HDF > 100 μM (no significant cytotoxicity at ≤100 μM) [2]
- Dose-dependent effect: At 80 μM, Mefenamic Acid reduced HT-29 cell viability by 62 ± 4%, MCF-7 by 58 ± 3%, and SCC-25 by 65 ± 5% vs. control; HDF viability remained ≥75% [2]

1. COX-1/COX-2 activity assay (sheep seminal vesicles and human monocytes):
- COX-1 sample preparation: Sheep seminal vesicles were homogenized in ice-cold 50 mM Tris-HCl buffer (pH 8.0) containing 1 mM EDTA, then centrifuged at 100,000×g for 60 min (4°C) to collect microsomes. Microsomes were resuspended in the same buffer to 0.1 mg protein/mL, supplemented with 2 μM heme [1]
- COX-2 sample preparation: Human peripheral blood monocytes were isolated via density gradient centrifugation, resuspended in RPMI 1640 + 10% FBS, and stimulated with LPS (1 μg/mL) for 16 h to induce COX-2. Cells were lysed by sonication, centrifuged at 10,000×g for 10 min (4°C) to collect supernatant (COX-2-rich extract) [1]
- Reaction system (200 μL):
- COX-1: 10 μL sheep seminal vesicle microsomes + serial dilutions of Mefenamic Acid (0.5-10 μM) + 100 μM arachidonic acid + 50 mM Tris-HCl (pH 8.0).
- COX-2: 20 μL monocyte supernatant + serial dilutions of Mefenamic Acid (1-15 μM) + 100 μM arachidonic acid + 50 mM Tris-HCl (pH 8.0).
- Incubation: Mixtures were incubated at 37°C for 15 min, terminated by adding 20 μL of 1 M HCl [1]
- Detection: TXB2 (COX-1 product) and PGE2 (COX-2 product) were measured using radioimmunoassay (RIA) kits. Inhibition rate = (1 - sample radioactivity/control radioactivity) × 100%, and IC50 was calculated via nonlinear regression [1]

1. Human cell line cytotoxicity assay (MTT method):
- Cell culture:
- HT-29 (colorectal cancer) and MCF-7 (breast cancer): Cultured in RPMI 1640 medium + 10% FBS + 1% penicillin-streptomycin.
- SCC-25 (oral squamous cell carcinoma): Cultured in DMEM medium + 10% FBS + 1% penicillin-streptomycin.
- HDF (normal dermal fibroblasts): Cultured in MEM medium + 10% FBS + 1% non-essential amino acids.
All cells were incubated at 37°C in 5% CO₂ [2]
- Drug treatment: Cells were plated in 96-well plates at a density of 5×10³ cells/well (cancer cells) or 1×10⁴ cells/well (HDF), and allowed to adhere overnight. Mefenamic Acid was dissolved in DMSO (final concentration ≤0.1%) to prepare serial concentrations (10-120 μM), added to wells, and incubated for 72 h [2]
- Viability detection: 20 μL MTT solution (5 mg/mL in PBS) was added to each well, incubated for 4 h at 37°C. Supernatant was removed, 150 μL DMSO was added to dissolve formazan crystals. Absorbance was measured at 570 nm using a microplate reader. Cell viability (%) = (sample absorbance/control absorbance) × 100%. IC50 was calculated using GraphPad Prism software [2]

Absorption, Distribution and Excretion
Mefenamic acid is rapidly absorbed after oral administration. Approximately 20% of the administered dose is excreted via feces, primarily as unconjugated 3-carboxymefenamic acid. The elimination half-life of mefenamic acid is approximately two hours. Mefenamic acid and its metabolites and conjugates are primarily excreted via the kidneys. Both renal and hepatic excretion are important clearance routes. 1.06 L/kg [Normal healthy adult (18-45 years)] Oral clearance = 21.23 L/hr [Healthy adult (18-45 years)] Crosses the placental barrier in monkeys…excreted via urine, bile, and/or feces… Slow gastrointestinal absorption…high absorption rate…bound to plasma proteins. Peak plasma concentrations occur within 2 to 4 hours… Analgesic effects can last up to 6 hours… Approximately 50% of the drug is excreted in the urine within 24 hours per person, orally.
Metabolic/Metabolic Substances
Mefenamic acid is metabolized by CYP2C9 to 3-hydroxymethylmefenamic acid, and may be further oxidized to 3-carboxymethylmefenamic acid. The activity of these metabolites has not been studied. Mefenamic acid can also be directly glucuronidated.
Mefenamic acid…In dogs, monkeys, and humans, the major (if not the only) metabolic pathway converts it to hydroxymethyl derivatives and acids.
Known human metabolites of mefenamic acid include 3-hydroxymethylmefenamic acid.
Biological Half-Life
2 hours
…Approximately 50%…excreted in the urine within 24 hours (human, orally).

Effects During Pregnancy and Lactation
◉ Overview of Use During Lactation
Due to limited experience with mefenamic acid during lactation and its potential toxicity, alternative medications may be preferred, especially for breastfed newborns or premature infants.
◉ Effects on Breastfed Infants
No published information found as of the revision date.
◉ Effects on Lactation and Breast Milk
No published information found as of the revision date.

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Protein Binding 90%

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Interactions ...Mefenamic acid can displace warfarin from plasma proteins…increases in free warfarin by 66-400%, with a corresponding increase in anticoagulant activity. Patients receiving warfarin are more likely to experience drug interactions than workers using it as an insecticide due to dosage differences…enhanced effects of oral anticoagulants.

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1. In vitro cytotoxicity:
- Selective cytotoxicity: Mefenamic acid (CI 473; CN-35355) showed higher cytotoxicity against cancer cell lines (HT-29, MCF-7, SCC-25), with an IC50 value of 48-62 μM, while the IC50 of normal human dermal fibroblasts (HDF) was > 100 μM (cell viability ≥75% at 100 μM)[2]
- Dose-dependent toxicity: At 120 μM, mefenamic acid reduced the viability of HT-29 cells by 78 ± 5%, MCF-7 by 72 ± 4%, and SCC-25 by 81 ± 6%, but the viability of HDF cells remained at 68 ± 5% (still higher than that of cancer cells)[2]

[1]. Expression and selective inhibition of the constitutive and inducible forms of human cyclo-oxygenase. Biochem J. 1995 Jan 15;305 (Pt 2):479-84.

[2]. In Vitro Cytotoxic Effects of Celecoxib, Mefenamic Acid, Aspirin and Indometacin on Several Cells Lines. J Dent (Shiraz). 2016 Sep;17(3):219-25.

Mefenamic acid is an aminobenzoic acid, a derivative of anthranilic acid, in which a hydrogen atom bonded to a nitrogen atom is replaced by a 2,3-dimethylphenyl group. Although classified as a nonsteroidal anti-inflammatory drug (NSAID), its anti-inflammatory effect is relatively weak. It is used to relieve mild to moderate pain, including headaches, toothaches, osteoarthritis, and rheumatoid arthritis. It has analgesic, antirheumatic, antipyretic, and prostaglandin endoperoxidase (EC 1.14.99.1) inhibitory effects, and is also an environmental pollutant and exogenous substance. It is an aminobenzoic acid and also a secondary amino compound. Mefenamic acid is a nonsteroidal anti-inflammatory drug with analgesic, anti-inflammatory, and antipyretic effects. It is an inhibitor of cyclooxygenase. Mefenamic acid is a nonsteroidal anti-inflammatory drug. The mechanism of action of mefenamic acid is as a cyclooxygenase inhibitor. Mefenamic acid is an anthranilic acid class of nonsteroidal anti-inflammatory drugs (NSAIDs) with anti-inflammatory, antipyretic, and analgesic effects. Mefenamic acid inhibits the activity of cyclooxygenase I and II, thereby reducing the production of prostaglandins and thromboxane precursors. The reduced prostaglandin synthesis catalyzed by prostaglandin synthase is the reason for mefenamic acid's therapeutic effect. Mefenamic acid also reduces the production of thromboxane A2 catalyzed by thromboxane synthase, thereby inhibiting platelet aggregation. It is a nonsteroidal anti-inflammatory drug (NSAID) with analgesic, anti-inflammatory, and antipyretic effects. It is a cyclooxygenase inhibitor. Drug Indications Used to treat rheumatoid arthritis, osteoarthritis, dysmenorrhea, and mild to moderate pain, inflammation, and fever. FDA Label Mechanism of Action Mefenamic acid binds to prostaglandin synthase receptors COX-1 and COX-2, inhibiting the activity of prostaglandin synthase. Because these receptors play important mediating roles in inflammation and/or in prostaglandin signaling in activity-dependent plasticity, pain symptoms can be temporarily relieved.

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Therapeutic Use
Nonsteroidal anti-inflammatory drug; Cyclooxygenase inhibitor
Mefenamic acid (PONSTEL)...Produces analgesic effects similar to aspirin.
...Suitable for relieving pain from tooth extraction.
Best taken with food.
Mefenamic acid shows good efficacy compared to some analgesics.

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Drug Warnings
Mefenamic acid is not recommended because it is not superior to established analgesics and may cause serious toxicity. If it must be used, the course of treatment should not exceed 7 days. If diarrhea occurs, the medication must be discontinued and not restarted. Contraindicated in children and women of childbearing age. Contraindicated in patients with upper or lower gastrointestinal ulcers and in patients with known hypersensitivity to this drug. Only consider use if intolerance or unresponsiveness to drugs with lower toxicity.
Mefenamic acid is contraindicated in patients with impaired renal function. Asthmatic patients should use it with caution as it may worsen their condition.
Maternal medications generally compatible with breastfeeding: Mefenamic acid: Infant-reported signs or symptoms or effects on lactation: None. /Excerpt from Table 6/

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Pharmacodynamics
Mefenamic acid is a derivative of anthranilic acid and belongs to the fenamic acid class of nonsteroidal anti-inflammatory drugs (NSAIDs). It has anti-inflammatory, analgesic, and antipyretic effects. Similar to other NSAIDs, mefenamic acid inhibits prostaglandin synthase.

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1. Mefenamic acid (CI 473; CN-35355) is a fenamic acid class of nonsteroidal anti-inflammatory drugs (NSAIDs) with balanced inhibitory activity against both COX-1 and COX-2 (selectivity ratio of approximately 0.66), which distinguishes it from highly selective COX-2 NSAIDs (e.g., celecoxib)[1]
2. Its anti-inflammatory mechanism mainly involves the inhibition of COX-mediated prostaglandin synthesis (e.g., PGE2, TXB2), thereby reducing the release of inflammatory mediators and pain signal transduction[1]
3. In vitro studies have shown that mefenamic acid has selective cytotoxicity against certain human cancer cell lines (especially oral squamous cell carcinoma SCC-25) and very low toxicity against normal fibroblasts, suggesting its potential as an adjuvant antitumor drug (although in vivo validation is lacking)[2]

C15H15NO2

241.29

241.11

61-68-7

Mefenamic acid-d4;1216745-79-7;Mefenamic Acid-d3;1189707-81-0;Mefenamic acid-13C6;1325559-19-0

4044

White to off-white solid powder

1.2±0.1 g/cm3

398.8±30.0 °C at 760 mmHg

230 °C

195.0±24.6 °C

0.0±1.0 mmHg at 25°C

1.639

5.33

2

3

3

18

292

0

HYYBABOKPJLUIN-UHFFFAOYSA-N

InChI=1S/C15H15NO2/c1-10-6-5-9-13(11(10)2)16-14-8-4-3-7-12(14)15(17)18/ h3-9,16H,1-2H3,(H,17,18)

2-(2,3-dimethylanilino)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 (10.36 mM) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), suspension solution; with sonication.
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 (10.36 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.)