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Mefenamic Acid (CI-473; CN-35355; CI 473; CN 35355; Mefenamic Acid; Ponstel; Parkemed; Coslan), a nonsteroidal anti-inflammatory drug (NSAID), is potent, nonselective, competitive inhibitor of the enzymes COX-1 and COX-2 with anti-inflammatory activity. Mefenamic acid displays a wide range of biological acitivity such as anti-inflammatory, analgesic, and antipyretic properties. It also demonstrated potent anti-proliferative activity in vitro against colorectal cancer cells.
Physicochemical Properties
| Molecular Formula | C15H15NO2 | |
| Molecular Weight | 241.29 | |
| Exact Mass | 241.11 | |
| CAS # | 61-68-7 | |
| Related CAS # | Mefenamic acid-d4;1216745-79-7;Mefenamic Acid-d3;1189707-81-0;Mefenamic acid-13C6;1325559-19-0 | |
| PubChem CID | 4044 | |
| Appearance | White to off-white solid powder | |
| Density | 1.2±0.1 g/cm3 | |
| Boiling Point | 398.8±30.0 °C at 760 mmHg | |
| Melting Point | 230 °C | |
| Flash Point | 195.0±24.6 °C | |
| Vapour Pressure | 0.0±1.0 mmHg at 25°C | |
| Index of Refraction | 1.639 | |
| LogP | 5.33 | |
| Hydrogen Bond Donor Count | 2 | |
| Hydrogen Bond Acceptor Count | 3 | |
| Rotatable Bond Count | 3 | |
| Heavy Atom Count | 18 | |
| Complexity | 292 | |
| Defined Atom Stereocenter Count | 0 | |
| InChi Key | HYYBABOKPJLUIN-UHFFFAOYSA-N | |
| InChi Code | 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) | |
| Chemical Name | 2-(2,3-dimethylanilino)benzoic acid | |
| Synonyms |
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| 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 |
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| 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 |
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] |
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| ln Vitro |
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]. 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] |
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| ln Vivo |
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| Enzyme Assay |
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] |
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| Cell Assay |
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] |
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| Animal Protocol |
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| ADME/Pharmacokinetics |
Absorption, Distribution and Excretion Mefenamic acid is rapidly absorbed after oral administration. The fecal route of elimination accounts for up to 20% of the dose, mainly in the form of unconjugated 3-carboxymefenamic acid.3 The elimination half-life of mefenamic acid is approximately two hours. Mefenamic acid, its metabolites and conjugates are primarily excreted by the kidneys. Both renal and hepatic excretion are significant pathways of elimination. 1.06 L/kg [Normal Healthy Adults (18-45 yr)] Oral cl=21.23 L/hr [Healthy adults (18-45 yrs)] CROSSES PLACENTAL BARRIER IN MONKEYS...EXCRETED IN URINE, BILE &/OR FECES... ABSORBED SLOWLY FROM GI TRACT...HIGH PERCENTAGE...BOUND TO PLASMA PROTEINS. PEAK PLASMA LEVELS APPEAR IN 2 TO 4 HR...ANALGESIA MAY PERSIST FOR UP TO 6 HR...ABOUT 50%...IS EXCRETED IN URINE WITHIN 24 HR /HUMAN, ORAL/ Metabolism / Metabolites Mefenamic acid undergoes metabolism by CYP2C9 to 3-hydroxymethyl mefenamic acid, and further oxidation to a 3-carboxymefenamic acid may occur. The activity of these metabolites has not been studied. Mefenamic acid is also glucuronidated directly. MEFENAMIC ACID...IS TRANSFORMED TO HYDROXYMETHYL DERIVATIVE & TO ACID BY MAJOR, IF NOT ONLY, METABOLIC PATHWAY IN DOG, MONKEY, & HUMAN. Mefenamic acid has known human metabolites that include 3-hydroxymethyl mefenamic aci. Biological Half-Life 2 hours ...ABOUT 50%...IS EXCRETED IN URINE WITHIN 24 HR /HUMAN, ORAL/ |
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| Toxicity/Toxicokinetics |
Effects During Pregnancy and Lactation ◉ Summary of Use during Lactation Because there is little published experience with mefenamic acid during breastfeeding and it is potentially toxic, other agents may be preferred, especially while nursing a newborn or preterm infant. ◉ Effects in Breastfed Infants Relevant published information was not found as of the revision date. ◉ Effects on Lactation and Breastmilk Relevant published information was not found as of the revision date. Protein Binding 90% Interactions ...WARFARIN CAN BE DISPLACED FROM PLASMA PROTEIN BY MEFENAMIC ACID...INCR IN FREE WARFARIN CAN RANGE FROM 66-400% WITH CORRESPONDING INCR IN ANTICOAGULANT ACTION. BECAUSE OF DOSAGES INVOLVED, INTERACTIONS ARE MORE LIKELY TO BE IMPORTANT IN PT TREATED WITH WARFARIN THAN IN WORKERS WHO USE IT AS PESTICIDE... EFFECTS OF ORAL ANTICOAGULANT AGENTS ARE ENHANCED. 1. In vitro cytotoxicity: - Selective cytotoxicity: Mefenamic Acid (CI 473; CN-35355) showed higher cytotoxicity toward cancer cell lines (HT-29, MCF-7, SCC-25) with IC50 values of 48-62 μM, while normal HDF cells had IC50 > 100 μM (viability ≥75% at 100 μM) [2] - Dose-dependent toxicity: At 120 μM, Mefenamic Acid reduced HT-29 viability by 78 ± 5%, MCF-7 by 72 ± 4%, SCC-25 by 81 ± 6%, but HDF viability remained at 68 ± 5% (still higher than cancer cells) [2] |
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| References |
[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. |
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| Additional Infomation |
Mefenamic acid is an aminobenzoic acid that is anthranilic acid in which one of the hydrogens attached to the nitrogen is replaced by a 2,3-dimethylphenyl group. Although classed as a non-steroidal anti-inflammatory drug, its anti-inflammatory properties are considered to be minor. It is used to relieve mild to moderate pain, including headaches, dental pain, osteoarthritis and rheumatoid arthritis. It has a role as an analgesic, an antirheumatic drug, a non-steroidal anti-inflammatory drug, an antipyretic, an EC 1.14.99.1 (prostaglandin-endoperoxide synthase) inhibitor, an environmental contaminant and a xenobiotic. It is an aminobenzoic acid and a secondary amino compound. A non-steroidal anti-inflammatory agent with analgesic, anti-inflammatory, and antipyretic properties. 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 and non-steroidal anti-inflammatory drug (NSAID) with anti-inflammatory, antipyretic and analgesic activities. Mefenamic acid inhibits the activity of the enzymes cyclo-oxygenase I and II, resulting in a decreased formation of precursors of prostaglandins and thromboxanes. The resulting decrease in prostaglandin synthesis, by prostaglandin synthase, is responsible for the therapeutic effects of mefenamic acid. Mefenamic acid also causes a decrease in the formation of thromboxane A2 synthesis, by thromboxane synthase, thereby inhibiting platelet aggregation. A non-steroidal anti-inflammatory agent with analgesic, anti-inflammatory, and antipyretic properties. It is an inhibitor of cyclooxygenase. Drug Indication For the treatment of rheumatoid arthritis, osteoarthritis, dysmenorrhea, and mild to moderate pain, inflammation, and fever. FDA Label Mechanism of Action Mefenamic acid binds the prostaglandin synthetase receptors COX-1 and COX-2, inhibiting the action of prostaglandin synthetase. As these receptors have a role as a major mediator of inflammation and/or a role for prostanoid signaling in activity-dependent plasticity, the symptoms of pain are temporarily reduced. Therapeutic Uses Anti-Inflammatory Agents, Non-Steroidal; Cyclooxygenase Inhibitors MEFENAMIC ACID (PONSTEL) ... PROVIDES ANALGESIA IN MAN SIMILAR TO THAT PRODUCED BY ASPIRIN. ...INDICATED FOR RELIEF OF PAIN RESULTING FROM DENTAL EXTRACTIONS. DRUG PREFERABLY SHOULD BE TAKEN WITH FOOD. MEFENAMIC ACID SHOWED FAVORABLE RESULTS RELATIVE TO SOME ANALGESICS. Drug Warnings SINCE IT IS NOT SUPERIOR TO ESTABLISHED ANALGESICS & CAN CAUSE SERIOUS TOXICITY, USE OF MEFENAMIC ACID IS NOT RECOMMENDED. IF IT IS USED, ADMIN SHOULD NOT BE EXTENDED BEYOND 7 DAYS. IF DIARRHEA OCCURS, DRUG MUST BE DISCONTINUED & NOT USED AGAIN. IT SHOULD NOT BE USED IN CHILDREN OR IN WOMEN OF CHILDBEARING AGE. IT IS CONTRAINDICATED IN PT WITH ULCERATION OF UPPER OR LOWER INTESTINAL TRACT...& PT KNOWN TO BE HYPERSENSITIVE TO DRUG. ...CONSIDER ITS USE ONLY IN CASES WHICH EITHER CAN NOT TOLERATE OR DO NOT RESPOND TO LESS TOXIC AGENTS. MEFENAMIC ACID IS CONTRAINDICATED IN PT...WITH IMPAIRED RENAL FUNCTION, & SHOULD BE USED WITH CAUTION IN ASTHMATICS BECAUSE IT MAY EXACERBATE CONDITION. Maternal Medication usually Compatible with Breast-Feeding: Mefenamic acid: Reported Sign or Symptom in Infant or Effect on Lactation: None. /from Table 6/ Pharmacodynamics Mefenamic acid, an anthranilic acid derivative, is a member of the fenamate group of nonsteroidal anti-inflammatory drugs (NSAIDs). It exhibits anti-inflammatory, analgesic, and antipyretic activities. Similar to other NSAIDs, mefenamic acid inhibits prostaglandin synthetase. 1. Mefenamic Acid (CI 473; CN-35355) is a non-steroidal anti-inflammatory drug (NSAID) of the fenamate class, with balanced inhibitory activity against both COX-1 and COX-2 (selectivity ratio ~0.66), distinguishing it from highly COX-2-selective NSAIDs (e.g., celecoxib) [1] 2. Its anti-inflammatory mechanism primarily involves inhibiting COX-mediated prostaglandin synthesis (e.g., PGE2, TXB2), which reduces inflammatory mediator release and pain signaling [1] 3. In vitro studies show that Mefenamic Acid exhibits selective cytotoxicity toward certain human cancer cell lines (especially oral squamous cell carcinoma SCC-25) with minimal toxicity to normal fibroblasts, suggesting potential as an adjuvant antitumor agent (though in vivo validation is lacking) [2] |
Solubility Data
| Solubility (In Vitro) |
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| Solubility (In Vivo) |
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. 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. (Please use freshly prepared in vivo formulations for optimal results.) |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 4.1444 mL | 20.7220 mL | 41.4439 mL | |
| 5 mM | 0.8289 mL | 4.1444 mL | 8.2888 mL | |
| 10 mM | 0.4144 mL | 2.0722 mL | 4.1444 mL |