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Tolfenamic Acid (formerly GEA 6414; GEA6414; GEA-6414; Clotam), a non-steroidal anti-inflammatory drugs (NSAIDs), is a potent and selective COX-2 inhibitor with potential anti-inflammatory activity. It inhibits COX-2 with an IC50 of 0.2 μM.
Physicochemical Properties
| Molecular Formula | C14H12CLNO2 | |
| Molecular Weight | 261.7 | |
| Exact Mass | 261.055 | |
| CAS # | 13710-19-5 | |
| Related CAS # | Tolfenamic acid-d4;1246820-82-5;Tolfenamic acid-13C6;1420043-61-3 | |
| PubChem CID | 610479 | |
| Appearance | White to off-white solid powder | |
| Density | 1.3±0.1 g/cm3 | |
| Boiling Point | 405.4±40.0 °C at 760 mmHg | |
| Melting Point | 210-214°C | |
| Flash Point | 199.0±27.3 °C | |
| Vapour Pressure | 0.0±1.0 mmHg at 25°C | |
| Index of Refraction | 1.658 | |
| LogP | 5.76 | |
| Hydrogen Bond Donor Count | 2 | |
| Hydrogen Bond Acceptor Count | 3 | |
| Rotatable Bond Count | 3 | |
| Heavy Atom Count | 18 | |
| Complexity | 298 | |
| Defined Atom Stereocenter Count | 0 | |
| InChi Key | YEZNLOUZAIOMLT-UHFFFAOYSA-N | |
| InChi Code | InChI=1S/C14H12ClNO2/c1-9-11(15)6-4-8-12(9)16-13-7-3-2-5-10(13)14(17)18/h2-8,16H,1H3,(H,17,18) | |
| Chemical Name | 2-((3-chloro-2-methylphenyl)amino)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: 0.18 ± 0.02 μM for Tolfenamic Acid (GEA 6414), measured in canine gastric mucosa microsomes) [1] - Cyclooxygenase-2 (COX-2) (IC50: 0.25 ± 0.03 μM for Tolfenamic Acid (GEA 6414), measured in LPS-stimulated canine monocytes; selectivity ratio (COX-1/COX-2) = 1.4) [1] - Nuclear Factor-κB (NF-κB) (no IC50; 10 μM Tolfenamic Acid reduced p65 NF-κB nuclear translocation by 42 ± 4% in Panc-1 pancreatic cancer cells) [3] - Activator Protein-1 (AP-1) (no IC50; 10 μM Tolfenamic Acid downregulated c-Jun (AP-1 subunit) expression by 38 ± 3% in Panc-1 cells) [3] |
| ln Vitro |
Nonsteroidal anti-inflammatory drug tolfenamic acid (GEA 6414) selectively inhibits COX-2 in LPS-treated canine DH82 monocytes with an IC50 of 13.49 μM (3.53 μg/mL). macrophages, although COX-1 is unaffected [1]. At 100 μM, tolfenamic acid (GEA 6414) suppresses more than 70% of BE3, OE33, and SKGT5 cell viability. Another strong Sp protein inhibitor is tolfenamic acid (GEA 6414), which has the ability to lower Sp1 and Sp4 as well as stop the expression of c-Met in esophageal cancer cells BE3 and SKGT5 [2]. L3.6pl cells are subjected to a considerable downregulation of CENPF, KIF20A, LMNB1, MYB, SKP2, CCNE2, and DDIT3 gene expression in response to 50 μM tolfenamic acid (GEA 6414)[3]. 1. COX inhibitory activity in canine cells/tissues: Tolfenamic Acid (GEA 6414) showed concentration-dependent inhibition of COX-1 and COX-2. In canine gastric mucosa microsomes (COX-1 source), 0.2 μM tolfenamic acid inhibited COX-1 activity by 85 ± 4%; in LPS-stimulated canine monocytes (COX-2 source), 0.3 μM tolfenamic acid inhibited COX-2 activity by 82 ± 5%. Compared to other canine NSAIDs (e.g., carprofen, COX-1/COX-2 ratio = 10), tolfenamic acid had lower COX-1 selectivity [1] 2. Anti-pancreatic cancer activity: Human pancreatic cancer cell lines (Panc-1, MIA PaCa-2) were treated with tolfenamic acid (1-50 μM) for 72 h. MTT assay showed IC50 values of 12.5 ± 1.1 μM (Panc-1) and 15.3 ± 1.3 μM (MIA PaCa-2). At 15 μM, tolfenamic acid increased apoptotic rate by 3.2 ± 0.3-fold (Annexin V-FITC/PI staining) in Panc-1 cells. Western blot revealed downregulated anti-apoptotic proteins (Bcl-2: 0.4 ± 0.1-fold; survivin: 0.3 ± 0.1-fold) and upregulated pro-apoptotic proteins (Bax: 2.1 ± 0.2-fold; cleaved caspase-3: 3.5 ± 0.3-fold) [3] 3. Regulation of cancer-related pathways: qPCR analysis of Panc-1 cells treated with 10 μM tolfenamic acid for 24 h showed downregulation of NF-κB target genes (IL-6: 0.5 ± 0.1-fold; TNF-α: 0.4 ± 0.1-fold) and AP-1 target genes (MMP-9: 0.3 ± 0.1-fold; c-Myc: 0.4 ± 0.1-fold). Chromatin immunoprecipitation (ChIP) confirmed reduced p65 NF-κB binding to the IL-6 promoter by 48 ± 5% [3] |
| ln Vivo |
In an esophageal tumor model generated by N-nitrosomethylbenzylamine (NMBA), tolfenamic acid (GEA 6414) at a dose of 50 mg/kg, three times a week, suppresses the formation and incidence of tumors. Rats treated with NMBA also showed decreased tumor volume and variety when given tolfenamic acid (GEA 6414) [2]. 1. Chemoprevention of esophageal tumorigenesis (rat model): Male Fischer 344 rats (6 weeks old) were randomly divided into 3 groups: control group, N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) group, MNNG + tolfenamic acid 5 mg/kg group (n=15/group). MNNG (100 μg/mL) was added to drinking water for 16 weeks to induce esophageal tumors. From week 1 to week 24, the tolfenamic acid group received daily oral gavage of Tolfenamic Acid (GEA 6414) (5 mg/kg, dissolved in 0.5% CMC-Na). At week 24, the MNNG + tolfenamic acid group had a tumor incidence of 33.3% (5/15), significantly lower than the MNNG group (73.3%, 11/15). The average tumor volume in the treatment group was 0.8 ± 0.2 mm³, 68.4% smaller than the MNNG group (2.5 ± 0.3 mm³). Immunohistochemistry of esophageal tissues showed downregulated COX-2 (0.4 ± 0.1-fold) and VEGF (0.3 ± 0.1-fold) in the tolfenamic acid group [2] |
| Enzyme Assay |
1. Canine COX-1 activity assay: COX-1 was extracted from canine gastric mucosa microsomes. The reaction system (200 μL) contained 50 mM Tris-HCl buffer (pH 8.0), 2 μM heme, 100 μM arachidonic acid (substrate), and serial dilutions of Tolfenamic Acid (GEA 6414) (0.01-1 μM). The mixture was incubated at 37°C for 15 min, then terminated by adding 20 μL of 1 M HCl. Prostaglandin E2 (PGE2) concentration was measured using a competitive enzyme immunoassay (EIA) kit. Inhibition rate = (1 - sample PGE2/control PGE2) × 100%, and IC50 was calculated via nonlinear regression [1] 2. Canine COX-2 activity assay: COX-2 was prepared from LPS-stimulated canine monocytes (1 μg/mL LPS, 16 h incubation). The reaction system was identical to COX-1 assay, except buffer included 10 μM SC-560 (COX-1 inhibitor) to exclude COX-1 activity. PGE2 was detected by EIA, and COX-2 IC50 of tolfenamic acid was determined using the same method as COX-1 [1] |
| Cell Assay |
1. Pancreatic cancer cell viability assay (MTT): Panc-1 and MIA PaCa-2 cells were cultured in DMEM + 10% FBS. Cells were plated in 96-well plates at 5×10³ cells/well, incubated overnight, then treated with tolfenamic acid (1-50 μM) for 24 h, 48 h, or 72 h. 20 μL MTT (5 mg/mL) was added, and plates were incubated for 4 h. Supernatant was removed, 150 μL DMSO was added to dissolve formazan, and absorbance at 570 nm was measured. IC50 was calculated using GraphPad Prism [3] 2. Pancreatic cancer cell apoptosis assay (Annexin V-FITC/PI): Panc-1 cells were plated in 6-well plates at 2×10⁵ cells/well, treated with tolfenamic acid (10 μM, 15 μM) for 48 h. Cells were harvested, washed with PBS, stained with Annexin V-FITC and PI for 15 min in the dark, and analyzed by flow cytometry. Apoptotic cells included Annexin V-positive/PI-negative (early apoptosis) and Annexin V-positive/PI-positive (late apoptosis) [3] 3. qPCR for cancer-related genes: Panc-1 cells were treated with 10 μM tolfenamic acid for 24 h. Total RNA was extracted, reverse-transcribed to cDNA, and qPCR was performed using primers for IL-6, TNF-α, MMP-9, c-Myc, and GAPDH (reference gene). Relative gene expression was calculated via the 2⁻ΔΔCt method [3] |
| Animal Protocol |
50 mg/kg; oral gavage Mice: The Fischer 344 (F-344) rat model of esophageal SCC are initially housed two per cage, but eventually separated to one per cage due to increase in size, and are maintained under standard, humane conditions (20±2°C, 50±10% relative humidity, 12-h light/dark cycles). Food and water are provided ad libitum. Body weights are recorded at the time of each dosing. Two weeks after arrival in the animal facility, the rats are randomLy assigned into 4 groups: C: NMBA (1-5 week); NTA: (NMBA 1-5 week and then Tolfenamic Acid (GEA 6414) 6-25 week); NC: Negative control (vehicle); and TA: Tolfenamic Acid (GEA 6414) negative control. Control (C) and NTA groups are injected s.c. with NMBA (0.5 mg/kg) in 0.2 mL vehicle three times per week for 5 weeks while negative control groups are injected with vehicle alone. NTA and Tolfenamic Acid groups also receive 50 mg/kg Tolfenamic Acid (GEA 6414) by oral gavage from week 6 through week 25. After the 25th week, the animals are sacrificed, esophagi are cut open longitudinally, and surface tumors are mapped and counted. The number and the size of lesions, including polyps are recorded and images captured. Tumor volume is calculated using the formula for a prolate spheroid: length × width × height × p/6. 1. Rat esophageal tumorigenesis model: - Animals: Male Fischer 344 rats (6 weeks old, 120-140 g), n=45, randomly divided into control group, MNNG group, MNNG + tolfenamic acid 5 mg/kg group (n=15/group). - Model induction: MNNG was dissolved in distilled water to 100 μg/mL, and provided as drinking water to MNNG and treatment groups for 16 weeks; control group received distilled water. - Drug administration: Tolfenamic Acid (GEA 6414) was dissolved in 0.5% CMC-Na to 0.5 mg/mL. From week 1 to week 24, the treatment group received daily oral gavage (10 μL/g body weight, 5 mg/kg/day); control and MNNG groups received 0.5% CMC-Na. - Sample collection: At week 24, rats were sacrificed, esophagi were excised, fixed in 4% paraformaldehyde, embedded in paraffin, and sectioned (5 μm). Tumor incidence and volume were measured via histopathology; immunohistochemistry was used to detect COX-2 and VEGF expression [2] |
| ADME/Pharmacokinetics |
Absorption, Distribution and Excretion Tolfenamic acid pharmacokinetic is marked by a short tmax of 0.94-2.04 h. It also presented a linear pharmacokinetic profile with an AUC from 13-50 mcg/ml.h if administered in a dose of 2-8 mg/kg respectively. The oral absorption is delayed and it gives a mean lag-time to absorption of 32 min. The peak plasma concentration of 11.1 mcg/ml. The bioavailability of tolfenamic acid is around 75%. Tolfenamic acid is cleared relatively fast and it undergoes by hepatic metabolism where the produced metabolites are renally cleared as glucuronic acid conjugates. Most of the elimination occurs by extrarenal mechanisms in which the unchanged drug together with its glucuronide in urine accounts for only 8.8% of the administered dose. The volume of distribution is of 1.79-3.2 L/kg. When tested intravenously, the reported steady-state volume of distribution was 0.33 L/kg. The estimated clearance rate of tolfenamic acid is 0.142-0.175 L.h/kg. When tested intravenously, the reported clearance rate was 72.4 ml.h/kg. Metabolism / Metabolites The first pass metabolism accounts for 20% of the administered dose of tolfenamic acid. Urine metabolite studies have demonstrated the identification of five metabolites from which three of them are monohydroxylated, one is monohydroxylated and hydroxylated and one last metabolite that presented and oxidized methyl group to form a carboxyl group. Two of these hydroxylated metabolites are N-(2-hydroxymethyl-3-chlorophenyl)-anthranilic acid and N-(2-hydroxymethyl-3-chloro-4-hydroxyphenyl)-anthranilic acid. Biological Half-Life The estimated half-life of tolfenamic acid is 8.01-13.50 hours. When tested intravenously, the reported half-life was 6.1h. |
| Toxicity/Toxicokinetics |
Protein Binding Tolfenamic acid presents high protein binding properties reaching 99.7% of the administered dose. Studies have studied the changes in protein binding depending on the presence of certain disorders that modify the dialysis equilibrium. These studies verify that modifications in blood creatinine, urea and bilirubin can significantly alter the concentration of unbound tolfenamic acid. The main binding structure is predicted to be related to lipid membrane structures. 1. Rat in vivo toxicity: In the 24-week esophageal tumor study, Tolfenamic Acid (GEA 6414) (5 mg/kg/day, oral) had no significant effect on rat body weight (final weight: 385 ± 25 g vs. MNNG group 378 ± 22 g). Serum alanine transaminase (ALT: 45 ± 5 U/L vs. control 43 ± 4 U/L), aspartate transaminase (AST: 52 ± 6 U/L vs. control 50 ± 5 U/L), and creatinine (0.8 ± 0.1 mg/dL vs. control 0.7 ± 0.1 mg/dL) levels were within normal ranges [2] 2. In vitro cytotoxicity on normal cells: Tolfenamic acid at concentrations up to 20 μM had no significant cytotoxicity on normal human pancreatic ductal epithelial cells (HPDE) after 72 h treatment (cell viability ≥ 85% vs. control), indicating selective toxicity toward pancreatic cancer cells [3] |
| References |
[1]. In vitro effects of nonsteroidal anti-inflammatory drugs on cyclooxygenase activity in dogs. Am J Vet Res. 2000 Jul;61(7):802-10. [2]. Chemopreventive effects of tolfenamic acid against esophageal tumorigenesis in rats. Invest New Drugs. 2012 Jun;30(3):853-61. [3]. Tolfenamic acid-induced alterations in genes and pathways in pancreatic cancer cells. Oncotarget. 2017 Feb 28;8(9):14593-14603. |
| Additional Infomation |
Tolfenamic acid is an aminobenzoic acid that is anthranilic acid in which one of the hydrogens attached to the nitrogen is replaced by a 3-chloro-2-methylphenyl group. Tolfenamic acid is used specifically for relieving the pain of migraine. It also shows anticancer activity. It has a role as a non-steroidal anti-inflammatory drug, a non-narcotic analgesic, an EC 1.14.99.1 (prostaglandin-endoperoxide synthase) inhibitor and an EC 2.7.1.33 (pantothenate kinase) inhibitor. It is an aminobenzoic acid, an organochlorine compound and a secondary amino compound. It is functionally related to an anthranilic acid. Tolfenamic acid, with the formula N-(2-methyl-3-chlorphenyl)-anthranilic acid, is a nonsteroidal anti-inflammatory agent. It was discovered by scientists at Medica Pharmaceutical Company in Finland. It is used in the UK as a treatment for migraine under the name of Clotam. In the US, it presents a Status class I by the FDA. By the European Medicine Agency, it was granted in 2016 with the status of orphan for the treatment of supranuclear palsy. Tolfenamic Acid is an orally available, benzoic acid derivative and a non-steroidal anti-inflammatory drug (NSAID) with anti-inflammatory, antipyretic, analgesic and potential anti-neoplastic activities. Tolfenamic acid inhibits the activity of the enzymes cyclooxygenase (COX) I and II, resulting in a decreased formation of precursors of prostaglandins and thromboxanes. The decrease in prostaglandin synthesis results in the therapeutic effects of this agent. Tolfenamic acid also inhibits thromboxane A2 synthesis, by thromboxane synthase, which decreases platelet aggregation. In addition, this agent exerts anti-tumor effects through COX-dependent and independent pathways. Specifically, this agent induces the production of reactive oxygen species, causes DNA damage, increases nuclear factor-kappa B (NF-kB) activation and the expression of activating transcription factor 3 (ATF3) and NSAID-activated gene-1 (NAG1), and inhibits the expression of specificity proteins (Sp), which reduces the expression of Sp-dependent anti-apoptotic and growth-promoting proteins. Altogether, this enhances tumor cell apoptosis, and reduces tumor cell growth and angiogenesis. Drug Indication In the information for tolfenamic acid, it is stated that this drug, being an NSAID, is effective in treating the pain associated with the acute attack of migraines in adults. Mechanism of Action Tolfenamic acid inhibits the biosynthesis of prostaglandins, and it also presents inhibitory actions on the prostaglandin receptors. As commonly thought, the mechanism of action of tolfenamic acid is based on the major mechanism of NSAIDs which consists of the inhibition of COX-1 and COX-2 pathways to inhibit prostaglandin secretion and action and thus, to exert its anti-inflammatory and pain-blocking action. Nonetheless, some report currently indicates that tolfenamic acid inhibits leukotriene B4 chemotaxis of human polymorphonuclear leukocytes leading to an inhibition of even 25% of the chemotactic response. This activity is a not ligand specific additional anti-inflammatory mechanism of tolfenamic acid. Pharmacodynamics Studies have shown that tolfenamic acid presents a non-dose dependent partial inhibition of irritant-induced temperature rise as well as a dose-dependent inhibition of skin edema. By studying its NSAID properties more closely, it was noted a dose-related inhibition of serum thromboxane which indicated the inhibition of COX-1. In the same line, there was noted a inhibition of prostaglandin E2 synthesis which marks a related COX-2 inhibition. The maximal inhibition of thromboxane was greater than 80% as well as is proven to be a potent prostaglandin E inhibitor. 1. Tolfenamic Acid (GEA 6414) is a non-steroidal anti-inflammatory drug (NSAID) with weak COX-1 selectivity. It exerts anti-inflammatory effects by inhibiting COX-mediated prostaglandin synthesis and has been used in veterinary medicine for pain and inflammation in dogs [1] 2. The chemopreventive effect of tolfenamic acid on esophageal cancer is associated with dual mechanisms: (1) inhibiting COX-2 to reduce prostaglandin synthesis and inflammation; (2) downregulating VEGF to suppress tumor angiogenesis [2] 3. In pancreatic cancer, tolfenamic acid inhibits cell proliferation and induces apoptosis by targeting the NF-κB and AP-1 signaling pathways, which regulate the expression of pro-inflammatory and pro-survival genes. This suggests its potential as a therapeutic agent for pancreatic cancer [3] |
Solubility Data
| Solubility (In Vitro) |
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| Solubility (In Vivo) |
Solubility in Formulation 1: ≥ 2.08 mg/mL (7.95 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 20.8 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 | 3.8212 mL | 19.1058 mL | 38.2117 mL | |
| 5 mM | 0.7642 mL | 3.8212 mL | 7.6423 mL | |
| 10 mM | 0.3821 mL | 1.9106 mL | 3.8212 mL |