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]

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]

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]

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]

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]

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]

Absorption, Distribution and Excretion
The pharmacokinetic characteristics of tofenamic acid include a short time to peak concentration, ranging from 0.94 to 2.04 hours. Its pharmacokinetics are linear, with an AUC of 13-50 mcg/ml·h (at doses of 2-8 mg/kg). Oral absorption is delayed, with a mean absorption lag time of 32 minutes. The peak plasma concentration is 11.1 mcg/ml. The bioavailability of tofenamic acid is approximately 75%. Tofenamic acid is cleared relatively quickly, primarily metabolized in the liver, with metabolites cleared by the kidneys as glucuronide conjugates. Most of the drug is cleared extrarenally, with only 8.8% of the administered dose in urine being the unchanged drug and its glucuronide. The volume of distribution is 1.79-3.2 L/kg. In intravenous administration studies, the steady-state volume of distribution was 0.33 L/kg. The estimated clearance of tofenamic acid is 0.142-0.175 L·h/kg. In intravenous administration studies, clearance was 72.4 ml·h/kg.

---

Metabolisms/Metabolites First-pass metabolism accounts for 20% of the tofenamic acid dose. Urinary metabolite studies have identified five metabolites, three of which are monohydroxylated metabolites, one is a monohydroxylated and hydroxylated metabolite, and the last metabolite is a methyl oxidation to carboxyl group. The two hydroxylated metabolites are N-(2-hydroxymethyl-3-chlorophenyl)-o-aminobenzoic acid and N-(2-hydroxymethyl-3-chloro-4-hydroxyphenyl)-o-aminobenzoic acid.

---

Biological Half-Life The estimated half-life of tofenamic acid is 8.01–13.50 hours. In intravenous administration studies, a half-life of 6.1 hours was reported.

Protein Binding
Tofenamic acid exhibits a high protein binding rate, reaching up to 99.7% of the administered dose. Studies have shown that certain diseases affecting dialysis balance can alter protein binding rates. These studies have confirmed that changes in blood creatinine, urea, and bilirubin significantly alter the concentration of free tofenamic acid. The primary binding structure is expected to be associated with lipid membrane structure. 1. In vivo toxicity in rats: In a 24-week esophageal tumor study, tofenamic acid (GEA 6414) (5 mg/kg/day, orally) had no significant effect on rat body weight (final body weight: 385 ± 25 g, 378 ± 22 g in the MNNG group). Serum alanine aminotransferase (ALT: 45 ± 5 U/L vs. control group 43 ± 4 U/L), aspartate aminotransferase (AST: 52 ± 6 U/L vs. control group 50 ± 5 U/L), and creatinine (0.8 ± 0.1 mg/dL vs. control group 0.7 ± 0.1 mg/dL) levels were all within the normal range [2]
2. In vitro cytotoxicity to normal cells: After treatment with tofenamic acid at a concentration as high as 20 μM for 72 hours, there was no significant cytotoxicity to normal human pancreatic ductal epithelial cells (HPDE) (cell viability ≥ 85% vs. control group), indicating that it has selective toxicity to pancreatic cancer cells [3]

[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.

Tofenamic acid is an aminobenzoic acid, a derivative of anthranilic acid, in which a hydrogen atom bonded to a nitrogen atom is replaced by a 3-chloro-2-methylphenyl group. Tofenamic acid is specifically used to relieve migraines and also has anticancer activity. It is a nonsteroidal anti-inflammatory drug (NSAID), a non-narcotic analgesic, and also an EC 1.14.99.1 (prostaglandin intraperoxidase) inhibitor and an EC 2.7.1.33 (pantothenic acid kinase) inhibitor. It is an aminobenzoic acid, an organochlorine compound, and a secondary amino compound, functionally related to anthranilic acid. Tofenamic acid, with the molecular formula N-(2-methyl-3-chlorophenyl)-anthranilic acid, is a nonsteroidal anti-inflammatory drug. It was discovered by scientists at the Finnish pharmaceutical company Medica. In the UK, tofenamic acid is used under the brand name Clotam for the treatment of migraines. In the US, it is classified as a Group 1 drug by the FDA. In 2016, the European Medicines Agency granted it orphan drug designation for the treatment of supranuclear paralysis. Tofenamic acid is an orally effective benzoic acid derivative belonging to the nonsteroidal anti-inflammatory drug (NSAID) class, possessing anti-inflammatory, antipyretic, analgesic, and potential antitumor activities. Tofenamic acid inhibits the activity of cyclooxygenase (COX) I and II, thereby reducing the production of prostaglandins and thromboxane precursors. This reduction in prostaglandin synthesis is the reason for its therapeutic effect. Tofenamic acid also inhibits thromboxane A2 synthesis by inhibiting thromboxane synthase, thereby reducing platelet aggregation. Furthermore, the drug exerts its antitumor effect through both COX-dependent and COX-independent pathways. Specifically, it induces the production of reactive oxygen species, causing DNA damage, enhancing the activation of nuclear factor-κB (NF-κB) and the expression of transcription factor 3 (ATF3) and NSAID-activated gene-1 (NAG1), and inhibiting the expression of specific proteins (Sp), thereby reducing the expression of Sp-dependent anti-apoptotic and growth-promoting proteins. In summary, this can enhance tumor cell apoptosis and reduce tumor cell growth and angiogenesis. Drug Indications The tofenamic acid package insert states that this drug, as a nonsteroidal anti-inflammatory drug (NSAID), is effective in treating pain caused by acute migraine attacks in adults. Mechanism of Action Tofenamic acid inhibits prostaglandin biosynthesis and has an inhibitory effect on prostaglandin receptors. It is generally believed that the mechanism of action of tofenamic acid is based on the main mechanism of action of NSAIDs, namely, inhibition of the COX-1 and COX-2 pathways, thereby inhibiting the secretion and action of prostaglandins, and thus exerting its anti-inflammatory and analgesic effects. However, some reports indicate that tofenamic acid can inhibit the leukotriene B4 chemotaxis of human polymorphonuclear leukocytes, resulting in an inhibition rate as high as 25%. This activity is an additional anti-inflammatory mechanism of tofenamic acid that is non-ligand specific. Pharmacodynamics Studies have shown that tofenamic acid has a non-dose-dependent partial inhibitory effect on stimuli-induced increases in body temperature, while also having a dose-dependent inhibitory effect on skin edema. Further investigation into its nonsteroidal anti-inflammatory drug (NSAID) properties revealed a dose-dependent inhibitory effect on serum thromboxane, indicating its inhibition of COX-1. Similarly, it was found to inhibit prostaglandin E2 synthesis, suggesting inhibition of the associated COX-2. The maximum inhibition rate of thromboxane exceeded 80%, and it has been confirmed as a potent prostaglandin E inhibitor.

---

1. Tofenamic acid (GEA 6414) is a nonsteroidal anti-inflammatory drug (NSAID) with weak selectivity for COX-1. It exerts its anti-inflammatory effect by inhibiting COX-mediated prostaglandin synthesis and has been used in veterinary medicine to treat pain and inflammation in dogs [1]. 2. The chemopreventive effect of tofenamic acid on esophageal cancer is related to a dual mechanism: (1) inhibiting COX-2 to reduce prostaglandin synthesis and inflammation; (2) downregulating VEGF to inhibit tumor angiogenesis [2]. 3. In pancreatic cancer, tofenamic 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 that tofenamic acid has the potential to be used as a therapeutic agent for pancreatic cancer [3].

C14H12CLNO2

261.7

261.055

13710-19-5

Tolfenamic acid-d4;1246820-82-5;Tolfenamic acid-13C6;1420043-61-3

610479

White to off-white solid powder

1.3±0.1 g/cm3

405.4±40.0 °C at 760 mmHg

210-214°C

199.0±27.3 °C

0.0±1.0 mmHg at 25°C

1.658

5.76

2

3

3

18

298

0

YEZNLOUZAIOMLT-UHFFFAOYSA-N

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)

2-((3-chloro-2-methylphenyl)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: ≥ 2.08 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.)