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].

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].

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.

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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.

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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.

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]. 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.

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.

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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.

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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.

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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.

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.

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 (Please use freshly prepared in vivo formulations for optimal results.)