Triflusal targets platelet cyclooxygenase-1 (Cox-1). It also has little inhibitory effect on Cox-2 expression. Additionally, it inhibits cAMP and cGMP phosphodiesterases.[1]

In vitro activity: The main Triflusal metabolite, HTB, preserves 6-keto-PGF1α synthesis in porcine aortic endothelial cells (PAEC) cells without a significant decline for up to 24 h even at the higher concentration. Triflusal at 10 mM, 100 mM and 1 M decreases LDH efflux in rat brain slices after anoxia/reoxygenation by 24%, 35% and 49% respectively. Triflusal also reduces inducible NO synthase activity by 18%, 21% and 30%.

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In vitro, the main metabolite of Triflusal, 2-hydroxy-4-trifluoromethylbenzoic acid (HTB), was tested on porcine aortic endothelial cells (PAEC). HTB preserved endothelial prostacyclin (PGI2) synthesis (measured as 6-keto-PGF1α) without a significant decline for up to 24 hours even at the highest concentration (300 μmol/L), whereas aspirin significantly reduced PGI2 production. Triflusal itself was also tested but HTB showed better preservation. All treated cells showed similar abundance of Cox-2 protein and mRNA expression levels (P = NS). [1]
In another in vitro experiment, increasing concentrations (37.5, 75, 300 μmol/L) of aspirin, Triflusal, and HTB were incubated with PAECs for 30 minutes, then PMA (100 nmol/L) was added for 4, 12, and 24 hours. HTB preserved 6-keto-PGF1α synthesis while aspirin caused a decline. [1]

Triflusal (10 mg/kg i.v.) reduces platelet deposition on subendothelium-induced primary thrombus by about 68% in rabbits. Triflusal (10 mg/kg i.v.) reduces platelet deposition on a fresh thrombus formed over tunica media by about 48% in rabbits. Triflusal (40 mg/kg p.o.) reduces platelet deposition on a primary thrombus triggered by subendothelium and tunica media by 53% in rabbits. Triflusal (40 mg/kg p.o.) significantly reduces Cox-2 mRNA levels and protein levels without influence Cox-1 mRNA levels on the vascular wall in rabbits. Triflusal (600 mg/day for 5 days) results in an increase in NO production by neutrophils and an increase in endothelial nitric oxide synthase (eNOS) protein expression in neutrophils in healthy volunteers. Triflusal (300 mg, twice-daily orally) shows a more important increase in total walking distance and in pain-free walking distance over the basal values than those treated with placebo, together with an improvement of the symptomatology correlated with claudication in patients with chronic peripheral arteriopathy. Triflusal (300 mg, twice-daily orally) shows an increase in the peak-flow recorded through strain-gauge plethysmography in patients with chronic peripheral arteriopathy. Triflusal (30 mg/kg) strongly decreases iNOS immunolabeling at both survival times analyzed, attenuating iNOS immunoreactivity in astroglial cells and infiltrated neutrophils in rats. Triflusal (30 mg/kg) decreases neuronal and microglial COX-2 expression at 10 and 24 hours after lesion and microglial and astroglial expression of IL-1beta and TNF-alpha at 24 hours after lesion in rats.

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In vivo, Triflusal (intravenous 10 mg/kg) and its metabolite HTB (intravenous 10 mg/kg) similarly and significantly (P<0.01) reduced secondary thrombus growth (platelet deposition) on a fresh primary thrombus triggered by subendothelium and tunica media under shear rates typical of mild carotid stenosis (212 s⁻¹). For subendothelium-triggered thrombus, Triflusal reduced platelet deposition by about 66% compared to placebo control; for tunica media-triggered thrombus, Triflusal reduced deposition by about 48% (P<0.001). [1]
Chronic oral treatment (8 days) with Triflusal (40 mg/kg/day) also significantly reduced platelet deposition on preformed thrombi under high stenotic conditions (1690 s⁻¹) triggered by subendothelium (53% reduction) and tunica media (similar reduction) compared to placebo control. Both aspirin and Triflusal similarly reduced secondary thrombus formation. [1]

No direct enzyme (protein/receptor) activity assay such as kinase activity, SPR, ITC, or HTRF is described in this paper. However, Cox-2 activity was indirectly assessed by measuring 6-keto-PGF1α (a stable hydrolysis product of PGI2) via radioimmunoassay following manufacturer's instructions. This assay was performed on cell culture supernatants from porcine aortic endothelial cells treated with test compounds and stimulated with PMA. [1]
Additionally, Cox-1 and Cox-2 protein expression in aortic vascular wall was evaluated by Western blotting. Proteins were extracted using the Tripure method, separated by electrophoresis, transferred to membranes, and incubated with monoclonal antibodies against Cox-2 and Cox-1. Signal was detected with Super Signal. Positive controls for Cox-1 and Cox-2 were run with samples. [1]

Porcine aortic endothelial cells (PAEC) were isolated from fresh pig aortas using a collagenase method. Cells were seeded in gelatin-precoated plates and grown in M199 medium supplemented with 5% fetal calf serum, 2 mmol/L L-glutamine, 100 U/mL penicillin G, and 100 μg/mL streptomycin at 37°C in 5% CO2. Near-confluent PAECs were placed in serum-free media for 24 hours. Then cells were incubated for 30 minutes with M199 media in the absence or presence of increasing concentrations (37.5, 75, 300 μmol/L) of aspirin, Triflusal, and HTB. Subsequently, Phorbol 12-Myristate 13-Acetate (PMA, 100 nmol/L) was added and maintained for 4, 12, and 24 hours. After incubation, media were collected for PGI2 measurement (6-keto-PGF1α radioimmunoassay). Cells were processed for protein and mRNA extraction using the Tripure method. mRNA was analyzed by real-time PCR (TaqMan) with specific primers and probe for porcine Cox-2, normalized to 18SrRNA. Western blot analysis was performed with monoclonal antibodies against Cox-2. [1]

10 mg/kg i.v. Rabbits

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Animal studies were performed on male New Zealand White rabbits (body weight 2.8 ± 1.7 kg). For acute intravenous (i.v.) treatment, rabbits received Triflusal at 10 mg/kg (2-acetyloxy-4-trifluoromethyl-benzoic acid) or its main metabolite HTB at 10 mg/kg. For chronic oral (p.o.) treatment, rabbits received Triflusal at 40 mg/kg/day for 8 days. Placebo control groups were included. The minimal oral dose required to exert significant antiplatelet effects was determined: 40 mg/kg/day for triflusal. [1]
In the ex vivo perfusion experiments, rabbits were anesthetized with intramuscular ketamine (35 mg/kg) and xylazine (5 mg/kg). The jugular vein and contralateral carotid artery were catheterized. Animals were heparinized with an i.v. bolus of 30 U/kg heparin. An arterio-venous shunt was established with a peristaltic pump at a fixed rate of 10 mL/min. Vessel wall segments (de-endothelialized rabbit subendothelium or porcine tunica media) were mounted in perfusion chambers with internal diameters of 0.2 and 0.1 cm to model shear rates of 212 s⁻¹ (mild stenosis) and 1690 s⁻¹ (severe stenosis). Blood from Rabbit A (unlabeled platelets) was perfused for 5 minutes to create a fresh thrombus, followed by blood from Rabbit B with autologous ¹¹¹In-labeled platelets (with or without drug treatment) perfused for another 5 minutes to measure secondary thrombus growth. Radioactivity was measured with a gamma-well counter and transformed to platelet number per surface unit. [1]

Absorption, Distribution and Excretion
Trifluralin is absorbed in the small intestine with a bioavailability of 83% to 100%. There is no significant difference in absorption between oral solutions and capsules. The peak plasma concentration (Cmax) of trifluralin is 11.6 mcg/ml, and the time to peak concentration (tmax) is 0.88 hours. The major metabolite of trifluralin exhibits different pharmacokinetic characteristics, with a Cmax of 92.7 mcg/ml and a time to peak concentration (tmax) of 4.96 hours, respectively. Trifluralin is primarily excreted via the kidneys. Urinary analysis shows the presence of unmetabolized trifluralin, HTB, and its glycine conjugates. The reported volume of distribution of trifluralin is 34 liters. The renal clearances of trifluralin and HTB are 0.8 ± 0.2 L/h and 0.18 ± 0.04 L/h, respectively.

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Metabolism/Metabolites
In the liver, trifluorosalicylate undergoes deacetylation to produce its major metabolite, 2-hydroxy-4-trifluoromethylbenzoic acid (HTB). This major metabolite appears to have significant antiplatelet activity in vitro.

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Biological Half-Life
In healthy humans, the half-life of trifluorosalicylate is 0.5 ± 0.1 hours, while the half-life of HTB is 34.3 ± 5.3 hours.
Biological Half-Life
In healthy humans, the half-life of trifluorosalicylate is 0.5 ± 0.1 hours, while the half-life of HTB is 34.3 ± 5.3 hours.

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Triflusal is rapidly converted to its principal metabolite 2-hydroxy-4-trifluoromethylbenzoic acid (HTB). It has a short half-life of around 30 minutes, whereas HTB lasts for at least 48 hours. [1]

Protein Binding
Trifluralin binds almost completely to plasma proteins, achieving 99% of the administered dose.

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J Thromb Haemost.2008 Aug;6(8):1385-92;Eur J Clin Invest.2000 Sep;30(9):811-7.

2-Acetoxy-4-(trifluoromethyl)benzoic acid belongs to the salicylates, carboxylates, and benzoic acids. Triflusal is a 2-acetoxy-4-trifluoromethylbenzoic acid molecule with a chemical structure similar to aspirin, but it is not a derivative of aspirin. Its advantage lies in that it does not affect the arachidonic acid pathway, promotes nitric oxide production, and increases the concentration of cyclic nucleotides on endothelial cells. The latter promotes peripheral vasodilation. Due to its low bleeding risk, triflusal is important in the secondary prevention of ischemic stroke. Developed by J. Uriach, it is marketed in several countries but has not yet been approved by the US FDA, EMA, or Health Canada. Indications: Triflusal is indicated for the prevention of thromboembolic diseases. It is registered in Spain and other countries in Europe, South America, and South Korea for the prevention of stroke and myocardial infarction. Mechanism of Action Trifluralin is chemically related to acetylsalicylic acid (ASA) and irreversibly inhibits cyclooxygenase-1 (COX-1) in platelets. Acetylation of the active group of COX-1 prevents the formation of thromboxane B2 in platelets. However, it is unique in that it does not affect the arachidonic acid metabolic pathway in endothelial cells. Furthermore, it promotes the production of the vasodilator nitric oxide. Pharmacodynamics Trifluralin is an antithrombotic and anticoagulant. It irreversibly inhibits the formation of thromboxane B2 in platelets by acetylation of cyclooxygenase-1. Trifluralin also affects many other targets, such as NF-κB, a regulator of gene expression of cyclooxygenase-α and cytokines. Numerous studies comparing the efficacy and safety (e.g., systemic bleeding) of trifluralin and acetylsalicylic acid have shown no significant difference in efficacy and safety, or that trifluralin may have better efficacy and safety. Studies have shown that trifluralin can inhibit lipid peroxidation during hypoxia-reoxygenation, thereby protecting brain tissue.

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Triflusal (also known as UR1501) is an antiplatelet agent that inhibits platelet Cox-1, preventing the formation of thromboxane A₂ (TXA₂), a potent aggregating and vasoconstrictor agent. Unlike aspirin, triflusal has little inhibitory effect on Cox-2 expression and its metabolite HTB preserves endothelial prostacyclin (PGI2) synthesis, which counteracts TXA₂ and provides vascular protection (vasodilation and platelet inhibition). The antithrombotic effect of triflusal has been demonstrated in clinical trials for prevention of cerebrovascular events (e.g., ischemic stroke) with better results than aspirin in prevention of fatal ischemic stroke according to a meta-analysis of five studies. The preservation of vascular prostacyclin may explain its favorable safety profile. [1]

C10H7F3O4

248.16

248.029

322-79-2

Triflusal-d3;2748541-63-9

9458

White to off-white solid powder

1.4±0.1 g/cm3

316.0±42.0 °C at 760 mmHg

115 °C

144.9±27.9 °C

0.0±0.7 mmHg at 25°C

1.484

2.9

1

7

3

17

313

0

RMWVZGDJPAKBDE-UHFFFAOYSA-N

InChI=1S/C10H7F3O4/c1-5(14)17-8-4-6(10(11,12)13)2-3-7(8)9(15)16/h2-4H,1H3,(H,15,16)

2-acetyloxy-4-(trifluoromethyl)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: ≥ 3 mg/mL (12.09 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (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 30.0 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: ≥ 3 mg/mL (12.09 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (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 30.0 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

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Solubility in Formulation 3: ≥ 3 mg/mL (12.09 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 30.0 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.)