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Purity: ≥98%
Teriflunomide (formerly HMR-1726; HMR1726; A771726; A-771726; trade name Aubagio), the active metabolite of the immunomodulator drug-leflunomide, is an orally bioavailable immunomodulatory agent with potential suppressing effects against immune systems. It acts by blocking the enzyme dihydroorotate dehydrogenase, thus inhibiting pyrimidine de novo synthesis. Teriflunomide has been approved for the treatment of arthritis and multiple sclerosis (MS). Teriflunomide inhibits rapidly dividing cells, including activated T cells, which are thought to drive the disease process in MS.
Targets |
Active metabolite of Leflunomide; dihydroorotate dehydrogenase (DHODH)
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ln Vitro |
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ln Vivo |
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Enzyme Assay |
Reduced glutathione (GSH) estimation[2]
1 ml of 0.3 M disodium hydrogen phosphate was introduced into 0.25 ml of the supernatant solution. Thereafter, 125 µl of 0.001 M 5, 5′-dithiol-bis- [2-nitrobenzoic acid] (DTNB) was added. The colored solution was assessed using spectrophotometry at a wavelength of 412 nm, and the outcomes were quantified in units of ‘µM’ per ‘mg’ of tissue. Thiobarbituric acid reactive substances (TBARS) estimation[2] The assay procedure consists of the interaction between lipid peroxidation byproducts, particularly malondialdehyde (MDA), and thiobarbituric acid (TBA), that results in the formation of MDA-TBA2 adducts. In this experiment, 0.25 ml of the supernatant was introduced into a solution consisting of 0.4 ml of a mixture containing 0.375% thiobarbituric acid (TBA), 15% trichloroacetic acid (TCA), and 0.25 N hydrochloric acid (HCL) in a 1:1:1 ratio. The mixture underwent heating at a temperature of 100°C for 15 minutes, after which it was subjected to cooling. After cooling, the mixture underwent centrifugation at 3000 rpm for 10 minutes. The color supernatant was collected and quantified at a wavelength of 535 nm. The findings were presented as ‘nM’ per ‘mg’ of tissue. Serum TNF-α estimation[2] The levels of TNF-α in the serum were quantified by using the TNF-α ELISA kit obtained from the Krishgen Biotech (Mumbai, India). The resultant was presented as picograms per milliliter (pg/ml). |
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Cell Assay |
Cell counting kit-8 assay[3]
The CCK-8 assay was conducted using a CCK-8 kit and commenced with seeding 100 μL of the cell suspension in each well of a 96-well plate, followed by a 24-h incubation at 37 °C in a 5 % CO2 incubator. Subsequently, 10 μL of CCK-8 solution was added directly to each well, with thorough mixing ensured. The plate was incubated for an additional 3 h, shaken for approximately 1 min, and the absorbance at 450 nm was measured using a microplate reader. This process facilitated the calculation of cell activity. Western blot analysis[3] Proteins were extracted from both whole hippocampal tissues of mice and the treated cell samples. The protein concentrations in the supernatants were determined using a BCA protein assay kit, with the manufacturer's instructions followed. The proteins were then separated via 10 % SDS-PAGE and transferred onto polyvinylidene difluoride membranes. The membranes were subsequently cut based on molecular weight in accordance with the protein marker and blocked with 5 % BSA in TBST (TBS containing 0.1 % Tween-20). Overnight incubation at 4 °C was carried out using a variety of primary antibodies: anti-MBP (1:800), anti-Bcl2 (1:800), anti-Bax (1:1000), anti-Caspase-3 (1:1000), anti-cleaved Caspase-3 (1:1000), anti-β-actin (1:40000), anti-PSD-95 (1:1000), Anti-Iba1 (1:4000) and anti-Synaptophysin (1:1000). This was followed by a 90-min incubation with anti-rabbit or anti-mouse secondary antibodies, and the membranes were subsequently scanned using the Tanon-5200 imaging system. After synaptophysin imaging, the membrane underwent immersion in an antibody eluent, facilitating antibody elution. Additionally, 1-h incubation with anti-β-actin was carried out at room temperature followed by a 90-min incubation with rabbit II antibody (1:1000) and subsequent imaging. ImageJ software was utilized for the quantification of all images. |
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Animal Protocol |
Mice were subjected to a nine-day protocol, during which they were given an intraperitoneal injection of scopolamine at a dosage of 2 mg/kg for the final three days to induce cognitive impairment. Animals were divided into 7 groups namely, Group 1 serves as vehicle control (0.1% CMC; p.o), Group 2 animals were treated with 0.1% CMC (p.o) + scopolamine (2 mg/kg; i.p). Group 3 received both donepezil (3 mg/kg i.p) and scopolamine. Group 4 animals were treated with teriflunomide (10 mg/kg; p.o) + scopolamine. Group 5 animals received teriflunomide (20 mg/kg; p.o) + scopolamine. Group 6 received PI3K inhibitor (LY294002) at a dose of 25 µg/kg. Lastly, group 7 animals were treated with both the PI3K inhibitor (LY294002) + teriflunomide (20 mg/kg; p.o). After training for five days, the donepezil, teriflunomide, and LY294002 treatments were given continuously for the next 9 days. On days 7, 8, and 9, donepezil and teriflunomide treatments were given 30 min before the scopolamine treatment. In group 7, teriflunomide was administered 30 min before the LY294002 treatment. As the scopolamine treatment was given for the last three days, therefore in the current study the behavioral analysis was done on day 7 (before scopolamine treatment) and after day 9 (one hour after scopolamine treatment). This was done to observe any change in the behavior of animals with the treatment regimens before impairing the memory with scopolamine. Therefore, on day 7, all the treatment groups were served as a control group as the scopolamine was administered after the behavioral analysis on day 7 and continued till day 9. Following a behavioral analysis on the ninth day, the animals were anesthetized with ketamine (50 mg/kg; i.p) and blood samples were obtained through cardiac puncture. Subsequently, the animals were euthanized, and brain samples were obtained for the quantification of oxidative stress[2].
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References |
[1]. Ther Clin Risk Manag.2013;9:177-90.
[2]. Mol Biol Rep . 2024 May 9;51(1):572. doi: 10.1007/s11033-024-09502-9. [3]. Heliyon . 2024 Apr 10;10(8):e29481. doi: 10.1016/j.heliyon.2024.e29481. |
Molecular Formula |
C12H9F3N2O2
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Molecular Weight |
270.21
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Exact Mass |
270.0616
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Elemental Analysis |
C, 53.34; H, 3.36; F, 21.09; N, 10.37; O, 11.84
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CAS # |
108605-62-5
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Related CAS # |
Teriflunomide-d4;1185240-22-5;Teriflunomide;163451-81-8
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Appearance |
White to off-white solid powder
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LogP |
0.71
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tPSA |
73.12
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SMILES |
C/C(O)=C(C#N)/C(NC1=CC=C(C(F)(F)F)C=C1)=O
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InChi Key |
UTNUDOFZCWSZMS-YFHOEESVSA-N
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InChi Code |
InChI=1S/C12H9F3N2O2/c1-7(18)10(6-16)11(19)17-9-4-2-8(3-5-9)12(13,14)15/h2-5,18H,1H3,(H,17,19)/b10-7-
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Chemical Name |
(Z)-2-cyano-3-hydroxy-N-(4-(trifluoromethyl)phenyl)but-2-enamide.
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Synonyms |
A77 1726, HMR-1726; A77 1726; Aubagio; A771726; A-771726; HMR1726; HMR 1726; teriflunomide; trade name: Aubagio.
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HS Tariff Code |
2934.99.9001
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Storage |
Powder -20°C 3 years 4°C 2 years In solvent -80°C 6 months -20°C 1 month |
Shipping Condition |
Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)
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Solubility (In Vitro) |
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Solubility (In Vivo) |
Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.
Injection Formulations
Injection Formulation 1: DMSO : Tween 80: Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)(e.g. IP/IV/IM/SC) *Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution. Injection Formulation 2: DMSO : PEG300 :Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline) Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil) Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals). View More
Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)] Oral Formulations
Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium) Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals). View More
Oral Formulation 3: Dissolved in PEG400  (Please use freshly prepared in vivo formulations for optimal results.) |
Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
1 mM | 3.7008 mL | 18.5041 mL | 37.0083 mL | |
5 mM | 0.7402 mL | 3.7008 mL | 7.4017 mL | |
10 mM | 0.3701 mL | 1.8504 mL | 3.7008 mL |
*Note: Please select an appropriate solvent for the preparation of stock solution based on your experiment needs. For most products, DMSO can be used for preparing stock solutions (e.g. 5 mM, 10 mM, or 20 mM concentration); some products with high aqueous solubility may be dissolved in water directly. Solubility information is available at the above Solubility Data section. Once the stock solution is prepared, aliquot it to routine usage volumes and store at -20°C or -80°C. Avoid repeated freeze and thaw cycles.
Calculation results
Working concentration: mg/mL;
Method for preparing DMSO stock solution: mg drug pre-dissolved in μL DMSO (stock solution concentration mg/mL). Please contact us first if the concentration exceeds the DMSO solubility of the batch of drug.
Method for preparing in vivo formulation::Take μL DMSO stock solution, next add μL PEG300, mix and clarify, next addμL Tween 80, mix and clarify, next add μL ddH2O,mix and clarify.
(1) Please be sure that the solution is clear before the addition of next solvent. Dissolution methods like vortex, ultrasound or warming and heat may be used to aid dissolving.
(2) Be sure to add the solvent(s) in order.
NCT Number | Recruitment | interventions | Conditions | Sponsor/Collaborators | Start Date | Phases |
NCT06190145 | Recruiting | Drug: Teriflunomide | Immune Thrombocytopenia | Peking University People's Hospital | December 5, 2023 | Phase 2 |
NCT04799288 | Recruiting | Drug: Teriflunomide | HAM/TSP | National Institute of Neurological Disorders and Stroke (NINDS) |
September 24, 2021 | Phase 1 Phase 2 |
NCT06176235 | Recruiting | Drug: Teriflunomide Drug: Dexamethasone |
Immune Thrombocytopenia | Peking University People's Hospital | December 19, 2023 | Phase 2 |
NCT03526224 | Completed | Drug: Dimethyl Fumarate Drug: Teriflunomide |
Tecfidera Teriflunomide |
University at Buffalo | June 14, 2018 |
Related chemical structures of leflunomide and teriflunomide. td> |
Mechanism of action of teriflunomide. td> |