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Purity: ≥98%
Leflunomide (formerly also known as HWA486; Leflunomidum, trade/brand name Arava) is a potent pyrimidine synthesis inhibitor belonging to the DMARD (disease-modifying antirheumatic drug), it is used as an immunosuppressant agent for treating inflammatory conditions such as RA-rheumatoid arthritis.
| Targets |
DHODH (dihydroorotate dehydrogenase)
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| ln Vitro |
It has been demonstrated that leflunomide, a prodrug, inhibits the growth of T- and mononuclear cells. Leflunomide exhibits IC50 values ranging from 30 mM to 100 mM in in vitro cellular and enzymatic studies, indicating its ability to inhibit several protein tyrosine kinases[1]. Leflunomide has the ability to prevent T cell proliferation that is driven by interleukin-2 (IL-2) and anti-CD3. Leflunomide has the ability to block the activity of p59fyn and p56lck in in vitro tyrosine kinase tests. Additionally, leflunomide prevents Ca2+ mobilization in Jurkat cells that are activated by anti-CD3 antibodies, but not in cells that are activated by ionomycin. Leflunomide also prevents the synthesis of IL-2 and the development of IL-2 receptors on human T cells, which are distal outcomes of anti-CD3 monoclonal antibody activation. Leflunomide also prevents CTLL-4 cells activated by IL-2 from phosphorylating tyrosine [2]. The immunomodulatory medication leflunomide may work by preventing the mitochondrial enzyme dihydroorotate dehydrogenase (DHODH) from doing its job. DHODH is essential for the de novo production of pyrimidine ribonucleotide uridine monophosphate (rUMP). Leflunomide inhibits the growth of autoimmune and activated lymphocytes by disrupting the cell cycle through mechanisms involving p53 and insufficient rUMP production[3].
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| ln Vivo |
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| Enzyme Assay |
Enzyme Activity Measurements. DHODase activity was measured by the DCIP colorimetric assay, as described by Copeland et al. (1995). This is a coupled assay in which oxidation of DHO and subsequent reduction of ubiquinone are stoichiometrically equivalent to the reduction of DCIP. Reduction of DCIP is accompanied by a loss of absorbance at 610 nm (ε = 21 500 M-1 cm-1). The assay was performed in a 96-well microtiter plate at ambient temperature (ca. 25 °C). Stock solutions of 10 mM leflunomide and A771726 were prepared in dimethyl sulfoxide (DMSO) and these were diluted with reaction buffer (100 mM Tris and 0.1 % Triton X-100, pH 8.0) to prepare working stocks of the inhibitors at varying concentrations. For each reaction, the well contained 10 nM DHODase, 68 μM DCIP, 0.16 mg/mL gelatin, the stated concentration of ubiquinone, 10 μL of an inhibitor working stock to give the stated final concentration, and reaction buffer. After a 5-min equilibration period, the reaction was initiated by addition of DHO to the stated final concentrations. The total volume of reaction mixture for each assay was 150 μL, and the final DMSO concentration was ≤ 0.01% (v/v). The reaction progress was followed by recording the loss of absorbance at 610 nm over a 10-min period (during which the velocity remained linear). Velocities are reported as the change in absorbance at 610 nm per minute (in units of mOD/min = 1000ΔA/min), and each reported value is the average of three replicates. In experiments where the DHO or ubiquinone concentration was varied, the other substrate was held constant at 200 μM. To determine the inhibitor potency of leflunomide and A771726, the effects of varying concentrations of the two compounds on the initial velocity of the DHODase reaction was measured over a concentration range of 0.01−1.0 μM. In these experiments the DHO and ubiquinone concentrations were held constant at 200 and 100 μM, respectively[1].
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| Cell Assay |
In vitro studies indicate that leflunomide is capable of inhibiting anti-CD3- and interleukin-2 (IL-2)-stimulated T cell proliferation. However, the biochemical mechanism for the inhibitory activity of leflunomide has not been elucidated. In this study, we characterized the inhibitory effects of leflunomide on Src family (p56lck and p59fyn)-mediated protein tyrosine phosphorylation. Leflunomide was able to inhibit p59fyn and p56lck activity in in vitro tyrosine kinase assays. The IC50 values for p59fyn (immunoprecipitated from either Jurkat or CTLL-4 cell lysate) autophosphorylation and phosphorylation of the exogenous substrate, histone 2B, were 125-175 and 22-40 microM respectively, while the IC50 values for p56lck (immunoprecipitated from Jurkat cell lysates) autophosphorylation and phosphorylation of histone 2B were 160 and 65 microM respectively. We also demonstrated the ability of leflunomide to inhibit protein tyrosine phosphorylation induced by anti-CD3 monoclonal antibody in Jurkat cells. The IC50 values for total intracellular tyrosine phosphorylation ranged from 5 to 45 microM, with the IC50 values for the zeta chain and phospholipase C isoform gamma 1 being 35 and 44 microM respectively. Leflunomide also inhibited Ca2+ mobilization in Jurkat cells stimulated by anti-CD3 antibody but not in those stimulated by ionomycin. Distal events of anti-CD3 monoclonal antibody stimulation, namely, IL-2 production and IL-2 receptor expression on human T lymphocytes, were also inhibited by leflunomide. Finally, tyrosine phosphorylation in CTLL-4 cells stimulated by IL-2 was also inhibited by leflunomide. These data collectively demonstrate the ability of leflunomide to inhibit tyrosine kinase activity in vitro, and suggest that inhibition of tyrosine phosphorylation events may be the mechanism by which leflunomide functions as an immunosuppressive agent[2].
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| Animal Protocol |
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| References |
[1]. Davis JP, et al. The immunosuppressive metabolite of leflunomide is a potent inhibitor of human dihydroorotate dehydrogenase. Biochemistry. 1996 Jan 30;35(4):1270-3.
[2]. Xu X, et al. Inhibition of protein tyrosine phosphorylation in T cells by a novel immunosuppressive agent, leflunomide. J Biol Chem. 1995 May 26;270(21):12398-403. [3]. Fox RI, et al. Mechanism of action for leflunomide in rheumatoid arthritis. Clin Immunol. 1999 Dec;93(3):198-208 |
| 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 # |
75706-12-6
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| Related CAS # |
Leflunomide-d4;1189987-23-2
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| Appearance |
White to off-white solid powder
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| LogP |
1.95
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| tPSA |
55.13
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| SMILES |
O=C(C1=C(C)ON=C1)NC2=CC=C(C(F)(F)F)C=C2
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| InChi Key |
VHOGYURTWQBHIL-UHFFFAOYSA-N
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| InChi Code |
InChI=1S/C12H9F3N2O2/c1-7-10(6-16-19-7)11(18)17-9-4-2-8(3-5-9)12(13,14)15/h2-6H,1H3,(H,17,18)
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| Chemical Name |
5-methyl-N-[4-(trifluoromethyl)phenyl]-1,2-oxazole-4-carboxamide
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| Synonyms |
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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 |
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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)
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| Solubility (In Vitro) |
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| Solubility (In Vivo) |
Solubility in Formulation 1: 2.5 mg/mL (9.25 mM) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), suspension solution; with heating and sonication.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 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. Solubility in Formulation 2: ≥ 2.5 mg/mL (9.25 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 25.0 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. View More
Solubility in Formulation 3: ≥ 2.5 mg/mL (9.25 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution. |
| 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 |
| NCT06228443 | Not yet recruiting | Drug: Leflunomide 20 mg Film-coated Tablet |
Healthy Volunteer | International Bio service | April 1, 2024 | Phase 1 |
| NCT04361214 | Terminated | Drug: Leflunomide | COVID-19 | University of Chicago | May 5, 2020 | Phase 1 |
| NCT05937191 | Recruiting | Drug: Leflunomide Drug: Steroid Drug |
Idiopathic Pulmonary Hemosiderosis Leflunomide |
Sun Yat-Sen Memorial Hospital of Sun Yat-Sen University |
June 1, 2023 | Phase 1 Phase 2 |
| NCT03709446 | Recruiting | Drug: Leflunomide | Breast Neoplasms Breast Diseases |
Joseph Sparano | April 16, 2019 | Phase 1 Phase 2 |
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