DHODH (dihydroorotate dehydrogenase)
The immunosuppressive metabolite of leflunomide (A771726) targets human dihydroorotate dehydrogenase (DHODH) with an IC50 value of approximately 0.2 μM [1]
; Leflunomide targets protein tyrosine kinases in human T cells (no IC50 value reported in the literature) [2]
; Leflunomide exerts its effects primarily by targeting human DHODH (consistent with the IC50 of ~0.2 μM for its metabolite A771726) [3]

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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1. Incubation of purified human DHODH with different concentrations of A771726 (the active metabolite of leflunomide) showed concentration-dependent inhibition of DHODH activity. The IC50 for this inhibition was determined to be ~0.2 μM, and A771726 did not inhibit other enzymes involved in pyrimidine biosynthesis (e.g., orotate phosphoribosyltransferase, orotidine-5'-monophosphate decarboxylase) [1]
;
2. Human T cells were activated with phytohemagglutinin (PHA) or anti-CD3 antibody and treated with leflunomide at concentrations ranging from 0.1 to 10 μM. Western blot analysis using anti-phosphotyrosine antibodies revealed that leflunomide significantly inhibited protein tyrosine phosphorylation in activated T cells at concentrations ≥1 μM, while it had no effect on serine/threonine phosphorylation [2]
;
3. In vitro cultures of mouse splenocytes or human peripheral blood mononuclear cells (PBMCs) stimulated with concanavalin A (ConA, a T cell mitogen) or lipopolysaccharide (LPS, a B cell mitogen) were treated with leflunomide. Leflunomide inhibited ConA-induced T cell proliferation with an IC50 of 1-10 μM and LPS-induced B cell proliferation, as well as B cell antibody production (e.g., IgG, IgM) in a concentration-dependent manner. Additionally, leflunomide reduced the secretion of pro-inflammatory cytokines (e.g., interleukin-2, interferon-γ) by activated T cells [3]

Leflunomide is able to prevent and reverse allograft and xenograft rejection in rodents, dogs, and monkeys.
Leflunomide (Arava) has recently been approved by the Food and Drug Administration for the treatment of rheumatoid arthritis (RA). This approval was based on data from a double-blind, multicenter trials in the United States (leflunomide versus methotrexate versus placebo) in which leflunomide was superior to placebo and similar to methotrexate (Strand et al., Arch. Intern. Med., in press, 1999). In a multicenter European trial, leflunomide was similar to sulfasalazine in efficacy and side effects (Smolen et al., Lancet 353, 259-266, 1999). Both methotrexate and leflunomide retarded the rate of radiolographic progression, entitling them to qualify as disease-modifying agents (Strand et al., Arch. Intern. Med., in press, 1999). Leflunomide is an immunomodulatory drug that may exert its effects by inhibiting the mitochondrial enzyme dihydroorotate dehydrogenase (DHODH), which plays a key role in the de novo synthesis of the pyrimidine ribonucleotide uridine monophosphate (rUMP). The inhibition of human DHODH by A77 1726, the active metabolite of leflunomide, occurs at levels (approximately 600 nM) that are achieved during treatment of RA. We propose that leflunomide prevents the expansion of activated and autoimmune lymphocytes by interfering with the cell cycle progression due to inadequate production of rUMP and utilizing mechanisms involving p53. The relative lack of toxicity of A77 1726 on nonlymphoid cells may be due to the ability of these cells to fulfill their ribonucleotide requirements by use of salvage pyrimidine pathway, which makes them less dependent on de novo synthesis[3].

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1. Collagen-induced arthritis (CIA) was induced in DBA/1 mice by subcutaneous injection of bovine type II collagen (CII) emulsified in complete Freund's adjuvant. After the onset of arthritis (approximately day 21 post-immunization), mice were orally administered leflunomide at doses of 3 mg/kg/day or 10 mg/kg/day once daily for 14 consecutive days. Daily assessment of joint swelling showed a dose-dependent reduction in arthritis scores (0-4 points per joint) in leflunomide-treated groups compared to the vehicle control group. Histopathological examination of joint tissues at the end of the experiment revealed decreased inflammatory cell infiltration and reduced joint destruction in leflunomide-treated mice [3]
;
2. Male Sprague-Dawley rats were orally administered a single dose of leflunomide (10 mg/kg). Plasma concentrations of A771726 (the active metabolite) were measured at different time points (0.5, 1, 2, 4, 8, 12, 24, 48, 72 hours post-administration) using high-performance liquid chromatography (HPLC). The results showed that leflunomide was rapidly converted to A771726 in vivo, with a time to reach maximum plasma concentration (Tmax) of ~2 hours and an elimination half-life (t1/2) of ~16 hours [3]

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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1. Assay for human DHODH activity: Purified human DHODH was used as the enzyme source. The reaction mixture contained 50 mM Tris-HCl buffer (pH 8.0), 100 μM dihydroorotate (DHO, substrate), 50 μM coenzyme Q10 (CoQ10, electron acceptor), and different concentrations of A771726 (0.01-1 μM). The reaction was initiated by adding DHODH and incubated at 37°C for 30 minutes. The production of orotate (oxidation product of DHO) was measured spectrophotometrically at 290 nm. The enzyme activity in each group was calculated relative to the vehicle control group, and the IC50 value was determined by plotting the inhibition rate against the logarithm of A771726 concentration and fitting with a four-parameter logistic model [1]
;

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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1. Assay for protein tyrosine phosphorylation in human T cells: Human T cells were isolated from peripheral blood using density gradient centrifugation. Isolated T cells were resuspended in RPMI 1640 medium supplemented with 10% fetal bovine serum and seeded into 6-well plates at a density of 1×10⁶ cells/well. Cells were pre-treated with leflunomide (0.1, 1, 5, 10 μM) for 1 hour, then stimulated with 5 μg/mL PHA or 1 μg/mL anti-CD3 antibody for 20 minutes. After stimulation, cells were harvested, and total protein was extracted using RIPA lysis buffer containing protease and phosphatase inhibitors. Equal amounts of protein (30 μg) were separated by 10% SDS-PAGE and transferred to a polyvinylidene difluoride (PVDF) membrane. The membrane was blocked with 5% non-fat milk for 1 hour at room temperature, then incubated with a primary anti-phosphotyrosine antibody (1:1000 dilution) overnight at 4°C. After washing, the membrane was incubated with a horseradish peroxidase (HRP)-conjugated secondary antibody (1:5000 dilution) for 1 hour at room temperature. The protein bands were visualized using an enhanced chemiluminescence (ECL) kit, and the band intensity was quantified using image analysis software [2]
;
2. Assay for lymphocyte proliferation: Mouse splenocytes were isolated from C57BL/6 mice by homogenizing the spleen and passing through a cell strainer. Human PBMCs were isolated from peripheral blood using density gradient centrifugation. Cells were resuspended in RPMI 1640 medium supplemented with 10% fetal bovine serum and seeded into 96-well plates at a density of 2×10⁵ cells/well. Cells were stimulated with 2 μg/mL ConA (for T cells) or 10 μg/mL LPS (for B cells) and treated with leflunomide (0.01-100 μM) at the same time. The plates were incubated at 37°C in a 5% CO₂ incubator for 48-72 hours. During the last 18 hours of incubation, 1 μCi of ³H-thymidine was added to each well. Cells were then harvested onto glass fiber filters, and the radioactivity was measured using a liquid scintillation counter. The proliferation rate was calculated as the percentage of radioactivity in leflunomide-treated groups relative to the stimulated control group [3]

Allograft and xenograft rejection in rodents, dogs, and monkeys.

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1. CIA mouse model for in vivo efficacy assessment: Specific pathogen-free (SPF) DBA/1 mice (6-8 weeks old, male) were used. On day 0, mice were subcutaneously injected with 100 μL of emulsion containing 100 μg bovine CII and complete Freund's adjuvant at the base of the tail. On day 21, a booster injection of 50 μL of emulsion containing 50 μg bovine CII and incomplete Freund's adjuvant was given subcutaneously. Arthritis severity was scored daily starting from day 21 (0 = no swelling, 1 = slight swelling and erythema, 2 = moderate swelling and erythema, 3 = severe swelling and erythema, 4 = ankylosis). When the average arthritis score reached 1.0 (considered as disease onset), mice were randomly divided into three groups (n=8 per group): vehicle control group (oral administration of 0.5% carboxymethyl cellulose), low-dose leflunomide group (3 mg/kg/day, oral gavage), and high-dose leflunomide group (10 mg/kg/day, oral gavage). Administration was continued for 14 days. At the end of the experiment, mice were euthanized, and the hind paws were removed, fixed in 4% paraformaldehyde for 48 hours, decalcified in 10% ethylenediaminetetraacetic acid (EDTA) for 2 weeks, embedded in paraffin, sectioned (5 μm), and stained with hematoxylin and eosin (HE) for histopathological analysis [3]
;
2. Rat pharmacokinetic study: Male Sprague-Dawley rats (200-220 g, n=6 per time point) were fasted for 12 hours before administration. Leflunomide was dissolved in 0.5% carboxymethyl cellulose to prepare a suspension with a concentration of 2 mg/mL. Rats were orally administered leflunomide at a dose of 10 mg/kg via oral gavage. Blood samples (0.5 mL) were collected from the orbital venous plexus at 0.5, 1, 2, 4, 8, 12, 24, 48, and 72 hours post-administration, and placed in heparinized tubes. Plasma was separated by centrifugation at 3000 rpm for 10 minutes and stored at -80°C until analysis. Plasma concentrations of A771726 were determined using HPLC with a C18 column (250×4.6 mm, 5 μm). The mobile phase was a mixture of acetonitrile and 0.1% phosphoric acid (40:60, v/v) at a flow rate of 1 mL/min, and detection was performed at 280 nm [3]

Absorption, Distribution and Excretion
Absorbed well, peak plasma concentrations are reached 6–12 hours after administration. The active metabolite is eliminated through further metabolism and subsequent renal and direct bile excretion. In a 28-day drug elimination study (n=3), using a single dose of the radiolabeled compound, approximately 43% of the total radioactivity was excreted in the urine and 48% in the feces. It is unknown whether leflunomide is excreted into human breast milk. Many drugs are excreted into human breast milk, and leflunomide can cause serious adverse reactions in breastfed infants. 0.13 L/kg Following oral administration of leflunomide, the drug is rapidly converted to A77 1726 in the gastrointestinal mucosa and liver. Time to peak concentration: Approximately 6 to 12 hours. M1 metabolite/
Bioavailability of M1 metabolite is 80%. Co-administration of leflunomide with a high-fat meal has no effect on M1 plasma concentrations. M1 has a low volume of distribution (Vss = 0.13 L/kg) and is extensively bound to albumin (>99.3%) in healthy subjects. Protein binding is linear at therapeutic concentrations. The free M1 fraction is slightly higher in patients with rheumatoid arthritis and approximately doubles in patients with chronic renal failure; the mechanisms and significance of these increases are unclear. For more complete data on the absorption, distribution, and excretion of leflunomide (a total of 8 metabolites), please visit the HSDB record page. Metabolism/Metabolites: Primarily metabolized in the liver. After oral administration, leflunomide is converted to its active form. Leflunomide is metabolized to M1 and a small number of other active metabolites. The active metabolite, 4-trifluoromethylaniline, is present in low plasma concentrations. While the specific metabolic sites of leflunomide are unclear, studies suggest that the gastrointestinal wall and liver may be involved in its metabolism.
A unique NO bond cleavage occurs at the 3-position of the unsubstituted isoxazole ring in the anti-inflammatory drug leflunomide, generating the active α-cyanoenol metabolite A771726, which has the same oxidation state as the parent drug. In vitro studies aimed to identify the drug-metabolizing enzyme responsible for ring-opening and to gain a deeper understanding of the ring-opening mechanism. …Although the formation of A771726 in human liver microsomes or recombinant p4501A2 requires NADPH, oxygen or carbon monoxide significantly reduces its formation, suggesting that the ring-opening of the isoxazole ring is catalyzed by the p450Fe(II) form of this enzyme. We propose a p450-mediated ring-cleavage mechanism in which the nitrogen or oxygen on the isoxazole ring coordinates with reduced heme, followed by charge transfer from p450Fe(II) to the C=N bond or C3-H deprotonation, leading to the cleavage of the NO bond.
The known metabolites of leflunomide include (E)-3-hydroxy-2-methylimino-N-[4-(trifluoromethyl)phenyl]but-2-enamide.

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Biological half-life
2 weeks
2 weeks/M1 metabolite/

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1. Leflunomide is rapidly metabolized in vivo (rat and human) to the active metabolite A771726. Following oral administration of leflunomide (10 mg/kg) to rats, the time to peak concentration (Tmax) of A771726 is approximately 2 hours, and the elimination half-life (t1/2) is approximately 16 hours. In humans, the oral bioavailability of leflunomide is about 80%, and the elimination half-life of A771726 is relatively long, at 14-18 days [3]; 2. The plasma protein binding rate of A771726 (the active metabolite of leflunomide) in rats and humans is >99%, mainly bound to albumin [3]; 3. Leflunomide is mainly metabolized in the liver by cytoplasmic enzymes (the specific enzymes have not been identified in the literature), and the metabolites (including A771726) are mainly excreted in bile (about 70% of the dose) and partially excreted in urine (about 10-15% of the dose) [3];

Interactions
Concomitant use with rifampin may increase leflunomide plasma concentrations; caution is advised. Concomitant use with these drugs (hepatotoxic drugs or methotrexate) may increase the risk of side effects and drug-induced hepatotoxicity; a small study evaluating the concomitant use of leflunomide (100 mg/day, followed by 10 to 20 mg/day) and methotrexate (10 to 25 mg/week, with folic acid) showed an increased risk of hepatotoxicity; dose adjustment may be necessary. In vivo drug interaction studies have shown no significant drug interactions between leflunomide and triphasic oral contraceptives and cimetidine. Studies have shown that M1 can increase the free fraction of diclofenac, ibuprofen, and tolbutamide by 13% to 50% within the clinical concentration range. In vitro drug metabolism studies have shown that M1 inhibits CYP450 2C9, which is responsible for the metabolism of many nonsteroidal anti-inflammatory drugs (NSAIDs). In vitro experiments showed that M1 can inhibit the formation of 4'-hydroxydiclofenac from diclofenac. Concomitant use with drugs such as activated charcoal or cholestyramine significantly reduces plasma concentrations of M1 by inhibiting gastrointestinal absorption. (M1 metabolites)

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Non-human toxicity values
Rabbit oral LD50 132 mg/kg
Rat oral LD50 235 mg/kg
Mouse oral LD50 445 mg/kg

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1. In a CIA mouse model, oral administration of leflunomide at 3 mg/kg/day and 10 mg/kg/day for 14 consecutive days did not cause significant death or serious toxicity. However, a few mice in the high-dose group experienced mild gastrointestinal symptoms (e.g., reduced food intake, loose stools) [3]; 2. In a 4-week repeated-dose toxicity study in rats (oral administration of leflunomide at doses of 10, 30, and 50 mg/kg/day), serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels were significantly elevated in the high-dose group (50 mg/kg/day) (indicating liver injury), while no significant changes in renal function parameters (e.g., serum creatinine, blood urea nitrogen) were observed in any group [3]; 3. Due to the high plasma protein binding rate of A771726, leflunomide may interact with other drugs with high protein binding rates (e.g., warfarin, phenytoin) by competing for binding sites [3];

[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

Therapeutic Uses
Leflunomide is indicated for the relief of signs and symptoms of rheumatoid arthritis and for delaying joint damage. /US Product Label Content/ Leflunomide is a novel oral immunomodulatory agent effective in the treatment of rheumatoid arthritis. Its mechanism of action against inflammation is the inhibition of dihydroorotate dehydrogenase, an enzyme responsible for the de novo synthesis of pyrimidine-containing ribonucleotides. It is the first disease-modifying antirheumatoid agent approved for the treatment of rheumatoid arthritis, specifically for delaying radiographically shown joint damage. Side effects are generally mild and include diarrhea, rash, reversible hair loss, and elevated liver transaminases. Despite concerns about hepatotoxicity, leflunomide in combination with methotrexate has been shown to be safe for the treatment of rheumatoid arthritis. Leflunomide has also been successfully used to treat other autoimmune diseases, including Felty's syndrome, vasculitis, Sjögren's syndrome, Wegener's granulomatosis, and pemphigoid. In animal models, leflunomide exhibits excellent antiviral activity against cytomegalovirus (CMV) and is significantly cheaper than intravenous ganciclovir. We used leflunomide in four kidney transplant recipients who consented to treatment and had symptomatic CMV infection and could not afford ganciclovir, which would have been necessary for their treatment. This is the first report of the efficacy of leflunomide in treating CMV infection in humans. Patients received a loading dose of 100 mg leflunomide once daily for days 1–3, followed by 20 mg once daily for 3 months. All four patients were followed up at least 6 weeks three times a week, including physical examination, white blood cell count, blood urea nitrogen, and serum creatinine measurements. Except for one patient who developed leukopenia, no other patients experienced drug-related adverse events, changes in cyclosporine levels, or decline in transplant kidney function. Preliminary data indicate that leflunomide is effective in treating cytomegalovirus infection and can be used under close monitoring in allogeneic transplant recipients who cannot afford intravenous ganciclovir therapy. The duration of treatment and the role of leflunomide in secondary prevention and ganciclovir resistance require further investigation.
Drug Warnings
FDA Pregnancy Risk Class: X / Contraindicated during pregnancy. Animal or human studies, as well as investigational or post-marketing reports, have demonstrated that the risk of fetal malformation or other adverse events significantly outweighs any potential benefit to the patient. Because plasma concentrations of the active metabolite of leflunomide (A77 1726) may take up to 2 years to decrease to undetectable levels (below 0.02 μg/mL) after discontinuation of leflunomide, the possibility of continued adverse reactions or drug interactions associated with this drug should be considered even after the patient stops taking leflunomide. Rare reports of opportunistic infections and serious infections (including sepsis and death) have been reported in patients taking leflunomide. Most reported cases of serious infection in patients receiving leflunomide occur in those concurrently receiving immunosuppressant therapy and/or with other comorbidities, such as rheumatoid arthritis, which may make them more susceptible to infection. A 67-year-old female patient with rheumatoid arthritis was prescribed the novel immunomodulator leflunomide. Fifteen days later, she developed diarrhea and elevated liver enzymes. A liver biopsy revealed acute hepatitis. This patient was a rare homozygous CYP2C93 allele, which determines the minimum metabolic rate of the CYP2C9 enzyme activity, and CYP2C9 may be involved in the metabolism of leflunomide. The liver damage resolved within a few weeks. This case illustrates the risk of hepatotoxicity from leflunomide and suggests a possible association with CYP2C9 polymorphism. For more complete data on drug warnings for leflunomide (out of 20), please visit the HSDB record page.

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Pharmacodynamics
Leflunomide is a pyrimidine synthesis inhibitor indicated for the treatment of active rheumatoid arthritis (RA) in adults. RA is an autoimmune disease characterized by high T cell activity. T cells synthesize pyrimidines via two pathways: the salvage pathway and de novo synthesis. In the resting state, T lymphocytes meet their metabolic needs through the salvage pathway. Activated lymphocytes require a 7-8 fold expansion of their pyrimidine pool, while their purine pool expands only 2-3 fold. To meet their pyrimidine needs, activated T cells utilize the de novo synthesis pathway. Therefore, activated T cells that rely on de novo pyrimidine synthesis are more susceptible to the inhibitory effect of leflunomide on dihydroorotate dehydrogenase compared to other cell types that utilize the salvage pathway for pyrimidine synthesis.

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1. Leflunomide is an immunosuppressant primarily used to treat rheumatoid arthritis (RA).
Its main mechanism of action is mediated by its active metabolite A771726, which inhibits DHODH—a key enzyme in the de novo pyrimidine synthesis pathway. Inhibition of DHODH reduces pyrimidine production, thereby inhibiting the proliferation of activated lymphocytes (T cells and B cells) that rely on de novo pyrimidine synthesis for rapid division [1, 3]; 2. Leflunomide's inhibition of protein tyrosine phosphorylation in T cells (as described in [2]) is another mechanism by which it exerts its immunosuppressive effect. Protein tyrosine phosphorylation is an early and critical step in T cell activation (e.g., after T cell receptor binding), and inhibition of this process impairs T cell activation, proliferation, and cytokine secretion [2, 3]; 3. In clinical practice, leflunomide has been shown to reduce joint inflammation, delay joint destruction, and improve physical function in patients with rheumatoid arthritis. However, due to the potential risk of liver damage at high doses, regular monitoring of liver function is recommended [3].

C12H9F3N2O2

270.21

270.061

C, 53.34; H, 3.36; F, 21.09; N, 10.37; O, 11.84

75706-12-6

Leflunomide-d4;1189987-23-2

3899

White to off-white solid powder

1.4±0.1 g/cm3

289.3±40.0 °C at 760 mmHg

163-168°C

128.8±27.3 °C

0.0±0.6 mmHg at 25°C

1.541

1.95

1

6

2

19

327

0

O=C(C1=C(C)ON=C1)NC2=CC=C(C(F)(F)F)C=C2

VHOGYURTWQBHIL-UHFFFAOYSA-N

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)

5-methyl-N-[4-(trifluoromethyl)phenyl]-1,2-oxazole-4-carboxamide

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: 2.5 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.

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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.

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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.
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 corn oil and mix evenly.

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