DHODH (dihydroorotate dehydrogenase)

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].

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].

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].

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].

Absorption, Distribution and Excretion
Well absorbed, peak plasma concentrations appear 6-12 hours after dosing
The active metabolite is eliminated by further metabolism and subsequent renal excretion as well as by direct biliary excretion. In a 28 day study of drug elimination (n=3) using a single dose of radiolabeled compound, approximately 43% of the total radioactivity was eliminated in the urine and 48% was eliminated in the feces. It is not known whether leflunomide is excreted in human milk. Many drugs are excreted in human milk, and there is a potential for serious adverse reactions in nursing infants from leflunomide.
0.13 L/kg
Following oral administration of leflunomide, the drug is rapidly converted to A77 1726 in the GI mucosa and liver.
Time to peak concentration: Approximately 6 to 12 hours. /M1 metabolite/
M1 metabolite is 80% bioavailable. Administration of leflunomide with a high-fat meal has no effect on the plasma concentration of M1. /M1 metabolite/
M1 has a low volume of distribution (Vss = 0.13 L/kg) and is extensively bound (>99.3%) to albumin in healthy subjects. Protein binding has been shown to be linear at therapeutic concentrations. The free fraction of M1 is slightly higher in patients with rheumatoid arthritis and approximately doubled in patients with chronic renal failure; the mechanism and significance of these increases are unknown.
For more Absorption, Distribution and Excretion (Complete) data for LEFLUNOMIDE (8 total), please visit the HSDB record page.

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Metabolism / Metabolites
Primarily hepatic. Leflunomide is converted to its active form following oral intake.
Leflunomide is metabolized to M1 and other minor active metabolites. An active metabolite, 4-trifluoromethylaniline, is present in plasma at low concentrations. Although the specific site of leflunomide metabolism is unknown, it has been suggested that the gastrointestinal wall and liver play a role in the metabolism.
The 3-unsubstituted isoxazole ring in the anti-inflammatory drug leflunomide undergoes a unique N-O bond cleavage to the active alpha-cyanoenol metabolite A771726, which resides in the same oxidation state as the parent. In vitro studies were conducted to characterize drug-metabolizing enzyme(s) responsible for ring opening and to gain insight into the mechanism of ring opening. ... Although A771726 formation in human liver microsomes or recombinant p4501A2 required NADPH, its formation was greatly reduced by oxygen or carbon monoxide, suggesting that the isoxazole ring opening was catalyzed by the p450Fe(II) form of the enzyme. A mechanism for the p450-mediated ring scission is proposed in which the isoxazole ring nitrogen or oxygen coordinates to the reduced form of the heme followed by charge transfer from p450Fe(II) to the C=N bond or deprotonation of the C3-H, which results in a cleavage of the N-O bond.
Leflunomide has known human metabolites that include (E)-3-Hydroxy-2-methanimidoyl-N-[4-(trifluoromethyl)phenyl]but-2-enamide.

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Biological Half-Life
2 weeks
2 weeks /M1 metabolite/

Interactions
Concurrent use with rifampin may increase the plasma concentration of leflunomide; caution is recommended.
Concurrent use with these medications /hepatotoxic medications or methotrexate/ may increase the risk of side effects and medication-induced hepatic toxicity; in a small study evaluating the concurrent use of leflunomide (100 mg/day followed by 10 to 20 mg/day) and methotrexate (10 to 25 mg/week with folate), an increased risk of hepatotoxicity was reported; dosage adjustment may be needed.
In vivo drug interaction studies have demonstrated a lack of a significant drug interaction between leflunomide and tri-phasic oral contraceptives, and cimetidine.
M1 was shown to cause increases ranging from 13-50% in the free fraction of diclofenac, ibuprofen and tolbutamide at concentrations in the clinical range. In vitro studies of drug metabolism indicate that M1 inhibits CYP 450 2C9, which is responsible for the metabolism of many NSAIDs. M1 has been shown to inhibit the formation of 4'-hydroxydiclofenac from diclofenac in vitro.
Concurrent use with these medications /activated charcoal, or cholestyramine/ will significantly decrease the plasma concentration of M1 by inhibiting gastrointestinal absorption. /M1 metabolite/

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Non-Human Toxicity Values
LD50 Rabbit oral 132 mg/kg
LD50 Rat oral 235 mg/kg
LD50 Mouse oral 445 mg/kg

[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
Antirheumatic
Leflunomide is indicated to alleviate the signs and symptoms of rheumatoid arthritis and to slow joint impairment. /Included in US product labeling/
Leflunomide, a new oral immunomodulatory agent, is effective for the treatment of rheumatoid arthritis. Its mechanism of action in suppressing inflammation is based in its inhibition of dihydroorotate dehydrogenase, an enzyme responsible for de novo synthesis of pyrimidine containing ribonucleotides. It is the first disease-modifying antirheumatic drug approved for treatment of rheumatoid arthritis with an indication for retardation of joint damage by radiography. Side effects are generally mild and include diarrhea, rashes, reversible alopecia, and elevation of hepatic transaminases. Despite the concern about hepatotoxicity, combination use with methotrexate in treating patients with rheumatoid arthritis has been shown to be safe. Other autoimmune diseases in which leflunomide has been used successfully include Felty syndrome, vasculitis, Sjogren syndrome, Wegener granulomatosis, and bullous pemphigoid.
Leflunomide has excellent antiviral activity against cytomegalovirus (CMV) in animal models and is considerably less expensive than intravenous ganciclovir. We used leflunomide in four consenting renal allograft recipients with symptomatic CMV disease, who were unable to afford ganciclovir and would otherwise remain untreated. This is the first report of efficacy of leflunomide in humans with CMV disease. They received loading dose of 100 mg of leflunomide once daily on days 1-3 and then 20 mg once daily for 3 months. All four patients were followed up three times weekly with physical examination, total leukocyte counts, blood urea and serum creatinine for a minimum period of 6 weeks. None of the patients showed drug related adverse events, alteration in cyclosporine levels, or decreased graft function, except one who developed leucopenia. Preliminary data presented suggests that leflunomide therapy for CMV disease is effective and could be used with careful monitoring in allograft recipients who cannot afford intravenous ganciclovir therapy. The duration of treatment and the role of leflunomide in secondary prophylaxis and in situations of ganciclovir resistance need to be studied further.

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Drug Warnings
FDA Pregnancy Risk Category: X /CONTRAINDICATED IN PREGNANCY. Studies in animals or humans, or investigational or post-marketing reports, have demonstrated positive evidence of fetal abnormalities or risk which clearly outweights any possible benefit to the patient./
Because it can take up to 2 years for plasma concentrations of the active metabolite of leflunomide (A77 1726) to decrease to undetectable concentrations (less than 0.02 ug/mL) following discontinuance of leflunomide, the possibility that adverse effects or drug interactions associated with the drug could continue to occur even thought the patient is no longer receiving leflunomide should be considered.
Opportunistic infections and serious infection, including sepsis and death, have been reported rarely in patients receiving leflunomide. Most serious infections reported in patients receiving leflunomide occurred in those receiving concomitant therapy with immunosuppressive agent and/or those with comorbid illness that, in addition to rheumatoid arthritis, could have predisposed them to infections.
Leflunomide, a new immunomodulatory agent, was prescribed to a 67-year-old female patient with rheumatoid arthritis. Fifteen days later she developed diarrhea and elevated liver enzymes. A liver biopsy showed a pattern of acute hepatitis. The patient was homozygous for the rare CYP2C9*3 allele, which determines the slowest metabolic rate for CYP2C9 enzymatic activity, that is probably involved in the metabolism of leflunomide. Liver damage subsided in few weeks. This case illustrates the risk of hepatotoxicity by leflunomide and suggests that it is possibly related to CYP2C9 polymorphism.
For more Drug Warnings (Complete) data for LEFLUNOMIDE (20 total), please visit the HSDB record page.

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Pharmacodynamics
Leflunomide is a pyrimidine synthesis inhibitor indicated in adults for the treatment of active rheumatoid arthritis (RA). RA is an auto-immune disease characterized by high T-cell activity. T cells have two pathways to synthesize pyrimidines: the salvage pathways and the de novo synthesis. At rest, T lymphocytes meet their metabolic requirements by the salvage pathway. Activated lymphocytes need to expand their pyrimidine pool 7- to 8-fold, while the purine pool is expanded only 2- to 3-fold. To meet the need for more pyrimidines, activated T cells use the de novo pathway for pyrimidine synthesis. Therefore, activated T cells, which are dependent on de novo pyrimidine synthesis, will be more affected by leflunomide's inhibition of dihydroorotate dehydrogenase than other cell types that use the salvage pathway of pyrimidine synthesis.

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.)