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PubMed1999

In vivo activity of leflunomide: pharmacokinetic analyses and mechanism of immunosuppression.

BACKGROUND: Leflunomide is an experimental drug with demonstrated ability to prevent and reverse acute allograft and xenograft rejection. The two biochemical activities reported for the active metabolite of leflunomide, A77 1726, are inhibition of tyrosine phosphorylation and inhibition of dihydroorotate dehydrogenase, an enzyme necessary for de novo pyrimidine synthesis. These activities can be distinctly separated in vitro by the use of uridine, which reverses the anti-proliferative effects of A77 1726 caused by inhibition of de novo pyrimidine synthesis. We report the effect of uridine on the in vivo immunosuppressive activities of leflunomide. METHODS: We first quantified the serum levels of A77 1726, the active metabolite of leflunomide, after a single treatment of leflunomide (5, 15, and 35 mg/kg). Additionally, we quantified the levels of serum uridine and of nucleotide triphosphates in the liver, spleen, and lymph nodes of Lewis rats after the administration of a single dose of uridine (500 mg/kg; i.p.). Lewis rats heterotopically transplanted with brown Norway or Golden Syrian hamster hearts were treated for 50 or 75 days with leflunomide (5, 15, and 35 mg/kg/day; gavage) alone or in combination with uridine (500 mg/ kg/day; i.p.). Hematocrits were determined and the levels of alloreactive or xenoreactive immunoglobulin (Ig)M and IgG were determined by flow cytometric analysis. The allograft and xenografts, small bowel, liver, kidney, and spleen were subjected to pathological examination. RESULTS: A linear relationship was observed between the serum A77 1726 concentrations in Lewis rats and the dose of leflunomide administered. Peak A77 1726 concentrations were 20.9, 71.8 and 129.3 mg/l (77.5, 266.1 and 478.8 microM) for the 5, 15, and 35 mg/kg doses of leflunomide, respectively. The concentration of uridine in the serum of normal Lewis rats is 6.5 microM; after i.p. administration of 500 mg/kg uridine, the serum uridine concentrations peaked at 384.1 microM in 15-30 min. The rapid elimination of uridine was not reflected in the lymphoid compartments, and the pharmacokinetics of pyrimidine nucleotides in the spleen resembled that of A77 1726. This dose of uridine, when administered daily (500 mg/kg/day, i.p.), weakly antagonized the immunosuppressive activities of leflunomide (5, 15, and 35 mg/kg/day) in the allotransplantation model. In contrast, in the xenotransplantation model, the same concentration of uridine completely antagonized the immunosuppressive activities of low-dose leflunomide (15 mg/kg/day) and partially antagonized the immunosuppressive activities of high-dose leflunomide (35 mg/kg/day). Toxicities associated with high-dose leflunomide (35 mg/kg/day) were anemia, diarrhea, and pathological changes in the small bowel and liver. These toxicities were significantly reduced by uridine co-administration. CONCLUSION: These studies reveal that the blood levels of A77 1726 in Lewis rats satisfy in vitro requirements for both inhibition of de novo pyrimidine synthesis and protein tyrosine kinase activity. Our data also illustrate that the in vivo mechanism of immunosuppression by leflunomide is complex and is affected by at least the following four factors: type and vigor of the immune response, availability of uridine for salvage by proliferating lymphocytes, species being investigated, and concentration of serum A77 1726.

Aniline Compounds↗
PubMed1999

Leflunomide: a review of its use in active rheumatoid arthritis.

UNLABELLED: A77 1726, the active metabolite of leflunomide, is an immunomodulator which inhibits cell proliferation in activated lymphocytes in patients with active rheumatoid arthritis. Because A77 1726 has a long half-life (approximately 2 weeks), treatment with oral leflunomide is initiated with a loading dose of 100mg once daily for 3 days and continued with 20mg once daily. Results of large randomised, double-blind, multicentre trials of up to 24 months' duration have shown that leflunomide is significantly superior to placebo and at least as effective as sulfasalazine in improving primary outcome measures, such as tender joint counts, swollen joint counts and physicians' and patients' global assessment, in adult patients with active rheumatoid arthritis. Whereas improvement in all primary outcome measures with leflunomide was similar to or significantly less than that with methotrexate after 12 months, the efficacy of both agents was similar after 24 months. The therapeutic effect of leflunomide appears earlier (at 4 weeks) than that of sulfasalazine or methotrexate, and reduction from baseline values in functional disability was significantly greater with leflunomide than with sulfasalazine, methotrexate or placebo at end-point. Leflunomide was at least as effective as sulfasalazine or methotrexate in delaying the rate of radiological progression of disease. The most common adverse events reported in patients receiving leflunomide in randomised double-blind, placebo-controlled trials were diarrhoea (27%), respiratory infections (21%), nausea (13%), headache (13%), rash (12%), increased serum hepatic aminotransferases (10%), dyspepsia (10%) and alopecia (9%). Leflunomide was as well tolerated as sulfasalazine or methotrexate in clinical trials. Monitoring of serum hepatic enzyme levels is recommended in patients receiving leflunomide. The drug is not recommended in female patients who are or may become pregnant. Drug treatment should be discontinued, and hastened drug elimination procedure should be considered, in male patients wishing to father a child. 16 potential cases of pancytopenia and 9 cases of serious skin reactions have been associated with the use of leflunomide in 76,000 patients to date. CONCLUSIONS: Leflunomide is a disease-modifying antirheumatic drug which reduces the signs and symptoms of inflammatory arthritis and delays the radiological progression of disease in adult patients with active rheumatoid arthritis. The drug appears to be as effective and as well tolerated as sulfasalazine or methotrexate, and represents a suitable alternative to these agents in adult patients with active rheumatoid arthritis. Benefits with leflunomide are evident within 4 weeks and efficacy is maintained for durations of up to 24 months.

Adult↗
PubMed1998

Leflunomide, a novel immunomodulating agent, prevents the development of allergic sensitization in an animal model of allergic asthma.

BACKGROUND: Leflunomide is a new anti-inflammatory and immunomodulating agent which is showing promise in several immune disorders, especially rheumatoid arthritis. Its activity profile suggests it may be of use in modulating allergic sensitization. OBJECTIVE: To investigate the effectiveness of leflunomide in preventing the development of allergic sensitization. METHODS: Fifty-three brown Norway rats were sensitized by intraperitoneal injection of ovalbumin and adjuvant (ricin) on day 0. To determine the ability of leflunomide to inhibit primary allergic sensitization six rats were treated with A77 1726, the active metabolite of leflunomide, from day 0 through day 5, six were treated from day 5 through day 10, and nine rats acted as controls. On day 14, ovalbumin-specific serum antibody levels and the magnitude of the early-phase airway response (EAR) after inhalation allergen challenge were assessed. To determine the ability of acute topical treatment with leflunomide to inhibit mast cell degranulation, three groups of five animals received either vehicle, 100 microg A77 1726, or 1000 g A77 1726 60 min prior to aerosol allergen challenge. To determine the effects of leflunomide treatment in vivo on mast cell function in vitro, mast cells were obtained by bronchoalveolar lavage from 17 rats (nine treated with leflunomide and eight controls). Allergen-specific and non-specific degranulation (48/80 induced) were studied. RESULTS: In the leflunomide treated rats both ovalbumin-specific IgE and IgG levels were significantly reduced, and the increases in lung resistance and lung elastance were essentially abolished, compared to those of the control group. Non significant differences were found in any of the parameters between the two leflunomide treated groups. Topical pre-treatment with leflunomide did not prevent the allergen-induced EAR. Treatment with leflunomide in vivo prevented allergen-induced mast cell degranulation in vitro because the mast cells lacked IgE on their surface. Non allergen-specific degranulation was normal and allergen-induced degranulation could be restored by passive sensitization. CONCLUSIONS: These data suggests that leflunomide can prevent primary allergic sensitization and prevent allergen-induced EAR by inhibiting production of allergen-specific IgE antibodies. Further studies in atopic conditions are warranted.

Allergens↗
PubMed1996

Effects of leflunomide and other immunosuppressive agents on T cell proliferation in vitro.

Leflunomide and its active metabolite, A771726, are structurally unrelated to immunosuppressive agents currently under investigation. Previous in vitro studies have revealed that leflunomide primarily inhibits interleukin-2-stimulated T cell proliferation. In the current study, we have extended our previous work and demonstrate that leflunomide prevents T cell progression induced by phytohemagglutinin into the S phase of the cell cycle. To discriminate further the action on T cells of leflunomide from other immunosuppressive agents, we performed kinetic studies where leflunomide was added either after the initiation of mixed lymphocyte cultures (MLC) or after interleukin-2 stimulation of CTLL-4 cell proliferation. These studies revealed that leflunomide acted comparably to rapamycin, but was distinct from brequinar sodium in the MLC, and from cyclosporine and mycophenolic acid in both MLC and CTLL-4. Although previous biochemical studies indicated that leflunomide can inhibit src-family tyrosine kinase activity, more recent studies have suggested that leflunomide can also inhibit pyrimidine synthesis. Our data demonstrate that the ability of leflunomide (25-100 microM) to inhibit MLC and CTLL-4 cell proliferation is partially antagonized by uridine (25-100 microM), and support the hypothesis that leflunomide inhibits pyrimidine synthesis in T cells. Unique molecular mechanisms of immunosuppression suggest that drug combinations may result in synergistic immunosuppression. Our in vitro studies revealed synergistic inhibition of T cell proliferation with the combinations of leflunomide with cyclosporine or with rapamycin. We have extended those studies to quantitate inhibition of MLC by the combinations of leflunomide and brequinar sodium or mycophenolic acid.

Cell Division↗
PubMed2005

Effects of a short course of leflunomide on T-independent B-lymphocyte xenoreactivity and on susceptibility of xenografts to acute or chronic rejection.

BACKGROUND: Leflunomide is a novel immunosuppressive agent with promising activity for xenotransplantation. It is not clear yet which mechanisms of action of leflunomide are responsible for that. METHODS: In a hamster-to-C57BL/6 nude mouse heart transplantation model, a 2-week course of leflunomide was used after transplantation or for pretreating donors. Nontolerant B lymphocytes were transferred to recipients after transplantation of first or second xenogeneic heart grafts that were transplanted with or without leflunomide treatment. RESULTS: Hamster xenogeneic hearts transplanted into athymic C57BL/6 nude mice receiving leflunomide did not induce immunoglobulin (Ig) M xenoantibodies (XAb) and survived without signs of chronic rejection. Second xenogeneic hearts transplanted 4 weeks after withdrawal of leflunomide survived without induction of XAb but developed chronic vascular lesions. After injection of naive B lymphocytes at 6 weeks after grafting a first or second hamster heart, only in the latter case were XAb induced. These were deposited in, and provoked acute rejection of, only the second grafts. Pretreatment of donors with leflunomide decreased the ex vivo xenoantibody deposition on the xenogeneic heart endothelia. CONCLUSIONS: A short posttransplant course of leflunomide induces T-independent B-lymphocyte xenotolerance. Leflunomide treatment also influences xenoantigen expression, as nontolerant B lymphocytes provoke IgM XAb formation and rejection of only second xenografts (transplanted without leflunomide) and not of first xenografts (transplanted with leflunomide treatment). The ex vivo experiments that show that XAb deposition is decreased in leflunomide-pretreated xenografts further confirm this. The latter may also explain the resistance of first and not second xenografts against chronic rejection.

Animals↗
PubMed2000

Leflunomide suppresses TNF-induced cellular responses: effects on NF-kappa B, activator protein-1, c-Jun N-terminal protein kinase, and apoptosis.

Leflunomide is a pyrimidine biosynthesis inhibitor that has recently been approved for treatment of rheumatoid arthritis. However, the mechanism of leflunomide's antiarthritis activity and is not fully understood. The critical role that TNF plays in rheumatoid arthritis led us to postulate that leflunomide blocks TNF signaling. Previously, we have demonstrated that leflunomide inhibits TNF-induced NF-kappaB activation by suppressing I-kappaBalpha (inhibitory subunit of NF-kappaB) degradation. We in this study show that leflunomide also blocks NF-kappaB reporter gene expression induced by TNFR1, TNFR-associated factor 2, and NF-kappaB-inducing kinase (NIK), but not that activated by the p65 subunit of NF-kappaB, suggesting that leflunomide acts downstream of NIK. Leflunomide suppressed TNF-induced phosphorylation of I-kappaBalpha, as well as activation of I-kappaBalpha kinase-beta located downstream to NIK. Leflunomide also inhibited TNF-induced activation of AP-1 and the c-Jun N-terminal protein kinase activation. TNF-mediated cytotoxicity and caspase-induced poly(ADP-ribose) polymerase cleavage were also completely abrogated by treatment of Jurkat T cells with leflunomide. Leflunomide suppressed TNF-induced reactive oxygen intermediate generation and lipid peroxidation, which may explain most of its effects on TNF signaling. The suppressive effects of leflunomide on TNF signaling were completely reversible by uridine, indicating a critical role for pyrimidine biosynthesis in TNF-mediated cellular responses. Overall, our results suggest that suppression of TNF signaling is one of the possible mechanisms for inhibitory activity of leflunomide against rheumatoid arthritis.

Apoptosis↗
PubMed2004

Leflunomide for chronic sarcoidosis.

BACKGROUND AND AIM: Leflunomide (Arava) is a cytotoxic drug which has been used as a single agent or in combination with methotrexate for the treatment of rheumatoid arthritis. It appears to have less toxicity than methotrexate. The use of leflunomide for sarcoidosis patients has not been systematically evaluated. METHODS: The records were reviewed from all patients treated at a tertiary sarcoidosis center from July 2002-July 2003, and information from patients treated with either leflunomide or methotrexate was analyzed for efficacy and toxicity. RESULTS: Thirty-two patients were treated with leflunomide; fifteen patients received concurrent therapy with methotrexate. The most common indications for therapy were ocular and lung disease. Complete or partial response to leflunomide was seen in 12 of 17 treated with leflunomide alone, and 13 of 15 treated with leflunomide plus methotrexate. There was no difference in the response rate for eye [23/28 (82%)] versus pulmonary disease [12/16 (75%)]. Seventeen patients were treated with leflunomide alone because of methotrexate toxicity (nausea in 12 and pulmonary symptoms in five). All but two tolerated leflunomide. Three patients experienced nausea leading to drug discontinuation, but no other serious adverse reaction was encountered with leflunomide. CONCLUSION: Leflunomide was well tolerated in patients with chronic sarcoidosis. It appears to be as effective as methotrexate, with less toxicity. It should be considered as an alternative in chronic sarcoidosis patients who cannot tolerate methotrexate.

Adult↗
PubMed1999

Leflunomide, a novel immunomodulator for the treatment of active rheumatoid arthritis.

Rheumatoid arthritis (RA) is a chronic disease affecting 0.8% of the population. Nonsteroidal anti-inflammatory drugs reduce the pain and inflammation of RA and improve mobility but do not slow the progression of joint damage. Disease-modifying antirheumatic drugs (DMARDs), which limit potentially irreversible joint damage, may influence the course of disease progression. This review describes the recently approved DMARD leflunomide, an isoxazole-based immunomodulator. Unlike other DMARDs, leflunomide arrests the growth of activated lymphocytes by inhibiting the enzyme dihydroorotate dehydrogenase, a critical link in the production of uridine monophosphate. Leflunomide is rapidly metabolized to the active major metabolite A77 1726, which is responsible for the drug's pharmacologic activity. Leflunomide has exerted inhibitory activity in animal models of RA. Its clinical efficacy has been demonstrated in a number of controlled trials. In two multinational 52-week studies and two 24-week studies, all leflunomide-treated patients received an initial loading dose of 100 mg for 3 days, followed by 20 mg/d. The effects on the signs and symptoms of RA were evaluated using the American College of Rheumatology (ACR) 20 responder index, tender and swollen joint counts and scores, patients' and physician's global assessments, and pain intensity index. Erosions and joint-space narrowing were assessed by radiography. Compared with placebo, leflunomide significantly improved the signs and symptoms of RA (41%-64% improvement) by ACR 20 responder criteria (P < 0.001). Leflunomide, methotrexate, and sulfasalazine were equally effective in terms of symptom outcomes. In terms of retarding the progression of disease, leflunomide was significantly superior to placebo, with no consistent difference from methotrexate or sulfasalazine. In a trial using a combination of leflunomide and methotrexate therapy, 53% of patients were responders by ACR 20 criteria. Adverse effects in RA patients receiving leflunomide included diarrhea, elevated liver enzymes, alopecia, and rash. Additional adverse events occurring with a frequency >5% included allergic reaction, asthenia, abdominal pain, back pain, and hypertension, among others. Thus leflunomide may be used in selected RA patients (ie, those starting RA therapy for the first time or failing earlier DMARD therapy). However, the product labeling requires monthly monitoring of liver enzymes until stable concentrations are reached. Other labeled warnings include a risk of immunosuppression and an increased risk of fetal death or teratogenic effects in pregnant women. Methotrexate, which is also hepatotoxic, is usually the initial DMARD recommended for use in patients with aggressive RA.

Adjuvants, Immunologic↗
PubMed1995

Inhibition of protein tyrosine phosphorylation in T cells by a novel immunosuppressive agent, leflunomide.

Leflunomide, a novel immunosuppressive drug, is able to prevent and reverse allograft and xenograft rejection in rodents, dogs, and monkeys. It is also effective in the treatment of several rodent models of arthritis and autoimmune disease. 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.

Animals↗
PubMed2001

Suppression of experimental autoimmune neuritis by leflunomide.

Leflunomide is a new immunosuppressive drug whose active metabolite, A77 1726, impairs cellular nucleotide metabolism by inhibiting the dihydroorotate dehydrogenase (DHODH), a rate-limiting enzyme of de novo pyrimidine synthesis. Furthermore, A77 1726 suppresses tyrosine kinases involved in signal transduction pathways. We investigated the immunosuppressive effects of leflunomide in experimental autoimmune neuritis (EAN) in rats, which is a model of immune-mediated neuropathies. In EAN that was actively induced by subcutaneous injection of peripheral nerve myelin, leflunomide completely prevented paraparesis if applied orally from the day of immunization. Leflunomide was much more effective than azathioprine, which did not mitigate EAN at all. Even when leflunomide was administered therapeutically after the appearance of the first neuropathical signs, it halted the progression and markedly reduced the severity and duration of EAN. Inflammatory infiltrates, demyelination and axonal degeneration in sciatic nerve sections of leflunomide-treated EAN rats were strongly reduced. Leflunomide-treated rats did not mount autoantibodies as specified by ELISA (enzyme-linked immunosorbent assay) with a mixture of peripheral myelin proteins, including P2 and myelin basic protein. In EAN that was adoptively transferred by injection of neuritogenic cells of a P2-specific T-helper line, application of leflunomide also clearly reduced signs of disease. Additional injection of uridine did not neutralize the effect of leflunomide. Similarly, transfer of neuritogenic P2-specific T cells, which were activated in the presence of A77 1726 plus uridine in vitro, still resulted in reduced severity of adoptive transfer EAN in vivo, although proliferation of these cells in vitro was identical to that of control cells. The T-cell receptor-mediated in vitro activatability of a P2-specific T-cell hybridoma was diminished by high concentrations of A77 1726, as evidenced by reduced Ca(2+) flux into the cytosol. Together with the findings in adoptive transfer EAN, this indicates that the antiproliferative effect is probably not the only mechanism of immunosuppressive action by leflunomide. In summary, leflunomide suppresses EAN efficiently and may constitute a promising therapy for immune-mediated neuropathies.

Adoptive Transfer↗
PubMed1996

Regulation of B cell function by the immunosuppressive agent leflunomide.

Leflunomide is an immunosuppressive drug capable of inhibiting cellular and humoral mediated responses in vivo. The mechanism responsible for suppression of B cell antibody responses in vivo has not been identified. In this study we demonstrate that leflunomide functions to inhibit murine B cell antibody production by directly acting on the B cell. Experiments performed in vivo showed that both T cell-dependent as well as T cell-independent antigen responses were suppressed by leflunomide. Initial in vitro experiments demonstrated that leflunomide inhibited B cell antibody production by decreasing B cell proliferation. The suppression of B cell proliferation induced by a variety of stimuli that use different signal cascade components suggested that leflunomide was acting on a common component required for B cell proliferation. Kinetic studies with LPS activated B cells revealed that leflunomide retained its inhibitory activity when added as late as 24 hr after stimulation in an 88-hr assay. By analyzing the cell cycle of LPS-stimulated B cells we observed that leflunomide targets two different stages in cell cycle transition: (1) from G1 to S phase and (2) from S phase to G2/M phase. Analysis of one of the cyclin-dependent kinases, Cdk2 protein, by Western blot revealed that Cdk2 levels were decreased, in the presence of leflunomide, 48 hr after stimulation. These data further confirmed that leflunomide inhibited B cell progression through the S phase. We also present evidence that the addition of exogenous uridine reversed the antiproliferative activity of leflunomide. This indicated that leflunomide acted as a pyrimidine synthesis inhibitor, thereby inhibiting B cell proliferation and cell cycle progression.

Animals↗
PubMedDate not supplied

Leflunomide prolongs pulmonary allograft and xenograft survival.

BACKGROUND: Leflunomide, an isoxazole derivative, has been shown to effectively prolong rodent allograft and cardiac xenograft survival. In vitro studies suggest that leflunomide inhibits the production of donor-specific antibodies and is capable of blocking both T- and B-cell proliferation. In light of the significant role that humoral immunity is believed to play in chronic pulmonary allograft rejection as well as hyperacute and accelerated acute xenograft rejection, we examined the efficacy of leflunomide in prolonging pulmonary allografts and xenografts and its effect on donor-specific antibody production. METHODS: Lungs from Brown Norway rats or Golden Syrian hamsters were orthotopically transplanted into Lewis rat recipients. Allograft recipients were treated daily for 14 days with vehicle, leflunomide (15 mg/kg/day orally), or cyclosporine (7.5 mg/kg/day orally) starting on the day of grafting (day 0). In xenograft recipients, leflunomide (20 mg/kg/day orally) or cyclosporine (7.5 mg/kg/day orally) treatment initiated on day 0 was continued until complete graft rejection; the leflunomide dosage was reduced to 10 mg/kg/day after day 14 because of weight loss and leukopenia. Graft viability was assessed with chest radiography in conjunction with open lung biopsies. Toxicity was monitored with body weight measurements, complete blood counts, and serum chemistries. Flow cytometric analysis of serum samples taken from graft recipients on day 7 was used to measure donor-specific immunoglobulin M and immunoglobulin G antibody titers. RESULTS: Allograft and xenograft control animals receiving vehicle yielded graft survival times of 6.0 +/- 0.0 and 5.4 +/- 0.6 days, respectively. Although xenograft recipients treated with cyclosporine (7.5 mg/kg/day orally) showed no significant graft prolongation, pulmonary allograft survival in recipients receiving cyclosporine alone was significantly prolonged to 28.2 +/- 0.7 days. Leflunomide-treated allograft (15 mg/kg/day orally) and xenograft (20 mg/kg/day orally) recipients displayed significant graft prolongation to 28.2 +/- 0.7 days and 15.8 +/- 3.3 days, respectively. Cyclosporine (7.5 mg/kg/day orally) enhanced the effect of leflunomide (20 mg/kg/day orally) in xenograft recipients with a mean graft survival time of 36.0 +/- 3.0 days achieved when both drugs were administered concomitantly. Cyclosporine significantly suppressed donor-specific immunoglobulin G antibody titers in both pulmonary allograft and xenograft recipients while not affecting immunoglobulin M levels. Leflunomide markedly suppressed both immunoglobulin G and immunoglobulin M donor-specific antibody titers in allograft and xenograft recipients. Except for mild leukopenia and anemia, both cyclosporine- and leflunomide-treated allograft recipients showed no evidence of toxic side effects after 14 days of therapy. However, leflunomide-treated xenograft recipients displayed significant weight loss, anemia, and leukopenia after 14 days of treatment with one death in each treatment group.

Administration, Oral↗
PubMed1998

Inhibition of JAK3 and STAT6 tyrosine phosphorylation by the immunosuppressive drug leflunomide leads to a block in IgG1 production.

Leflunomide is an immunosuppressive drug capable of inhibiting T and B cell responses in vivo. A number of studies demonstrate that leflunomide functions both as a pyrimidine synthesis inhibitor and as a tyrosine kinase inhibitor. We previously reported that leflunomide inhibits LPS-stimulated B cell proliferation, cell cycle progression, and IgM secretion. This inhibition can be reversed by the addition of exogenous uridine, suggesting that leflunomide functions as a pyrimidine synthesis inhibitor in B cells. We report here that while the addition of uridine restored proliferation and IgM secretion to leflunomide-treated LPS-stimulated B cells, as determined by metabolic labeling and immunoprecipitation, it did not completely restore secretion of IgG Ab. We hypothesized that leflunomide inhibits LPS-induced IgG secretion by inhibiting tyrosine kinase activity required for isotype switch. We tested this hypothesis in a well-defined model of isotype switch, LPS plus IL-4 induction of IgG1. Leflunomide inhibited IgG1 secretion in this model in a dose-dependent manner. The signal transduction pathway utilized by IL-4 to induce IgG1 involves tyrosine phosphorylation of the IL-4 receptor, JAK1, JAK3, and STAT6 proteins induced by IL-4 binding to the IL-4R. Leflunomide diminished the tyrosine phosphorylation of JAK3 and STAT6 in the absence or presence of uridine. In gel mobility shift studies, STAT6 binding to the STAT6 DNA binding site in the IgG1 promoter decreased in the presence of leflunomide or leflunomide plus uridine. Taken together, these data suggest that leflunomide acts as a tyrosine kinase inhibitor to block IgG1 production.

Animals↗
PubMed1999

In vitro and in vivo antitumor activity of a novel immunomodulatory drug, leflunomide: mechanisms of action.

Leflunomide, a novel immunomodulatory drug, has two biochemical activities: inhibition of tyrosine phosphorylation and inhibition of pyrimidine nucleotide synthesis. In the present study, we first showed that A77 1726 [N-(4-trifluoromethylphenyl-2-cyano-3-hydroxycrotoamide)], the active metabolite of leflunomide, was more effective at inhibiting the tyrosine kinase activity of platelet-derived growth factor (PDGF) receptor than that of epidermal growth factor (EGF) receptor, and had no effect on the tyrosine kinase activity of the fibroblast growth factor receptor. In the presence of exogenous uridine, A77 1726 was more effective at inhibiting the PDGF-stimulated proliferation of PDGF receptor-overexpressing C6 glioma than the EGF-stimulated proliferation of EGF receptor-overexpressing A431 cells. In vivo studies demonstrated that leflunomide treatment strongly inhibited the growth of the C6 glioma but had only a modest effect on the growth of the A431 tumor. Uridine co-administered with leflunomide did not reverse the antitumor activity of leflunomide on C6 and A431 tumors significantly. Quantitation of nucleotide levels in the tumor tissue revealed that leflunomide treatment significantly reduced pyrimidine nucleotide levels in the fast-growing C6 glioma but had no effect on the relatively slow-growing A431 tumor. Whereas uridine co-administration normalized pyrimidine nucleotide levels, it had minimal effects on the antitumor activity of leflunomide in both tumor models. Immunohistochemical analysis revealed that leflunomide treatment significantly reduced the number of proliferating cell nuclear antigen-positive cells in C6 glioma, and that uridine only partially reversed this inhibition. These results collectively suggest that the in vivo antitumor effect of leflunomide is largely independent of its inhibitory effect on pyrimidine nucleotide synthesis. The possibility that leflunomide exerts its antitumor activity by inhibition of tyrosine phosphorylation or by a yet unidentified mode of action is discussed.

3T3 Cells↗
PubMed1998

The effects of leflunomide and cyclosporin A on rejection of cardiac allografts in the rat.

Leflunomide is a new low molecular weight immunosuppressive drug which inhibits the enzymes dehydroorotate-dehydrogenase and protein tyrosine kinase, both of which are important components in the immune response. As the mechanisms of action of leflunomide and cyclosporin A (CsA) are different, we postulated a synergistic effect of the two drugs and tested graft survival following leflunomide administration alone or in combination with CsA in a rat cardiac transplantation model. Low- and high-responder rat strain combinations were used in parallel and the experiments were performed both with and without challenge with Linomide, an immunomodulator which promotes graft rejection in this model. In the low-responder rat strain combination (Piebald Virol Glaxo graft to Dark Agouti recipient; PVG to DA), graft survival appeared to be a dichotomous variable, being characterized by tolerance or early rejection. Leflunomide (10 or 5 mg/kg) given for 10 days induced tolerance and CsA did likewise; the addition of Linomide abolished the immunosuppressive effect of leflunomide but not that of CsA. In the high-responder combination (DA to PVG), no tolerance was seen and graft survival was moderately prolonged both after leflunomide and after CsA treatment; the addition of Linomide to CsA or to leflunomide (5 mg/kg) abolished the immunosuppressive effect of the drugs. However, when CsA-Linomide or leflunomide-Linomide were supplemented with the second immunosuppressive drug, leflunomide or CsA respectively, graft survival was significantly prolonged (P < 0.001 in both cases). This suggests leflunomide and CsA have additive potential.

Animals↗
PubMed1993

Leflunomide, a novel immunosuppressive agent. The mechanism of inhibition of T cell proliferation.

Leflunomide is a novel immunomodulating drug that has recently been demonstrated to prevent acute rejection and reverse ongoing rejection of kidney and cardiac allografts in rats. In vitro studies here demonstrate that leflunomide suppresses proliferation of human PBL stimulated with (1) allogeneic PBL in a one-way MLR (50% inhibition with 50-25 microM); (2) anti-CD3 mABs plus PMA (50% inhibition with 70 microM leflunomide); and (3) anti-CD28 mABs plus PMA (50% inhibition with 65 microM leflunomide). In contrast, CsA only inhibited T cell proliferation stimulated by anti-CD3 plus PMA. Leflunomide partially inhibited IL-2 production of T cells stimulated with anti-CD3 plus PMA or anti-CD28 plus PMA, whereas CsA completely inhibited IL-2 production by T cells stimulated by the CD3 pathway and only partially inhibited IL-2 production by T cells stimulated by the CD28 pathway. Because comparable levels of IL-2 were produced by CD28-stimulated T cells treated with either CsA or leflunomide, but no inhibition of proliferation was observed in the CsA-treated cultures, we hypothesized that the lowering of IL-2 levels was not the mechanism by which leflunomide inhibited T cell proliferation. This hypothesis was supported by the observations that exogenous IL-2 failed to restore the T cell proliferation in the presence of leflunomide. Loss of T cell responsiveness to IL-2 in the presence of leflunomide was not due loss of expression of IL-2 receptors. Collectively, our data suggest that inhibition of T cell proliferation by leflunomide occurs via inhibition of responsiveness to IL-2.

Antigens, CD↗
PubMed2004

Leflunomide in active rheumatoid arthritis: a prospective study in daily practice.

AIMS: We prospectively studied the efficacy, incidence of adverse drug reactions and withdrawal from leflunomide in an outpatient population with rheumatoid arthritis in a setting of care-as-usual. METHODS: In this prospective case series study, from outpatient medical records a standard dataset was collected including patient and disease characteristics, data on leflunomide use and adverse drug reactions. RESULTS: During the study period 136 rheumatoid arthritis patients started leflunomide. Median (range) follow-up duration was 317 (11-911) days. Sixty-five percent of patients experienced at least one adverse drug reaction related to leflunomide. During follow-up 76 patients (56%) withdrew from leflunomide treatment, mainly because of adverse drug reactions (29%) or lack of efficacy (13%). The overall incidence density for withdrawal from leflunomide was 56.2 per 100 patient-years. Complete data for calculating efficacy using a validated disease activity score on 28 joints (DAS(28)) was available for 48, 36, and 35% of patients at 2, 6, and 12 months follow-up, respectively. Within a 12-month period after start of leflunomide treatment 76% of the evaluable patients were classified as moderate or good responders according to the DAS(28) response criteria. CONCLUSIONS: In the setting of care-as-usual, rheumatoid arthritis patients starting leflunomide frequently experienced adverse drug reactions. More than half of the patients withdrew from leflunomide treatment within a year after start of leflunomide treatment, mainly because of adverse drug reactions.

Adult↗
PubMed2004

Leflunomide in active rheumatoid arthritis: a prospective study in daily practice.

AIMS: We prospectively studied the efficacy, incidence of adverse drug reactions and withdrawal from leflunomide in an outpatient population with rheumatoid arthritis in a setting of care-as-usual. METHODS: In this prospective case series study, a standard dataset was collected from outpatient medical records, including patient and disease characteristics, data on leflunomide use and adverse drug reactions. RESULTS: During the study period 136 rheumatoid arthritis patients started leflunomide. Median (range) follow-up duration was 317 (11-911) days. Sixty-five percent of patients experienced at least one adverse drug reaction related to leflunomide. During follow-up 76 patients (56%) withdrew from leflunomide treatment, mainly because of adverse drug reactions (29%) or lack of efficacy (13%). The overall incidence density for withdrawal from leflunomide was 56.2 per 100 patient years. Complete data for calculating efficacy using a validated disease activity score on 28 joints (DAS(28)) was available for 48, 36, and 35% of patients at 2, 6, and 12 months follow-up, respectively. Within a 12-month period after start of leflunomide treatment 76% of the evaluable patients were classified as moderate or good responders according to the DAS(28) response criteria. CONCLUSIONS: In the setting of care-as-usual rheumatoid arthritis patients starting leflunomide frequently experienced adverse drug reactions. More than half of the patients withdrew from leflunomide treatment within 1 year of starting leflunomide treatment, mainly because of adverse drug reactions.

Adult↗