Active Metabolite Of Leflunomide Research Articles

Inhibition of HIV Replication by A77 1726, the Active Metabolite of Leflunomide, in Combination With Pyrimidine Nucleoside Reverse Transcriptase Inhibitors

Inhibition of HIV Replication by A77 1726, the Active Metabolite of Leflunomide, in Combination With Pyrimidine Nucleoside Reverse Transcriptase Inhibitors

Flow cytometric quantitation of calcium-dependent and -independent mitogen-stimulation of T cell functions in whole blood: inhibition by immunosuppressive drugs in vitro

We have optimized assays to measure mitogen-stimulated rat lymphocyte activation in whole blood and have used these assays to quantitate the potencies of immunosuppressive drugs with different mechanisms of action. To define the optimal conditions for measuring T cell functions in whole blood, the effects of different concentrations of mitogens that activate T cells through calcium-dependent and -independent pathways were measured over time. Proliferation was measured by tritium-labeled thymidine ([ 3H]-TdR) incorporation and by flow cytometric analysis of proliferating cell nuclear antigen (PCNA)/DNA content. Furthermore, we detected the increases in percent expression of cell-surface activation antigens (CD25, CD134, CD71, CD11a and CD54). Concanavalin A (Con A) stimulated maximum lymphocyte proliferation and expression of T cell surface activations by 72–96 h, which was 48 h later than stimulation by phorbol 12-myristate 13-acetate (PMA) plus anti-CD28 monoclonal antibody (mAb) or PMA plus ionomycin (IONO). Addition of sirolimus, tacrolimus, cyclosporine or the active metabolite of leflunomide, A77 1726, to mitogen-stimulated whole blood produced drug concentration-dependent inhibitions of lymphocyte proliferation and expression of cell surface activation antigen expression. From these data, we determined drug potencies (inhibitory concentration of 50%, IC 50) and drug concentrations causing maximum inhibition of T cell functions ( I max). We developed simple and reproducible assays to measure different lymphocyte functions in whole blood cultures. These assays were used to investigate the mechanisms of different immunosuppressive drugs. These methods can be exploited to measure T cell functions in blood collected from subjects treated with immunosuppressants in vivo.

Leflunomide induces apoptosis of thymocytes and T-cell hybridoma: differences in sensitivity and signaling pathways

LEFLUNOMIDE is a novel antiphlogistic and immunomodulating agent that has been shown to be efficacious in preventing and healing autoimmune disorders and reactions leading to organ transplantation rejection. However, the mechanisms of its action are not sufficiently elucidated. It has been postulated that the immunomodulatory effect of the drug is probably a consequence of reversible inhibition of T-cell proliferation by blocking the early stage of T-cell activation and preventing the expression of interleukin 2 receptor (IL-2R). The effect is partly dependent on inhibition of certain protein tyrosine kinases (PTK). In this work we show evidence that A77 1726, an active metabolite of leflunomide, induces apoptosis of rat thymocytes and rat thymocyte hybridoma in vitro. Experiments were done on thymocytes of AO rats (9 weeks old) and BWRT8 hybridoma cells. The following chemicals were obtained from ICN (Costa Mesa, Calif, USA); RPMI 1640 medium; fetal calf serum (FCS); Z-PheAla-CH2F, an IL-1b converting enzyme (ICE)-like inhibitor; Z-Val-Ala-Asp(Ome)-CH2F, a caspase inhibitor; and dexamethasone. Anti-rat abT-cell receptor (TCR) monoclonal antibody (mAb) was purchased from Serotec (Oxford, UK). Merocyanine (MC) 540, propidium iodide (PI), phorbol myristate acetate (PMA), genistein, and herbimycin A were obtained from Sigma (Munich, Germany). Cyclosporin A was obtained from Novartis (Basel, Switzerland), whereas A77 1726 was obtained from Hoechst, AG (Wiesbaden, Germany). Apoptosis induced by A77 1726, genistein, herbimycin A, dexamethasone, or immobilized R73 mAb was measured after cultivation of the cells for 20 hours (RPMI 1 10% FCS) using morphological criteria, MC 540, and PI staining. The percentage of specific apoptosis was determined as previously described. We found that A77 1726 induced apoptosis of both thymocytes and BWRT8 hybridoma cells, as judged by staining with MC 540 (Fig 1A), morphological analysis of nuclei (chromatin condensation, pyknosis, and kariorexis) (Fig 1B), and staining with PI (Fig 1C). All the methods gave comparable results. As shown in Fig 1A and 1B, A77 1726 at concentrations between 10 mmol/L and 100 mmol/L induced apoptosis of thymocytes in a dose-dependent manner. Approximately 10 times lower concentrations of the metabolite were necessary for apoptosis induction in BWRT8 cells. This is the first report showing direct proapoptotic effect of leflunomide on thymocytes and T-cell hybridoma. Peripheral T cells are more resistant, but the compound significantly modulates activation-induced death of these cells (Colic et al, (manuscript in preparation). It is well known that glucocorticoids and TCR crosslinking promote apoptosis of thymocytes. A similar phenomenon was published for BWRT8 cells. Therefore, in the next experiments we studied the interference of A77 1726 with dexamethasone and an mAb to rat abTCR (R73) immobilized to plastic in inducing apoptosis. As shown in Fig 1, the leflunomide metabolite increased apoptosis both of thymocytes and BWRT8 cells triggered by these agents. The finding may be relevant for selection processes in the thymus because the glucocorticoid-mediated apoptosis is supposed to be a model of “death by neglect,” whereas TCR cross-linking mimics the processes of negative selection. When we studied the effect of PMA, a PKC stimulator, and CsA, an inhibitor of calcineurin, the agents known to modulate apoptosis of thymocytes, it was found that PMA potentiated apoptosis of thymocytes but significantly inhibited apoptosis of BWRT8 cells. In contrast, CsA slightly inhibited the hybridoma cell death, but did not modulate apoptosis of thymocytes, either alone or in combination with A77 1726 (Fig 1C). PMA has been shown to either promote or inhibit apoptosis of T cells. The effect probably depends on whether this compound activates and/or subsequently depletes PKC and also on the type of cells or their activation state. In our experiments, a shortterm preincubation of the cells with PMA probably reflects activation of PKC, and the differences between the two cell types might be related to the differences in their activation state and intracellular signaling pathways. This is in agreement with the requirements for calcineurin, as shown using CsA. CsA has been found to prevent the activation-induced cell death (AICD) by inhibiting the Fas-L expression. However, it is not efficacious in modulating apoptosis of

Multiple inhibitor analysis of the brequinar and leflunomide binding sites on human dihydroorotate dehydrogenase.

Brequinar and the active metabolite of leflunomide, A77 1726, have been clearly shown to inhibit human dihydroorotate dehydrogenase (DHODH), but conflicting mechanisms for their inhibition have been reported. DHODH catalyses the conversion of dihydroorotate (DHO) to orotate concurrent with the reduction of ubiquinone. This study presents data that indicates brequinar is a competitive inhibitor versus ubiquinone; A77 1726 is noncompetitive versus ubiquinone and both are uncompetitive versus DHO. 2-Phenyl 5-quinolinecarboxylic acid (PQC), the core moiety of brequinar also shows competitive inhibition versus ubiquinone. Multiple inhibition experiments indicate that PQC (and thus brequinar) and A77 1726 have overlapping binding sites. Both PQC and A77 1726 are also mutually exclusive with barbituric acid (a competitive inhibitor versus DHO). In addition, we failed to observe brequinar binding to E.orotate by isothermal titration calorimetry (ITC). These results indicate that the E.DHO.inhibitor and E.orotate.inhibitor ternary complexes do not form. The absence of these complexes is consistent with the two-site ping-pong mechanism reported for DHODH. This kinetic data suggests that recent crystal structures of human DHODH complexed with orotate and A77 1726 or brequinar may not represent the relevant physiological binding sites for these inhibitors [Liu, S., Neidhardt, E. A., Grossman, T. H., Ocain, T., and Clardy J. (2000) Structure 8, 25-33].

Inhibition of herpes simplex virus type 1 by the experimental immunosuppressive agent leflunomide.

Despite advances in antiviral chemotherapy, herpes simplex virus type 1 (HSV-1), continues to complicate the clinical course of many allograft recipients. We have previously demonstrated that the experimental immunosuppressive agent leflunomide inhibits production of cytomegalovirus by interference with virion assembly. We test the hypothesis that this agent exerts similar antiviral activity against HSV-1. Plaque assay of virus yield from endothelial or Vero cells after inoculation with each of four clinical HSV-1 isolates demonstrated a dose-dependent reduction of virus production in the presence of pharmacologic concentrations of A77 1726, the active metabolite of leflunomide. DNA dot blot and biochemical assay of viral DNA polymerase activity indicated that A77 does not inhibit viral DNA synthesis. Rather, as visualized by transmission electron microscopic method, this agent seems to disrupt virion assembly by preventing nucleocapsid tegumentation. These findings, in demonstrating that leflunomide exerts antiviral activity against HSV-1 by mechanisms similar to those we have previously shown with cytomegalovirus, imply that this agent may possess broad spectrum activity against other herpesviruses.

Cell-Specific Inhibition of Inducible Nitric Oxide Synthase Activation by Leflunomide

The influence of a novel immunomodulating drug, leflunomide, on iNOS-dependent nitric oxide (NO) production in rodent macrophages and fibroblasts was investigated. Leflunomide's active metabolite A77 1726 caused a dose-dependent decrease of NO production in IFN-γ-treated L929 fibroblasts. The observed effect was cell-specific, as well as stimulus-specific, since A77 1726 did not affect NO production in IFN-γ-stimulated murine peritoneal macrophages or db-cAMP-treated L929 cells. A77 1726 reduced expression of IFN-γ-induced iNOS and IRF-1 mRNA in L929 cells, while iNOS enzymatic activity remained unchanged. Specific inhibitor of MAP kinase kinase (MEK), PD98059, but not unselective protein kinase inhibitor genistein, completely mimicked cell-type-specific and stimulus-specific NO-inhibitory action of leflunomide. Therefore, the recently described inhibition of MEK/MAP pathway by leflunomide could present a possible mechanism for its suppression of iNOS activation in L929 fibroblasts. Finally, a similar inhibitory effect of A77 1726 on both NO production and iNOS mRNA expression was observed also in IFN-γ + LPS-activated murine and rat primary fibroblasts.

Identification and characterization of potential new therapeutic targets in inflammatory and autoimmune diseases.

The isoxazole derivative Leflunomide (HWA 486) is a novel immunoregulatory and anti-inflammatory drug. Affinity chromatography was used to purify and identify Leflunomide binding proteins, which might play a role as potential cellular targets in the molecular mode of action. The Leflunomide derivative A 0273 was covalently coupled to a Fractogel(R) matrix. This column was used to separate a cytosolic protein extract of the macrophage cell line RAW 264.7 by several selected and specific gradient elution steps. Proteins that were specifically eluted through the active metabolite of Leflunomide, A 1726, were identified by subsequent protein sequence analysis. This allowed us to specify 10 cytosolic proteins, which bind with high affinity to this matrix. Three of them, glyceraldehyde 3-phosphate dehydrogenase, pyruvate kinase and phosphoglycerate mutase belong to the second part of the glycolytic pathway. The binding specificity of these protein/drug interactions was further evaluated using BIAcore(R) analysis. Kd values of glyceraldehyde 3-phosphate dehydrogenase, pyruvate kinase and lactic dehydrogenase were similar to the Kd value of a known Leflunomide target protein, dihydroorotate dehydrogenase. In order to elucidate the features as well as the overall relevance of these results, cytosolic fractions of three additional cell lines MOLT-4, A20.2J, HeLa were compared using the same chromatographic protocol. The elution profiles as well as subsequent Western blot analyses confirmed the data obtained previously for the macrophage cell line RAW 264.7.

Five analogs of the active metabolite of leflunomide

The title compounds, 2-cyano-3-hydroxy-N-(4-bromo-phenyl)but-2-enamide, C 11 H 9 BrN 2 O 2 (LFM-A1), 2-cyano-3-hydroxy-N-(2-fluorophenyl)but-2-enamide, C 11 H 9 FN 2 O 2 (LFM-A7), 2-cyano-3-hydroxy-N-(3-bromophenyl)but-2-enamide, C 11 H 9 BrN 2 O 2 (LFM-A9), 2-cyano-3 -hydroxy-N-(3 -chlorophenyl)but-2-enamide, C 11 H 9 ClN 2 O 2 (LFM-A10), and 2-cyano-3-hydroxy-N-(3-fluorophenyl)but-2-enamide, C 11 H 9 FN 2 O 2 (LFM-A11), are analogs of A77 1726, the active metabolite of the immunosupressive drug leflunomide, which is known to act in part by inhibiting the tyrosine kinase epidermal growth factor receptor (EGFR) [Mattar, Kochhar, Bartlett, Bremer & Finnegan (1993). FEBS Lett. 334, 161-164]. The molecular structures of the title compounds are very similar and they display similar crystal packing and hydrogen-bonding networks. All five molecules are approximately planar; the dihedral angles between the phenyl ring and the plane defined by the N-C-C=C-CH 3 group are 4.8(8)° for LFM-A1, 12.5 (2)° for LFM-A7, 6.2(6)° for LFM-A9, 5.5 (3)° for LFM-A10 and 4.4(3)° for LFM-A11. The intramolecular hydrogen bond between the O atoms observed in all the compounds locks them into a planar conformation and may contribute to a conformation which is favorable for binding the shallow ATP-binding pocket of EGFR.

Mechanism of action for leflunomide in rheumatoid arthritis.

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.

Disease-modifying activity of malononitrilamides, derivatives of leflunomide's active metabolite, on models of rheumatoid arthritis.

Disease-modifying activity of malononitrilamides, derivatives of leflunomide's active metabolite, on models of rheumatoid arthritis.

In vivo lymphoproliferation in the popliteal lymph node (PLN) assay can be inhibited by leflunomide's active metabolite A77 1726.

In vivo lymphoproliferation in the popliteal lymph node (PLN) assay can be inhibited by leflunomide's active metabolite A77 1726.

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.

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

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. 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. 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. 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.

Inhibition of CMV in vitro and in vivo by the experimental immunosuppressive agent leflunomide

Lupus nephritis (LN) is a challenging problem that affects 50% of patients with systemic lupus erythematosus (SLE) without effective therapy. Here, we report that A77 1726, the active metabolite of leflunomide, effectively inhibits development of LN and attenuates the generalized autoimmune features. A77 1726 suppresses the expansion of double negative (DN) T cells, and inhibits T and B cell activation. Intriguingly, A77 1726 treatment significantly increases CD4+Foxp3+ regulatory T cells but suppresses potential “pathogenic” IL-17-producing DN T cells in lymph nodes. In vitro experiment shows that A77 1726 potentiates the conversion of naive CD4+CD25− T cells into CD4+CD25+Foxp3+ inducible regulatory T cells (iTregs) by inhibiting Akt. Taken together, our data indicate that the therapeutic effects of A77 1726 in murine LN are mediated, at least in part, by augmenting iTregs which suppress pathogenic IL-17-producing DN T cells through an Akt-dependent mechanism.

MALONONITRILAMIDES (MNAs) EXERT THEIR IMMUNOSUPPRESSIVE ACTIVITY PRIMARILY BY INHIBITION OF DIHYDROOROTATE DEHYDROGENASE (DHODH).

216 Purpose: MNAs, a new generation of low molecular weight immunosuppressants belong to the derivatives of leflunomide's active metabolite A771726 and have been shown to prevent and reverse acute allograft rejection and effectively control xenograft survival in various rodent transplantation models. In this study, we examined the molecular mechanism of action of the MNAs' immunosuppressive activity. Methods: In vitro, MNAs bind to and inhibit the mitochondrial enzyme DHODH, which is responsible for the conversion of dihydroorotate (DHO) to orotate during the de novo pyrimidine synthesis. The resultant accumulation of DHO levels in cell cultures was determined by a new sensitive and specific chromatographic separation method as described by Milbert et al. (Rheumatol. Eur. 27:109, 1998) . Results: Various MNAs (A77 1726, HMR 1279, HMR 1715 and HR 325) inhibited the proliferation of different cell lines in a dose-dependent manner, and concomitantly reduced the pyrimidine ribonucleotide pools, because this anti-proliferative effect could be reversed by addition of uridine or cytidine to replenish the nucleotides. In parallel experiments, a rapid accumulation of DHO, due to DHODH inhibition could be demonstrated in the cell cultures after incubation with MNAs. These intracellular DHO concentrations correlated with the cell numbers and were time and dose dependent on the used concentrations of MNAs. Using brequinar (BQR), a well known inhibitor of the de novo pyrimidine biosynthesis at the level of DHODH, has shown comparable results, although significant differences between the MNAs and BQR could be demonstrated in "washing out" experiments. Conclusion: MNAs primarily inhibit DHODH, which plays a key role in the de novo pyrimidine synthesis. The resultant lowering of pyrimidine ribonucleotides causes inhibition of further cell cycle progression and inhibited the proliferation of various cell lines. Furthermore, inhibition of DHODH, which is responsible for the conversion of DHO to orotate during the de novo pyrimidine biosynthesis, results in a rapid, time, and dose dependent accumulation of DHO in cell cultures.

Flow cytometric analysis of the molecular mechanisms of immunosuppressive action of the active metabolite of leflunomide and its malononitrilamide analogues in a novel whole blood assay

Malononitrilamides (MNAs) are a new class of immunomodulatory drug highly effective in in vivo models of allo- and xenotransplantation. Knowledge of their effects on immune cells, however, is limited and has been derived solely from investigations using isolated mononuclear cells. This use of purified cells to investigate drug activity is not ideal, so we have combined the analytical power of flow cytometry with our mitogen-driven, whole blood lymphocyte activation and proliferation assays to investigate the in vitro mechanism of action of MNAs. We first show that MNAs (A77 1726, HMR1279, and HMR1715), as well as brequinar (BQR) and cyclosporine (CsA), effectively inhibit cell activation antigen expression and lymphocyte proliferation. We next show that the inhibitory effects of MNAs and BQR, but not CsA, are reversed by the addition of uridine to the culture. These results suggest that inhibition of pyrimidine biosynthesis may be a mechanism by which MNAs suppress both lymphocyte activation and proliferation since these effects were reversed when uridine nucleotide pools were replenished. This novel finding of suppression of activation antigen expression by MNAs in whole blood expands our understanding of the effects of this new class of drug.

Leflunomide

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. 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.

How Does Leflunomide Modulate the Immune Response in Rheumatoid Arthritis?

Leflunomide has recently been approved by the US Food and Drug Administration for the treatment of rheumatoid arthritis. This approval was based on data from double-blind multicentre trials in the US (US 301; leflunomide versus methotrexate versus placebo) and multicentre European trials (leflunomide versus sulfasalazine versus placebo, and leflunomide versus methotrexate versus placebo). In these trials, leflunomide was superior to placebo and similar to methotrexate or sulfasalazine in efficacy and adverse effects. Both methotrexate and leflunomide retarded the rate of radiological progression, entitling them to qualify as disease-modifying agents (DMARDs). Leflunomide is an immunomodulatory drug that may exert its effects by inhibiting the mitochondrial enzyme dihydro-orotate dehydrogenase (DHO-DH), which plays a key role in the de novo synthesis of the pyrimidine ribonucleotide uridine monophosphate (rUMP). The inhibition of human DHO-DH by A77-1726, the active metabolite of leflunomide, occurs at concentrations (approximately 600 nmol/L) that are achieved during treatment of rheumatoid arthritis. We propose that leflunomide prevents the expansion of activated and autoimmune lymphocytes by interfering with cell cycle progression. This is mediated by inadequate production of rUMP and utilises mechanisms involving the sensor protein p53. The relative lack of toxicity of A77-1726 on nonlymphoid cells may be due to the ability of these cells to fulfil their ribonucleotide requirements by use of the salvage pyrimidine pathway, which makes them less dependent on de novo synthesis.

Purification of human dihydro-orotate dehydrogenase and its inhibition by A77 1726, the active metabolite of leflunomide.

Leflunomide is currently in phase-III clinical trials for the treatment of rheumatoid arthritis. In this study, we have focused our efforts on the study of the mechanism of action of the active metabolite of leflunomide, A77 1726, in cells and tissue of human origin. The human high-affinity binding protein for radiolabelled A77 1726 was purified from solubilized U937 membranes by following the binding activity through the purification process and was characterized as the mitochondrial enzyme dihydro-orotate dehydrogenase (DHO-DH). The human and murine enzyme displayed identical pI and molecular mass values on SDS/PAGE (43 kDa), which contrasts notably with previous reports suggesting a molecular mass of 50 kDa for the human enzyme. DHO-DH activity was inhibited by A77 1726 and its analogue HR325 with similar potency in U937 and human spleen membrane preparations. HR325 was found to be anti-proliferative for phytohaemagglutinin-stimulated human peripheral blood mononuclear cells, at the same concentrations that caused accumulation of DHO and depletion of uridine. Supplementation of the cultures with exogenous uridine led to partial abrogation of the anti-proliferative effect. This is in line with our recent demonstration that the anti-proliferative effect in vitro of A77 1726 on lipopolysaccharide-stimulated mouse spleen cells was mediated by DHO-DH inhibition [Williamson, Yea, Robson, Curnock, Gadher, Hambleton, Woodward, Bruneau, Hambleton, Moss et al., (1995) J. Biol. Chem. 270, 22467-22472]. Thus, DHO-DH inhibition by A77 1726 and its analogues is responsible for the anti-proliferative effects in vitro of the compounds on human cells and is likely to be responsible for some of its effects in vivo.

Dose-dependent enhancing and inhibitory effects of A77 1726 (leflunomide) on cytotoxic T lymphocyte induction

Leflunomide is an immunosuppressive prodrug which prevents allograft rejection in several animal model systems and may, therefore, have clinical application in organ transplant recipients. Although cytotoxic T lymphocytes (CTL) are an important component of the allograft rejection response, the effect of leflunomide on CTL development has not been thoroughly explored. In this study we have determined the effect of A77 1726, the active metabolite of leflunomide, on CTL induction in C57BL/6 mouse T cell cultures stimulated with anti-CD3 monoclonal antibody. Conjugate formation with P815 target cells, granzyme B enzymatic activity in CTL lysates, and P815 cytolysis in a 51Cr-release assay were used as determinants of in vitro CTL function. At high concentrations (10–20 μM), A77 1726 strongly inhibited CTL generation. In contrast, a low concentration (0.5 μM) of A77 1726 promoted CTL development. These dose-dependent opposing effects of A77 1726 on CTL induction could not be attributed to alterations in CD8 + lymphocyte percentages, interleukin-2 or CD25 expression, or the ability to conjugate with P815 target cells. However, both interferon- γ and granzyme B expression were significantly decreased when CTL were induced in the presence of 10-20 μM A77 1726, and were slightly, but not always significantly, elevated in the presence of 0.5 μM A77 1726. We conclude that at high concentrations A77 1726 is a potent inhibitor of CTL induction, but a low concentration of A77 1726 enhances CTL development.