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Treating MRL/1pr mice, which spontaneously develop systemic lupus erythematosus and rheumatoid arthritis, with 15-DOS resulted in a decrease in the amount of autoantibodies and inhibited proteinuria of the developing glomerulonephritis with an improved survival rate of these autoimmune mice. 15-DOS treatment also lowered the percentage of animals with swollen lymph nodes and inhibited the development of splenomegaly. In the established disease 15-DOS returned urine-protein values and renal function (serum urea and creatinine) to normal levels. Circulating rheumatoid factor and autoantibodies to double-stranded DNA were reduced and the increase in paw volume (signs of a polyarthritis) was inhibited.

Leflunomide has been shown to be very effective in preventing and curing several autoimmune animal diseases. Further, this agent is as effective as cyclosporin A in preventing the rejection of skin and kidney transplants in rats. Preliminary results from patients suffering from severe cases of rheumatoid arthritis demonstrated that clinical and immunological parameters could be improved with leflunomide therapy. Mode of action studies revealed that this substance antagonizes the proliferation inducing activity of several cytokines and is cytostatic for certain cell types. In this light, we could show that tyrosine phosphorylation of the RR-SRC peptide substrate and the autophosphorylation of the epidermal growth factor (EGF) receptor were, dose dependently, inhibited by leflunomide. EGF activates the intrinsic tyrosine kinase of its receptor, which stimulates the phosphorylation of a variety of peptides, the amino acid residue in all cases is tyrosine. These results indicate that much of leflunomide's activity could be due to the inhibition of tyrosine-kinase(s), which is an important general mechanism for the proliferation of various cell types. Thus, leflunomide, which is effective against autoimmune diseases and reactions leading to graft rejection, would seem to have a mode of action separating it from known immunosuppressive drugs.

The effect of leflunomide (HWA 486) was tested in proteoglycan-induced arthritis in an autoimmune animal model showing many similarities to human rheumatoid arthritis and ankylosing spondylitis. The development of the disease in genetically susceptible BALB/c mice is dependent upon the expression of both cell-mediated and humoral immunity to host mouse cartilage proteoglycan. Arthritic and control (non-arthritic) animals were treated with 35 mg leflunomide/kg body weight/day for 12 weeks to suppress inflammatory events and antibody titers. Leflunomide suppressed acute inflammatory events, protected animals from new inflammatory episodes and acute exacerbations, slightly reduced the stiffness in joints and blocked the degradation of cartilage. The suppressive effect of leflunomide in proteoglycan-induced arthritis is due primarily to the suppression of autoantibody formation.

We examined the therapeutic effect of leflunomide in the two models of acute and chronic relapsing EAE in Lewis rats. In the first model, sensitization of adult rats with guinea pig spinal cord resulted in an acute clinical episode of severe EAE, and by day 15 all animals died. Treatment of these sensitized Lewis rats with leflunomide was most effective in delaying and reducing the onset of clinical symptoms and mortality was prevented. The protection afforded by leflunomide was long-lasting and no subsequent relapse has been observed. In the second model of chronic relapsing EAE, aged Lewis rats (6–8 months old) were immunized with rabbit myelin basic protein, and all untreated animals developed a disease with up to three relapses. The second and third episodes were both milder and shorter in duration than the first. All animals treated with leflunomide survived the first attack, which was also delayed, in comparison to untreated controls, and relapses did not occur. Inhibition of pathological signs and prevention of relapses were observed even when leflunomide treatment was started after the first appearance of clinical symptoms of chronic relapsing EAE.

Leflunomide (HWA 486), an isoxazol derivative, has been shown to be very effective in combating autoimmune diseases and transplantation rejection in a great number of animal models. The main metabolite of leflunomide, A77 1726, is a potent antiproliferative compound. To further elucidate this affect, lymphocytes of healthy human donors were cultured for 24, 48 or 72h in the presence of PHA or immobilized anti-CD3 antibody. A77 1726 was added at concentrations between 10 and 100 μM. Flow cytometric evaluation of early activation or proliferation markers (IL-2 and transferrin receptors, respectively) showed that their expression was inhibited in a dose-dependent manner by A77 1726. Together with previous data, these experiments indicate that leflunomide not only inhibits the expansion of already proliferating lymphocytes, but also impairs PHA and anti-CD3 antibody triggered activation of quiescent cells. Thus, this compound may exert its effects through influencing two important aspects of an immune response, that is, activation and proliferation of lymphocytes.

Leflunomide has been shown to combat effectively autoimmune diseases in a number of animal models, as well as chronic polyarthritis of humans. Here we report on the effects of this novel drug on the adherence of leukocytes to endothelium, an essential event in establishment and maintenance of inflammation. The entry of cells into tissues is dependent on interactions of adhesion molecules. The process of diapedesis, which these molecules control, involves three phases: tethering, triggering of receptors on endothelial cells and firm attachment of leukocytes to these cells. The interaction of LECAM-1 (constitutively expressed on circulating leukocytes) and P- and E-selectins on the vessel wll leads to rolling of leukocytes along the endothelium. Using anin vivo system (rat peritoneum) we found that leflunomide effectively inhibits the rolling of leukocytes along the vessel wall after formyl-methionyl peptide (FMLP) induction. Leflunomide's inhibition of FMLP-induced rolling does not appear to be due to its effects on either LECAM-1 or CD11b, because their modulation was unaltered by this drug. We did, though, find that leflunomide strongly inhibited the FMLP-induced induction of the oxidative burst in these cells. Although we have not yet obtained data on leflunomide's effects on the other phases of diapedesis, our results indicate that this drug has effects on leukocyte activation and adhesion, key events in the initiation and maintenance of chronic inflammation and pathological tissue damage.

Studies were conducted to evaluate the use of a murine SLE-like disease for the discovery of novel drugs: This disease is the result of a chronic form of a graft vs host (GVH) reaction. Using prednisolone (Pr), cyclophosphamide (Cy), indomethacin (Indo), and the isoxazol derivative, HWA 486, we found that only Indo was ineffective in inhibiting the SLE symptoms. Interestingly, HWA 486, which did not display any immunosuppressive activity, restored the suppressed T-cell response to the same level as found in healthy mice. We feel that this murine model of SLE could be of value for discovering substances with novel antirheumatic, and/or immunomodulating activities.

Without abstract

Leflunomide is a novel immunomodulatory drug shown to be very effective in animal models of autoimmune diseases and transplantation rejection, as well as in human rheumatoid arthritis. Leflunomide's main metabolite, A77 1726, has been shown to be reversibly antiproliferativein vitro. Pursuing this, we performed cell cycle analysis by flow cytometry of a B-cell lymphoma line and found that at concentrations >2.5 μM cells accumulated in the early S-phase. In order to determine A77 1726's effects on cell activation, human peripheral blood lymphocytes (PBL) were cultured in the presence of PHA or OKT 3 antibody. Flow cytometric evaluation of IL-2 and transferrin receptor expression exhibited a dose-dependent inhibition of these activation markers (10–100 μM). Further, using the polymerase chain reaction, we investigated the ability of leflunomide to impair the transcription of the IL-2-R gene. We found that A77 1726 did not significantly decrease IL-2-R α-chain mRNA expression regardless of stimulation. It seems that leflunomide's main metabolite did not affect IL-2-R at the level of gene transcription, and thus its effects could be due to impairment of post-translational events. Taken together, these studies demonstrate that leflunomide: (1) impairs activation of quiescent lymphocytes (reduction of IL-2-R expression), without inhibiting IL-2-R mRNA formation; (2) reversibly inhibits proliferation by holding resting lymphocytes in the G0-phase and activated cells in the early S-phase of the cell cycle. Thus, this compound may exert its effects through influencing two important aspects of an immune response, i.e. activation and proliferation of lymphocytes.