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.

Leflunomide (HWA 486), a novel immunomodulating compound for the treatment of autoimmune disorders and reactions leading to transplantation rejection.

A solution of an apparently nonimmunogenic synthetic compound, N,N-dioctadecyl-N',N'-bis(2-hydroxyethyl) propanediamine (CP-20961), suspended in mineral oil or olive oil (50 mg/ml), induced an acute, as well as a chronic, polyarthritis when single intradermal injections (0.2 ml) were made in the tail or hindpaw of Lewis rats. The polyarthritis was morphologically almost indistinguishable from classic adjuvant arthritis induced by Freund's complete adjuvant (FCA), a disease generally thought to be the result of a delayed hyper-sensitivity reaction to a constituent(s) of the injected tubercle bacilli. The disease induced by CP-20961 and that induced by Freund's complete adjuvant followed the same time course and almost identical pattern of development of clinical and histopathologic features. Like the classic adjuvant arthritis, CP-20961 induced arthritis is suppressed by an immunosuppressive agent (cyclophos-phamide) or an antiinflammatory drug (phenylbutazone). The alkyldiamine (CP-20961) was found to be a potent adjuvant; a dispersion or a solution of the compound in mineral oil administered intraperitoneally enhanced the development of both the cell-mediated and the humoral immune responses to EL4 cells in the rat. These findings suggest that the immunogen responsible for the development of adjuvant arthritis is endogenous, e.g., a constituent of host tissue, a viral protein, or some complex of the two.

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Adjuvant polyarthritis. IV. Induction by a synthetic adjuvant: immunologic, histopathologic, and other studies.

Objective. To determine the effects of peptidyl fluoromethyl ketones on the in vitro activity of purified cathepsins B and L, on tissue cysteine proteinase activity, and on cartilage and bone destruction in experimental arthritis.



Methods. The effects of the fluoroketones on cathepsins B and L in vitro and the effects of oral administration of fluoroketones on ex vivo cysteine proteinase activity in tissue homogenates were determined by measuring the inhibition of fluorogenic substrate cleavage. To determine the effects on arthritis, animals were injected with adjuvant or type II collagen, treated orally with the fluoroketones, and the severity of arthritis was assessed by clinical, histologic, and radiologic methods.



Results. All of the fluoroketones tested were potent inhibitors of purified cathepsins B and L activity. Oral administration of the fluoroketones reduced tissue cysteine proteinase activity by up to 77%. In addition, fluoroketone treatment significantly reduced the severity of clinical joint disease and decreased the destruction of articular cartilage and bone. Quantitative analysis of radiographic images indicated that treatment significantly reduced soft tissue changes, periosteal proliferation, and bone erosion, but only partially reduced juxtaarticular osteoporosis.



Conclusion. These studies suggest that cysteine proteinase inhibitors may limit tissue destruction in diseases such as rheumatoid arthritis.

Cysteine Proteinase Inhibitors Decrease Articular Cartilage and Bone Destruction in Chronic Inflammatory Arthritis

Immunopharmacological profile of a novel isoxazol derivative, HWA 486, with potential antirheumatic activity--I. Disease modifying action on adjuvant arthritis of the rat.

Leflunomide is an antiphlogistic and immunomodulating agent that has been shown to be effective in preventing and healing autoimmune disorders and reactions leading to organ graft rejection. From our preliminary clinical data [4], we now have hopes that these effects, observed in experimental animals, can truly be transferred to humans. Although we are far from understanding the mode of action of leflunomide, we are slowly gathering some insight. A good many of the immunosuppressive effects of leflunomide can be attributed to the antagonistic effects it has on responses to many cytokines, most likely through receptor expression and signal transduction (tyrosine kinase inhibition). The inhibition of transplant rejection could be explained by interference with the activity of IL-2 and IL-4, i.e. the interference of cytotoxic T cell formation. Considering, further, that increased IL-3-like activity has been reported in autoimmune MRL/lpr mice [23], and that it is felt that this amplified activity may contribute to the pathology of their illness [23], then the interference of leflunomide with IL-3 may, together with the antagonistic activity of TRF and specifically IL-4, explain some of the disease modifying properties of this drug in animals with SLE-like and other autoimmune diseases. Also, interference with responses to IL-6 (Germann, personal communication) could be responsible for the suppression of acute-phase proteins observed in adjuvant-diseased rats [24]. Our data concerning tyrosine kinase inhibition as a hypothetical mechanism for the non-cytotoxic and reversible antiproliferative activity of A77 1726 are in many ways, intriguing. First of all, many known receptors for growth factors are associated with tyrosine kinase, i.e. EGF [35], IL-2 (the high binding, 75 kDa chain) [21], IL-3 [26], G-CSF, GM-CSF and TNF-α [9]. Leflunomide antagonizes all of these mediators. On the other hand, IL-1, which is not antagonized by leflunomide, is not associated with tyrosine kinase, but with threonine and serine kinase [11]. Much more work must be conducted before we can be sure that tyrosine kinase inhibition is important for the mode of action of leflunomide. Another important aspect of this drug is its inhibitory effect on the release of histamine from basophils and mast cells, because of its role in inflammatory reactions. Relating to our findings on the activity of leflunomide on murine SLE-like disorders, it has been reported recently that SLE patients often exhibit abnormal production of antibodies to IgE, and that these autoantibodies may, by activating mast cells and basophils, play a consequential part in the release of vasoactive amines, thus leading to generalized tissue injury [15]. We are confident that leflunomide will prove to be an effective drug in combating human autoimmune disorders. Indeed, we already have preliminary evidence for this, from studies of its effects on humans suffering from autoimmune diseases. Moreover, this drug provides a tool for gaining new insights into both the mechanisms leading to such ailments and their therapeutic control, and as such may facilitate the discovery of even more proficient drugs or other means to modulate these malfunctioning immune reactions.

Effects of leflunomide on immune responses and models of inflammation

SummaryVarious methods for inducing AA in rats were examined using different rat strains, adjuvant preparations, and sites of injection. The time course of the development of arthritis in the injected and nonin-jected extremity was determined and methods for inducing a disease with limited variability and a high incidence was demonstrated. Standard compounds were tested in two models of AA with special reference to denning a method for discriminating between inhibition due to immunosuppression (IS) and that due to anti-inflammatory (AI) effects. 12 daily doses of IS agents, started on the day of adjuvant injection, totally prevented the disease in the noninjected extremity when measurements were made on day 21. Standard AI agents were ineffective. This test (nonestablished arthritis) was therefore capable of detecting compounds with immunosuppressive potential. The same standard compounds were administered daily for 12 days after the peak of the disease had been reached (established arthritis). In this te...

H. Schroder

Papers

The Use of a Standardized Adjuvant Arthritis Assay to Differentiate Between Anti-inflammatory and Immunosuppressive Agents