Inhibition of murine IgE and immediate cutaneous hypersensitivity responses to ovalbumin by the immunomodulatory agent leflunomide.

Leflunomide has been identified as an immunoregulatory and anti-inflammatory compound. Allergic disease is characterized by elevated serum IgE levels, production of allergen-specific IgE and the release of inflammatory mediators from mast cells and granulocytes. Here we demonstrate, using an in vivo murine model, the ability of leflunomide to down-regulate levels of total and allergen-specific serum IgE production. Mice receiving leflunomide (45 mg/kg) orally at the time of primary immunization with ovalbumin adsorbed to aluminium hydroxide adjuvant, showed a reduction in total serum IgE levels of 95%, 41% and 32% following primary, secondary and tertiary immunizations, respectively (P < 0.05). When leflunomide was administered both at the time of primary and subsequent immunizations, reductions in total and specific serum IgE levels of > 80% and > 38%, respectively, were observed (P < 0.05). Administration of leflunomide to mice which had already developed an IgE response resulted in reductions in total and specific serum IgE levels of > 80% and > 45%, respectively (P < 0.05). Following leflunomide treatment, animals failed to develop immediate cutaneous hypersensitivity responses when challenged intradermally with allergen. Down-regulation of immunoglobulin production was not restricted to IgE, since levels of allergen-specific IgG1 and IgG2a in serum were also reduced. The finding of significant reductions in total and allergen-specific IgM suggests that the mechanism of action does not involve selective inhibition of immunoglobulin class switching. A loss in production of the T helper cell-derived B cell differentiation factor IL-5 may account for the reduction in immunoglobulin levels. In adoptive transfer experiments leflunomide did not induce tolerance in allergen-reactive Th2 populations, contrary to animal disease models of transplantation and autoimmunity, where leflunomide was shown to induce tolerance in the effector T cell population.

The new immunosuppressants, the malononitrilamides MNA 279 and MNA 715, inhibit various graft-vs.-host diseases (GvHD) in rodents.

The use of inbred mouse strains of defined genetic background has allowed for the development of systems capable of reproducibly generating either an acute or chronic graft-versus-host disease (GvHD). The malononitrilamides MNA 279 and MNA 715, analogues of the main metabolite of leflunomide, have been shown to directly inhibit T-cell proliferation and B-cell functions. Therefore, they have been studied in a local GvH reaction in the popliteal lymph node (PLN) assay in LBN rats, on the development of an acute and lethal GvHD in B6C3F1 mice and on a chronic autoimmune GvHD in BDF1 hybrid mice. In the PLN assay an oral administration of various concentrations (7.5 to 50 mg/kg) of both MNAs inhibited the localized GvH reaction dose-dependently and suppressed the lymph node hyperplasia. Both MNAs also acted therapeutically in this assay when they were given as late as day 4 or 5 after challenge. In the model of an acute lethal GvHD the treatment of the GvH-B6C3F1 hybrid mice with the MNAs (2.5 to 20 mg/kg/day) shortly after disease induction on days 3 to 12 resulted in a dose-dependently improved survival rate. With 20 mg/kg of drugs, mortality of this life-threatening GvHD was completely prevented and also other parameters like splenomegaly, erythrocyte counts and hematocrit values were strongly suppressed. Treatment of sensitized GvH-BDF1 hybrid mice in the chronic autoimmune-like model with the MNAs (30 mg/kg/day), given on days 3 to 36 by oral gavage, resulted in an improved survival rate, inhibited lymphadenopathy and splenomegaly, reduced the levels of autoantibodies and other immunoglobulins like IgE and IgG1, prevented proteinuria and the development of glomerulonephritis. Both MNA 279 and MNA 715 can inhibit ongoing aberrant immune responses in animals suffering from GvHD.

Therapeutic activity of malononitrilamides (MNA 279 and MNA 715) on acute and chronic, relapsing, experimental, allergic encephalomyelitis (EAE).

Due to their immunosuppressive mode of action, we examined the therapeutic effects of the malononitrilamides MNA 279 and MNA 715 in acute EAE, and two models of chronic relapsing EAE in Lewis rats and Biozzi mice. In the first model, sensitization of adult Lewis rats with guinea pig spinal cords results in an acute clinical episode of severe EAE, and by day 15 all animals had died. Treatment of these sensitized rats with the MNAs was most effective in delaying and reducing the onset of clinical symptoms, and mortality of acute EAE was prevented in a dose-dependent manner. The protection afforded by the two MNAs was long-lasting and no subsequent relapse was observed. Similarly, in the chronic relapsing disease, aged Lewis rats 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 the MNAs survived the first attack, which also was delayed. Pathological signs were reduced and relapses did not occur. Inhibition of chronic relapsing EAE in aged Lewis rats was observed, even when the MNA-treatment was started after the first appearance of clinical symptoms. All treated animals recovered completely and mortality was prevented. Also in the second model of chronic relapsing EAE in Biozzi AB/H mice, MNA treated animals showed only one acute and delayed episode and no further relapses. All these results qualify both MNA 279 and MNA 715 as powerful immunosuppressants with therapeutic potential in human multiple sclerosis (MS).