Agents and Actions, vol. 32, 1/2 (1991)
`
`0065-4299/91/020010-12 $1.50+0.20/0
`© 1991 Birkhiiuser Verlag, Basel
`
`Leflunonlide (HWA 486), a novel immunomodulating compound
`for the treatment of autoimmune disorders
`and reactions leading to transplantation rejection
`
`, T. Mattar 3
`R.R. Bartlett!, M. Dimitrijevic 2
`, T. Zielinski 3, T. Germann \ E. Riide 3 , G.H. Thoenes 4 ,
`C.C.A. Kiichle 4
`, H.-U. Schorlemmer 5
`, E. Bremer 6
`, A. Finnegan 6 andR. Schleyerbach 1
`
`'Pharmacological Research, Hoechst AG Werk Albert, W-6200 Wiesbaden 12, FRG, 2Immunology Research Center, Belgrade,
`Yugoslavia, 3 Immunology, Johannes Gutenberg University, W-6500 Mainz, FRG, 4Medical Clinic, Ludwig-Maximilians University,
`W-8000 Munich 2, FRG, 5 Behring Research Laboratories, W-3550 Marburg, FRG, 6 Department of Medicine, Rush Medical Center,
`Chicago, Ill, USA
`
`Abstract
`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 pep tides, the amino
`acid residue in all cases is tyrosine. These results indicate that much of leflunomide's activity could be
`due to the inhibition oftyrosine-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 immunosup(cid:173)
`pressive drugs.
`
`Introduction
`Leflunomide (HWA 486) an isoxazol derivative
`with antiphlogistic and novel immunomodulating
`properties, would seem to be a universal drug to
`combat autoimmune disorders [1-10]. Although
`the pharmacological profile of this substance has
`recently been reviewed [1], so much more data has
`
`, Address for correspondence.
`
`been generated that a new review is warranted.
`Here we will briefly cover the already published
`results and present some new and preliminary data
`dealing with leflunomide's effects in animal models
`of autoimmunity and organ transplantation, as
`well as some of our most recent in vitro findings
`concerning the mode of action of its active
`metabolite, A 771726. Further, very preliminary
`clinical data concerning leflunomide's effects on
`the immune response of patients with rheumatoid
`arthritis will be presented.
`
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`Agents and Actions, vol. 32, 1/2 (1991)
`
`o
`H
`g-NH-@-CF.
`~CH' Leflunomide
`
`Autoimmune animal studies
`Leflunomide inhibits the adjuvant disease of rats
`
`Studies dealing with effects of leflunomide on the
`adjuvant arthritic disorder, of Lewis rats, offered
`us the initial clues that leflunomide may have anti(cid:173)
`inflammatory and immunomodulating properties.
`Using the "standardized arthritic assay" described
`by Peper et al. [11] to differentiate between non(cid:173)
`steroidal anti-inflammatory drugs and immuno(cid:173)
`suppressive agents, we found that not only was
`leflunomide able to arrest the development of ad(cid:173)
`juvant arthritis, but, unlike immunosuppressive
`agents that were considered to be exclusively active
`in this assay, it could restore the diminished mito(cid:173)
`gen induced lymphocyte response of the diseased
`animals [2]. Further, although effective in this as(cid:173)
`say, to our surprise, leflunomide did not demon(cid:173)
`strate immunosuppressive activity, at least con(cid:173)
`cerning the ex-vivo response of lymphocytes to
`mitogens in healthy rats [2].
`These results were independently confirmed by
`Pasternak et al. [6], as well as Hambleton and McMa(cid:173)
`hon [8]. Pasternak found further that leflunomide
`significantly reduced edema, fibrinogen levels, and
`erythrocyte sedimentation rates. The antiarthritic
`effects of this agent were more sustained than
`those observed after cyclosporin A (CSA) therapy.
`Whereas both leflunomide and CSA could reduce
`the delayed type hypersensitivity (DTH) response
`to mycobacterial antigen on day 9 followed by a
`rebound to an enhanced DTH response on day 21,
`only leflunomide was able to restore the sup(cid:173)
`pressed mitogenic response of splenocytes to phy(cid:173)
`tohemagglutinin (PHA) , a T-cell mitogen. Ham(cid:173)
`bleton and McMahan confirmed the suppressive
`effects of leflunomide and CSA on the early (day
`10) DTH-reaction, with no effect, on this reaction,
`when tested on day 15. Similar results were ob(cid:173)
`served when these animals were treated with pred-
`
`11
`
`nisolone (PRED), whereas neither indomethacin
`nor tiaprofenic acid influenced the DTH reaction.
`
`Leflunomide arrests murine systemic lupus erythe(cid:173)
`matosus (SLE)-like disease of MRL/lpr-mice
`SLE is an autoimmune disease that affects multiple
`body organs and is characterized by the develop(cid:173)
`ment of certain types of self antigens. Primarily,
`the antibodies formed against double-stranded
`DNA (dsDNA), the most prevalent in this ailment,
`complex together and, with complement, deposit
`in the small blood vessels, leading to widespread
`vasculitis. MRL Mpf Ipr/lpr (MRL/lpr)-mice
`spontaneously develop a severe disease with many
`symptoms very similar to human SLE, i.e. hyper(cid:173)
`gammaglobulinaemia, and glomerulonephritis [3,
`12, 13]. These mice may equally well serve as a
`model for human rheumatoid arthritis, especially
`considering the articular involvement, such as
`swelling of the pawns, pannus formation, prolifer(cid:173)
`ation of synovial tissue, degradation of articular
`cartilage, and the presence of circulating rheuma(cid:173)
`toid factor (RF) [3, 12-14].
`Treatment of MRL/lpr mice with leflunomide dose
`dependently arrested disease progression and pre(cid:173)
`vented the development of glomerulonephritis [3,
`4]. This was due to the suppression of circulating
`immune complexes (Table 1), which was a direct
`result of the greatly lowered autoantibody forma(cid:173)
`tion, such as those to dsDNA or to immunoglobu(cid:173)
`lins (RF) [3,4]. Further, the tremendous number
`of
`lymphocytes which accumulate
`in
`the
`Iymphnodes and spleens of MRL/lpr-mice could
`be greatly suppressed, depressing the amount of
`the double negative T-cells, i.e. T-Iymphocytes
`possessing neither CD4 (T-helper cell phenotype)
`nor CD8 (T -suppressor cell phenotype) differential
`antigens (Table 1). At the same time the ratio of
`CD4/CD8-T-cells, which is greatly increased in
`these mice, was restored to normal values. These
`effects were also observed after treatment of these
`animals with CSA (Table 1). Furthermore, lefluno(cid:173)
`mide therapy could restore not only the suppressed
`proliferative response oflymphocytes to T-cell mi(cid:173)
`togens (PH A and concanavalin A (Con A)), but
`also the depressed activity of macrophages to
`phorbolmyristenacetate (PMA) [4].
`The disease inhiting effects of leflunomide were not
`limited to prophylactic activity [7]. MRL(lpr mice
`that had elevated levels of protein in their urine
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`12
`
`Table 1
`Effects of drugs on MRL/Jpr-disease.
`
`Agents and Actions, vol. 32, 1/2 (1991)
`
`Mouse strain
`
`Drug
`
`Dose
`mg/kg/day
`
`No. double
`neg. T -cells 1
`
`CD4/CD8
`ratio
`
`Proteinuria
`mg/mouse
`
`Circulating immune
`complexes 2
`
`C3H
`MRL/lpr
`MRL/lpr
`MRL/lpr
`
`none
`none
`HWA486
`CSA
`
`0
`0
`35
`100
`
`30
`1000
`100
`50
`
`2.5
`7.0
`2.0
`1.9
`
`<0.1
`1.4
`0.1
`1.7
`
`0.1
`2.0
`0.5
`0.5
`
`Therapy was initiated when the animals were 10 weeks old and terminated when the animals were 22 weeks of age. The data given
`was obtained 6 weeks after the last drug application.
`1 Double negative T-cell are those whithout CD4 or CD8 differential antigens (double negative/lymphnode).
`2 Data is given as OD at a serum titer of 1/3200.
`
`were aministered, for 10 weeks, either leflunomide,
`CSA, or prednisolone (PRED). At the end of the
`medication period, 90% of the animals receiving
`leflunomide were still alive, whereas 50% of the
`non-treated control mice and only 40% of the CSA
`or PRED medicated animals survived. Although
`all of the surviving mice had normal urine-protein
`levels, only the leflunomide medicated rodents had
`significantly reduced levels of RF and autoanti(cid:173)
`bodies to dsDNA. CSA and PRED treatment re(cid:173)
`sulted in amplified titers of autoantibody to
`dsDNA. It would appear that leflunomide is better
`suited to combat the established affliction of
`MRL/lpr mice than either CSA or PRED.
`As to the question about what happens when
`leflunomide therapy is terminated after the ani(cid:173)
`mals have become "healthy", we have published
`results offering a good answer [1]. We treated
`MRL/lpr mice with either 35 mg/kg leflunomide
`or 20 mg/kg azathioprine, starting when the ani(cid:173)
`mals were 10 weeks old. After 9 weeks (19 weeks of
`age), the therapy was discontinued and the disease
`development followed. The progression of the ail(cid:173)
`ment in animals given azothioprine could be
`slowed down, but, even before the therapy was
`terminated, the symptoms of the disorder ad(cid:173)
`vanced to the same level as that of non-treated
`MRL/lpr-mice. Leflunomide therapy, on the other
`hand, not only prevented the appearance of symp(cid:173)
`toms, but 20 weeks after the treatment was ended,
`no signs of the illness could be detected [1], al(cid:173)
`though they did slowly appear somewhat later.
`
`Leflunomide therapy prevents paralysis
`in experimental allergic encephalomyelitis (EAE)
`EAE is a T cell mediated, neurologic autoimmune
`disease that develops in susceptible animals follow-
`
`ing senslllzation with either spinal cord ho(cid:173)
`mogenate, or myelin basic protein [15]. Although
`the induction of EAE is essentially due to cellular
`immune reactions [16], there is i1).creasing evidence
`for an additional role of humoral factors in the
`pathogenesis of this illness [17]. EAE in animals is
`considered to be an appropriate model for multiple
`sclerosis (MS) in man [18]. In Lewis rats, clinical
`EAE is characterized by the development of tran(cid:173)
`sient hindquarter paralysis [1].
`Studying the effects of leflunomide on the preven(cid:173)
`tion of paralysis in an acute form of EAE in Lewis
`rats, we found that this agent was as effective as
`CY [1]. Yet, contrary to the effects of CY, spleno(cid:173)
`cytes from animals treated with leflunomide re(cid:173)
`sponded normally to T and B cell mitogens [1].
`
`Leflunomide prevents organ specific nephritic
`diseases
`Examples of leflunomide's effects on organ(cid:173)
`specific autoimmunity have been very recently re(cid:173)
`ported from two independent laboratories using
`two different animal models of nephritic disorders.
`Thoenes et al. [10] demonstrated that this agent is
`very effective in preventing experimental tubu(cid:173)
`lointerstitial nephritis (TIN) in rats. TIN is in(cid:173)
`duced by immunizing animals with either ho(cid:173)
`mologous or heterologous tubular basement mem(cid:173)
`branes (TBM) in Freund's complete adjuvants. In
`rats, TIN commences at about 10 days after TBM
`(from sheep) stimulation, leading to serious dam(cid:173)
`age to the kidney cortex and decreased kidney
`function [19]. In the above mentioned study [10], it
`was found that leflunomide was just as effective as
`CSA, but more efficacious than PRED, naproxen,
`or indomethacin in preventing disease develop(cid:173)
`ment. Regarding the inhibition of autoantibody
`
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`Agents and Actions, vol. 32, 1/2 (1991)
`
`formation to TIN, leflunomide was much more
`effective than the other drugs tested.
`Using an antibasement membrane antibody in(cid:173)
`duced glomerulonephritis in rats, Ogawa et al. [9]
`could show that an oral dose of 2 mgjkgjd lefluno(cid:173)
`midc resulted in significant decrease in total uri(cid:173)
`nary protein, plasma cholesterol and fibrogen, as
`well as decreased incidences of fibrin, IgG and C 3
`deposits. This was the case for both preventive
`(two days before disease induction and ending on
`day 20) as well as curative (five days after induc(cid:173)
`tion and ending on day 20) drug therapy.
`
`Effects of leflunomide on inflammatory
`and allergic reactions
`Concerning antiallergic activity, we have observed
`that leflunomide effectively inhibits the edema for(cid:173)
`mation in the skin of guinea pigs sensitized with
`specific IgE (passive cutaneous anaphylaxis test)
`[1]. Further, this drug was as effective as phenylbu(cid:173)
`tazone in inhibiting the inflammatory reaction in(cid:173)
`duced by carrageenan [1].
`
`Leflunomide suppresses reactions leading
`to organ transplantation rejection
`and graft versus host diseases
`
`Effects of leflunomide on mice undergoing
`a chronic graft-versus-host (CGVH) reaction
`
`The intravenous injection of a mixture of parental
`splenocytes into healthy inbred F 1-mice results in
`graft-versus-host (GVH) induced immune abnor(cid:173)
`malities. This is due to T-Iymphocytes in the donor
`inoculum that recognize the major histocompati(cid:173)
`bility alloantigens (murine H 2-antigens) expressed
`by the F 1-animals. The host FIT-cells are geneti(cid:173)
`cally unable to recognize antigens of the parental
`donor as foreign, thus the response involves only
`donor recognition of host and non host recognition
`of donor. The ensuing immune abnormalities de(cid:173)
`pend on the parental and F 1 strain combinations
`used. For example, the inoculation of C57BLj6
`spleen cells into (C57BLj6 x DBAj2) F I-mice, fur(cid:173)
`ther referred to as DF I-mice, leads to the develop(cid:173)
`ment of an acute GVH (AGVH))-disease resulting
`in profound immunodeficiency, anemia, hypo(cid:173)
`gammaglobulinemia, the appearance of suppres(cid:173)
`sor cells [20] and the development of cytotoxic
`T -lymphocytes (CTL) specific to BD F 1 -alloantigens
`
`13
`
`[21]. In contrast, inoculation of DBAj2 cells into
`BDF I-mice results in a chronic GVH (CGVH)-re(cid:173)
`action in which lymphoid hyperplasia, autoanti(cid:173)
`body production, immune complex glomerulon(cid:173)
`ephritis [21,22] and the failure to form CTL to
`BDF ralloantigens [20], i.e., an illness resembling
`human systemic Lupus erythematosus (SLE).
`First, we studied the effects of leflunomide on the
`chronic graft versus host (CGVH) disease of mice,
`i.e. animals undergoing a disease displaying symp(cid:173)
`toms very similar to SLE. Comparing the protec(cid:173)
`tive effects of this agent to those of CY, PRED,
`and indomethacin, we found that when therapy
`was started 4 weeks after disease induction (shortly
`before the first appearance of proteinuria), only
`indomethacin was ineffective in inhibiting the
`[5]. Curiously, although
`SLE-like symptoms
`PRED could prevent the development of glomeru(cid:173)
`lonephritis and thus proteinuria, it did not inhibit
`the deposition of immune complexes on the
`glomeruli [5]. This may be due to the mode of
`action of steroids, which have been reported to
`inhibit complement, as well as the production of
`interleukin-l (IL-1) [1]. Interestingly, although
`leflunomide is not a cytotoxic agent, as is CY [23],
`and to some extent PRED, the splenomegaly of the
`CGVH-diseased mice was dose-dependently inhib(cid:173)
`ited after therapy with this agent [5].
`As the case in both adjuvant- and MRLjlpr-dis(cid:173)
`eased animals, mice undergoing a CGVH-reaction
`lymphocyte re(cid:173)
`have significantly suppressed
`sponses to T cell mitogens (Con A and PHA).
`Treatment with leflunomide restored these re(cid:173)
`sponses, whereas neither indomethacin nor PRED
`displayed any positive effects. Depending on the
`dose, CY partly restored or inhibited these mito(cid:173)
`gen induced responses of T cells [5, 23].
`
`Prevention of skin and kidney graft rejection
`by leflunomide
`In the prevention of reactions leading to transplant
`rejection, leflunomide initially seemed to be com(cid:173)
`pletely ineffectual [1]. Although successful in pre(cid:173)
`venting the chronic graft-versus-host (CGVH)-dis(cid:173)
`ease [5], this agent was first reported not to have
`any protective activity in the runting illness
`brought on by an acute GVH reaction [1]. Due to
`the results obtained from the effects of this agent
`on the murine CGVH-disease, and considering
`that all studies we [1-7] and others [6, 8, 9] had
`
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`
`14
`
`Agents and Actions, vol. 32, 1/2 (1991)
`
`Table 2
`Effects of drug therapy on allogenic kidney transplantation.
`
`Transplant
`
`n
`
`Drug
`
`Dosage
`(mg/kg/d)
`
`Plasma-creatine levels on day (mg/dl±SD)
`
`8
`
`32
`
`40
`
`50
`
`60
`
`Syngenic
`Allogenic
`Allogenic
`Allogenic
`Allogenic
`Allogenic
`Allogenic
`
`7
`6
`12
`13
`13
`7
`7
`
`none
`none
`HWA486
`HWA486
`CSA
`AZA
`PRED
`
`0
`0
`5 (po)
`10 (po)
`10 (po)
`5 (iv)
`5 (iv)
`
`O.H 1.2
`7.0± 16.1
`0.7± 0.7
`O.H 0.7
`0.8 ± 0.4
`7.9± 17.4
`6.9± 4.6
`
`0.6±0.4
`
`0.6±0.3
`
`0.6±0.2
`
`0.6±0.3
`
`0.7 ±0.8
`0.7±0.7
`0.7±0.5
`
`0.7±0.6
`0.7±0.7
`0.7 ±0.8
`
`0.7 ±0.3
`0.7±0.8
`0.7 ±0.9
`
`0.7 ±0.3
`0.HO.8
`0.7 ±0.5
`
`Survival
`rate (d±SD)
`
`>60
`8.0±0.3
`>60
`>60
`>60
`8 ±O
`7.9±0.6
`
`Lewis rats were transplanted with either syngenic (Lewis) or allogenic (BN) kidneys. Drug therapy was initiated on day -1 and
`terminated on day 30 as indicated. AZA = azathioprine; CSA = cyclosporin A; PRED = prednisolone; HWA486 = leflunomide;
`po = per os; iv = interveinous. From Kiichle et aJ. [24].
`
`Table 3
`Effects of leflunomide therapy on allogenic skin transplantation.
`
`Transplant
`
`DA-->LEWIS
`DA-->LEWIS
`DA-->LEWIS
`DA-+LEWIS
`DA-->LEWIS
`LEWIS --> FISHER
`LEWIS -+ FISHER
`LEWIS --> FISHER
`LEWIS--> FISHER
`LEWIS--> FISHER
`
`n
`
`10
`10
`10
`10
`10
`10
`10
`10
`10
`10
`
`Dosage
`(mg/kg/d)
`
`Graft survival
`time (d/SD)
`
`0.0
`2.5
`5.0
`10.0
`20.0
`0.0
`2.5
`5.0
`10.0
`20.0
`
`10.5 ± 1.1
`19.7±1.9
`23.7±2.0
`27.0± 1.1
`29.1 ± 1.8
`16.2± 1.0
`22.6±2.2
`25.9±2.1
`28.9 ± 1.7
`33.8±2.8
`
`Rats were treated from day 1 to 10 with leflunomide (per os) after
`tail skin was transplanted. From Kiichle, et al. [24].
`
`conducted demonstrated that leflunomide was just
`as effective as CSA in the therapy of various
`autoimmune disorders, we reasoned that this drug
`must also be efficient in preventing transplantation
`rejection reactions.
`Using Lewis rats (RT 1I), as host animals, kidneys
`from BN rats were transplanted. In the untreated
`rats, these allografts were rejected within eight
`days, whereas treatment with leflunomide, for 30
`days, prolonged the graft and thus the animal
`survival of all of these animals for the duration of
`the experiment (more than 60 days) (Table 2).
`Following the serum-creatinine levels we could
`determine that the transplanted kidneys func(cid:173)
`tioned normally (Table 2), and the histological
`studies revealed virtually no signs of chronic rejec(cid:173)
`tion [24]. The results we obtained from CSA ther-
`
`apy were very similar to those observed after
`leflunomide, whereas, in our experiment, neither
`azathioprine nor prednisolone offered any protec(cid:173)
`tion (Table 2).
`Looking further, we found that leflunomide was
`not only efficacious in suppressing kidney but also
`skin rejection reactions in rats. Using two different
`strain combinations for our studies, DA/Lewis
`(MHC and non-MHC different) and Lewis/Fisher
`(non-MHC different), tail skin from the donQr an(cid:173)
`imals was grafted to the hosts. Therapy with
`leflunomide was started one day after transplanta(cid:173)
`tion and terminated on day ten. Using this proto(cid:173)
`col, a dose dependent depression of the rejection
`time could be observed in both transplant combi(cid:173)
`nations (Table 3). With this protocol, i.e. starting
`drug application a day after exposure to foreign
`antigen, CSA is not efficient, at least in our hands.
`This is because CSA is much better suited to sup(cid:173)
`press primary reactions, before they are initiated,
`and is much less efficient in inhibiting ongoing
`immune reactions [1, 7, 25]. This indicates a much
`different mode of action of leflunomide than that
`of CSA.
`
`Studies concerning the mode of action
`ofleflunomide
`Ex vivo and in vivo studies
`For a long time, we felt that leflunomide did not
`have any or very little influence on T-cells. It
`seemed that this drug asserted its effects chiefly on
`B-cells, or perhaps T-cell products mediating B(cid:173)
`cell activity. This was based on our findings that
`
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`
`leflunomide seemed to be only effective in diseases
`in which primarily B-cells and autoantibodies
`played a major role, i.e. SLE, etc., but did not seem
`to be effective in AGVH-reactions, which is mainly
`ineffectiveness of
`T-cells mediated. That the
`leflunomide in the murine AGVH-disease was a
`matter of dosage, we discovered later (Bartlett,
`unpublished results). Of course, SLE and other
`autoimmune diseases are T-cell mediated, thus we
`could have realized much earlier that this agent
`must assert some effects on these lymphocytes.
`That T -cells are affected by this agent has become
`very obvious from our results concerning trans(cid:173)
`plantation rejection. Perhaps one of the primary,
`at the time misleading, results originated from the
`ex-vivo experiments using healthy mice [26]. Com(cid:173)
`paring the effects of leflunomide therapy on the
`immune response to those of cyclophosphamide
`(CY), prednisolone (PRED), and CSA, we found
`that like the other three agents, leflunomide arrest(cid:173)
`ed the development of antibody producing cells to
`the T-cell antigen SRBC, and, thus, suppressed the
`production of their specific antibody. Yet, unlike
`CY and PRED, neither CSA nor leflunomide had
`inhibitory effects on the proliferative response of
`lymphocytes to T-cell independent B-cell mito(cid:173)
`gens, i.e. lipopolysaccharide (LPS) and dextransul(cid:173)
`fate (DXS). Contrary to the activities of the other
`drugs, leflunomide did not inhibit the response of
`lymphocytes to the T-cell mitogens concanavalin
`A (Con A), phytohemagglutinin (PHA), nor, un(cid:173)
`expectedly, to the preimmunized T-cell antigen
`sheep red blood cells (SRBC). The reaction to
`SRBC was unanticipated, because of the lack of
`antibody response to this specific antigen, yet the
`proliferative response was still detectable. Further,
`not only was the oxidative burst of peritoneal
`macrophages, after PMA induction, not inhibited,
`as was the case after treatment with the three im(cid:173)
`munosuppressive substances, but this response
`was greatly enhanced after therapy with lefluno(cid:173)
`mide [26].
`These results suggested that leflunomide has, on
`the one hand, suppressive activity on the develop(cid:173)
`ment of T -lymphocyte dependent antibody pro(cid:173)
`ducing cells and the formation of their specific
`antibody, yet, on the other hand, does not interfere
`with the proliferative response of lymphocytes to
`this antigen. Due to the fact that this agent did not
`inhibit T-cell responsiveness to mitogens, it seemed
`that leflunomide may influence, somehow, the T-B
`
`15
`
`lymphocyte cooperation, without negatively influ(cid:173)
`encing general T-cell or other leukocyte coopera(cid:173)
`tion and function. That this interpretation was not
`fully correct became obvious later when we ob(cid:173)
`tained data indicating that T-cells are most likely
`inhibited in their activity, i.e. inhibitory effects of
`leflunomide on T-cell mediated DTH-reaction to
`mycobacterial antigens of rats with adjuvant
`arthritis [6, 8,], and suppression of skin and kidney
`transplantation rejection was observed [24].
`
`In vitro studies
`For the in vitro studies, the primary metabolite of
`leflunomide, A 771726 was used. This metabolite is
`the molecule, which is very stable and makes up
`more than 90% of compound found in serum of
`animals and humans and is responsible for the
`disease modifying activity of this drug. This has
`been determined in the adjuvant arthritis-disorder,
`MRL/lpr-disease, and the murine CGVH-illness
`[1 ].
`
`.effects on mediator release of inflammatory cells
`Hypersensitivity reactions are inflammatory re(cid:173)
`sponses resulting from the release of mediators
`from tissue mast cells or basophilic leukocytes. In
`allergic individuals, this release is initiated by an
`antigen (in this case allergen) cross-linking two IgE
`molecules affixed to their receptors in the cell's
`plasma membrane. Mast cells and basophils also
`have receptors for other immunoglobulins, i.e.
`IgG 1, which mediate anaphylactic responses with a
`short sensitization period (not more than a few
`hours). Not only immunologic induction of media(cid:173)
`tor release is possible, but also non-immunologic
`means can lead to such liberation, for instance
`calcium ionophores [27], and basic polypeptides
`[28] have been reported to provoke the discharge
`of mediators from mast cells.
`
`o
`CN-n-~-NH-@-CF.
`A771726
`
`\CH
`
`3
`
`C
`
`HO/
`
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`

`
`16
`
`An important primary mediator of hypersensitiv(cid:173)
`ity is histamine, which binds to target cells (smooth
`muscle cells, endothelial cells of blood vessels) via
`specific receptors. We have found that lefluno(cid:173)
`mide's primary metabolite, A 771726, can inhibit
`the release of histamine from isolated human
`basophils, rat peritoneal mast cells, and a murine
`bone marrow derived mast cell line [1]. This is true
`for both calcium-ionophore (A23187) and IgE in(cid:173)
`duced release. The IDso values, for both types of
`induction, were found to be about 0.7 11M [1].
`Not only histamine release, but also the formation
`and liberation of biologically active metabolites of
`arachidonic acid were shown, to varying degrees,
`to be inhibited by leflunomides's primary metabo(cid:173)
`lite. For example, the calcium-ionophore induced
`release of 5-HETE was strongly inhibited (IDso~
`311M), leukotriene B4 (LTB4) considerably less
`suppressed (ID 50 ~ 100 11M) and PGEz formation
`was not affected [1]. Despite the limited ability to
`inhibit the generation of mediators from arachi(cid:173)
`donic acid, antagonism of contractive activity of
`one such mediator, prostaglandin Fzcx (PGFzcx),
`was detected [1]. Further, we have observed that
`the generation of oxygen radicals, during mediator
`liberation, can be effectively prevented by treat(cid:173)
`ment with leflunomide or its primary metabolite,
`A771726 [1].
`Taken together, many of the antiphlogistic effects
`of leflunomide observed in animal models surely
`have to do with the activity discovered in vitro on
`various isolated inflammatory cells and their medi(cid:173)
`ators. Perhaps, also, these effects may explain
`some of the immunorestoring characteristics of
`this agent. For instance, it has been shown that
`LTB4 has many effects on various immune cells,
`including T-Iymphocytes and natural killer func(cid:173)
`tion [29].
`
`Effects on lymphocytes and cytokines
`Due to leflunomide's obvious immunomodulating
`properties, our interest has focused on its influence
`on various mediators of immunocompetent cells.
`As already mentioned, we could demonstrate that
`T -dependent antibody production of healthy mice
`[26], and the formation of self-reactive antibodies
`(T-dependent) in autoimmune animals, can be ef(cid:173)
`fectively suppressed, both preventively and cura(cid:173)
`tively, by oral treatment with this drug [1-9]. Fur(cid:173)
`ther, disorders requiring T-T cell cooperation, i.e.
`
`Agents and Actions, vol. 32, 1/2 (1991)
`
`transplantation rejection, could be inhibited by
`leflunomide therapy. Thus, it was our feeling that
`leflunomide must somehow interfere in the interac(cid:173)
`tion of lymphocytes, especially T and B cells, and
`that this interference is not limited to primary im(cid:173)
`mune responses, but is effective on secondary im(cid:173)
`mune reactions as well.
`We first studied the effects of leflunomide's prima(cid:173)
`ry metabolite (A 771726) on the in vitro PFC-assay,
`to determine if we could observe the same effects
`we were seeing in vivo. We found that this molecule
`could dose dependently block the formation of
`PFC to the T cell dependent antigen SRBC, even
`when it was given into the culture as late as 4 days
`after they were set up and assayed on day 5 [1].
`This paralleled our experience in animal experi(cid:173)
`ments [1]. This inhibition ofPFC-formation could,
`though, be partially overcome if fairly high con(cid:173)
`centrations of Con A supernatant (containing sev(cid:173)
`eral T-cell growth factors) were added into the
`cultures on day 2 [1]. This data indicated that
`something in Con A supernatant could replace
`what leflunomide had somehow blocked. This
`could be either through inhibition of mediator pro(cid:173)
`duction or interference of their activity.
`Our interest then fixed on leflunomide's effects on
`cytokine production. Confirming and adding to
`our already published results concerning IL-1, IL-2,
`IL-3 production [1], Germann, et al. (manuscript
`in preparation) have found that the formation and
`release of cytokines formed by either Thl or Thz
`cells, i.e. IL-l, IL-2, IL-3, IL-4, TRF-cx, or IFN-y,
`are not inhibited by this agent. Thus, if this drug
`does have effects on cytokines, then it must some(cid:173)
`how interfere in their activity.
`The first evidence that leflunomide does antago(cid:173)
`nize T cell products was obtained in an assay sys(cid:173)
`tem for T helper cell replacing factor (TRF) [30].
`Athymic lymphocytes from nu/nu mice are unable
`to generate plasma cells producing antibody to
`sheep red blood cells (SRBC) because they lack T
`lymphocytes. It is possible to restore this humoral
`response by replacing helper cell function through
`the addition of T cell products that act directly on
`B cells. When supernatant from Con A stimulated
`T cells, containing TRF, is given into a culture of
`nu/nu-splenocytes and SRBC on day 2, large num(cid:173)
`bers of PFC to this antigen can be detected on day
`5. When A771726 was given into such cultures on
`day 0 or 2, a dose dependent inhibition of specific
`plaque formation could be observed [1].
`
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`
`Agents and Actions, vol. 32, 1/2 (1991)
`
`The activity of TRF has been attributed to inter(cid:173)
`leukin 5 (IL-5), yet, as tested in the above described
`system, it is difficult to ascribe these results using
`Con A supernatant to one interleukin alone, be(cid:173)
`cause this activity is most likely resulting from a
`combination of distinct factors interacting with
`each other.
`With the coming of recombinant interleukines, it
`has become possible to study these individually.
`Leflunomide seems, to varying degrees, to interfere
`in the activities of several interleukines. We have
`found, for instance, that IL-3 induced prolifera(cid:173)
`tion can be strongly antagonized by A 771726 [1],
`with an 1D50 of about 311M. IL-3 is a sort of
`universal colony stimulating factor with varying
`effects on several haematopoietic cells, i.e. the ac(cid:173)
`tivity attributed to such factors as mast cell growth
`factor (MCGF), P-cell stimulating factor (PSF),
`burst promoting activity (BPA) , haematopoietic
`growth factor (ECSF), megakaryocyte colony
`stimulating factor (MEG-CSF), and eosinophile
`colony stimulating factor (Eo-CSF). IL-3 has been
`reported to support the growth of murine pre-B
`cell clones [31], enhance immune responses to T
`cell-dependent antigens [32], and strongly inhibit
`the generation of NK cells in vivo [33]. Recently, it
`has been demonstrated that the sera of autoim(cid:173)
`mune MRLjlpr mice contain antibodies against
`the IL-3 receptor, inducing IL-3 like activity,
`which may play an important role in the pathology
`of these animals.
`Yet, not only is IL-3 strongly antagonized, but also
`IL-4 to almost exactly the same degree [1]. IL-4
`(BSF-1) also