Leflunomide: Mode of Action in the Treatment of Rheumatoid Arthritis

Ann Rheum Dis 2000;59:841–849 841 Ann Rheum Dis: first published as 10.1136/ard.59.11.841 on 1 November 2000. Downloaded from

REVIEW

Leﬂunomide: mode of action in the treatment of rheumatoid arthritis

F C Breedveld, J-M Dayer

Abstract may downregulate the glycosylation of Leflunomide is a selective inhibitor of de adhesion molecules, eVectively reducing novo pyrimidine synthesis. In phase II and cell-cell contact activation during inflam- III clinical trials of active rheumatoid mation. arthritis, leflunomide was shown to im- (Ann Rheum Dis 2000;59:841–849) prove primary and secondary outcome measures with a satisfactory safety profile. The active metabolite of leflunomide, Although the pathogenic mechanisms of rheu- A77 1726, at low, therapeutically applica- matoid arthritis (RA) remain elusive, advances ble doses, reversibly inhibits dihydro- in both molecular biology and clinical research orotate dehydrogenase (DHODH), the have identified a unique orchestration of immune system cell subsets, cell surface markers,

rate limiting step in the de novo synthesis http://ard.bmj.com/ of pyrimidines. Unlike other cells, acti- and soluble cell products that have a role in the vated lymphocytes expand their pyrimi- process of inflammation associated with RA. dine pool by approximately eightfold Inflammation and subsequent degradation of during proliferation; purine pools are the synovial tissue are initiated by the influx of increased only twofold. To meet this lymphocytes (B cells, CD4+, and CD8+ demand, lymphocytes must use both sal- T cells) into the synovial tissue. In the simplest vage and de novo synthesis pathways. model, CD4+ T lymphocytes are activated by Thus the inhibition of DHODH by antigens in the joint and stimulate plasma cells, on September 30, 2021 by guest. Protected copyright. A77 1726 prevents lymphocytes from ac- mast cells, macrophages, and synovial fibro- Academic Ziekenhuis cumulating suYcient pyrimidines to sup- blasts to produce inflammatory mediators (tu- Leiden, Stafcentrum port DNA synthesis. At higher doses, mour necrosis factor á (TNFá) and interleukin Reumatologie, Leiden, A77 1726 inhibits tyrosine kinases respon- 1 (IL1)), which stimulate matrix degradation.1–3 The Netherlands sible for early T cell and B cell signalling Refining our knowledge of the immunoregula- F C Breedveld tory function of the various T cell subsets, as in the G0/G1 phase of the cell cycle. well as knowledge of the e ects of macrophages Division of Because the immunoregulatory eVects of V Immunology and A77 1726 occur at doses that inhibit and monocytes at the site of inflammation, may Allergy, Geneva DHODH but not tyrosine kinases, the lead to the development of eVective and targeted University Hospital, interruption of de novo pyrimidine syn- treatments for RA. Geneva, Switzerland thesis may be the primary mode of action. The T cell activation associated with RA has J-M Dayer Recent evidence suggests that the ob- focused much attention on identifying drugs Correspondence to: served anti-inflammatory eVects of that regulate T cell progression through the cell Professor F C Breedveld, A77 1726 may relate to its ability to cycle in an attempt to modulate proliferation.45 Academic Ziekenhuis One such agent, leflunomide (N-(4-trifluoro- Leiden, Stafcentrum suppress interleukin 1 and tumour necro- Reumatologie, Gebouw 1, C sis factor á selectively over their inhibitors methylphenyl)- 5 - methylisoxazol-4-carbox- 4–R, NL–2300 RC Leiden, in T lymphocyte/monocyte contact activa- amide), is a low molecular weight (270), The Netherlands tion. A77 1726 has also been shown to sup- synthetic isoxazol derivative. Interest in leflu- f.c.breedveld@ rheumatology. press the activation of nuclear factor êB, a nomide as an antirheumatic drug is due to its medfac.leidenuniv.nl potent mediator of inflammation when unique ability to regulate progression through Accepted for publication stimulated by inflammatory agents. Con- the cell cycle by inhibiting de novo pyrimidine 1 May 2000 tinuing research indicates that A77 1726 ribonucleotide biosynthesis.6–17

www.annrheumdis.com 842 Breedveld, Dayer Ann Rheum Dis: first published as 10.1136/ard.59.11.841 on 1 November 2000. Downloaded from O 4-trifluoromethyl group of the aromatic ring Leflunomide was replaced by more lipophilic groups. H C N CF3 Replacement of the aromatic ring by straight H chain carbon groups decreased drug eYcacy. N A phase II clinical trial with patients with RA OH– O showed that A77 1726 was highly bound to plasma protein (>99%) and had a half life of between 15 and 18 days. The total plasma O clearance was 0.3 ml/kg/h.20 A somewhat A77 1726 N C CC N CF3 shorter half life of about 11 days was reported Active in studies of healthy subjects.21 The majority of metabolite C H A77 1726 (60% to 70%) is eventually metabo- lised into trifluoromethylaniline-oxanilic acid HO CH 3 and excreted in the urine. Figure 1 Chemical structure of leflunomide and its active metabolite A77 1726. A77 1726 regulates lymphocyte proliferation To be classified as a disease modifying A77 1726 has been shown to prevent skin, antirheumatic drug (DMARD), a pharma- heart, and kidney graft rejection in animal ceutical agent must show eYcacy and safety, models.22–24 Leflunomide is also eVective in as well as the ability to slow the progression various experimental models of autoimmune of RA. Leflunomide (active metabolite diseases, including a spontaneous syndrome in A77 1726) is a unique DMARD with immuno- MRL/lpr mice that resembles human systemic modulatory properties. EVective treatment for lupus erythematosus,25 an anti-acetylcholine RA requires a thorough knowledge of the mode receptor induced model of myasthenia gravis in of action of the therapeutic regimen chosen. In rats,26 and a T cell mediated allergic encephalo- addition, with the current interest in combina- myelitis, which models human multiple sclero- tion treatment, the specific interaction between sis.27 More important for its role as an agents must be considered. Therefore, the antirheumatic drug is the demonstration that purpose of this review is to describe the leflunomide is highly eVective in the treatment immunomodulatory actions of leflunomide in of animal models of both adjuvant28–31 and col- the treatment of RA at the cellular and lagen induced arthritis.19 molecular levels. In addition, the eVects of A77 1726 has been shown to regulate lym- combination treatment will be considered. phocyte proliferation both in vitro7 9 12 13 32–36 and in vivo.14 31 36 37 In in vitro studies, A77 1726 is an Leflunomide chemistry and metabolism equally eVective immunoregulator of T cell pro- Leflunomide is a prodrug that is rapidly liferation when stimulated by cell surface recep- converted in the gastrointestinal tract and tor mediated mitogens or by mitogens, such as plasma to its active, open ring metabolite, phorbol ester plus ionomycin, which bypass ini- 91234 the malononitrilamide, A77 1726 (2- cyano- tial signal transduction events. T cell http://ard.bmj.com/ 3-hydroxy-N-(4-trifluoromethylphenyl) buten- dependent B cell formation of autoantibodies, amide) (fig 1). Structure-activity studies have including IgA and IgG isotypes, is also inhibited shown how modifications to A77 1726 aVect by A77 1726.13 The dose of A77 1726 needed to its immunoregulatory activity.10 18 19 In general, modulate lymphocyte proliferation can vary increased drug eYcacy was observed when the greatly between species.12 34 In an in vitro study comparing proliferation of rat, mouse, and Activating stimulus human lymphocytes, the mean 50% inhibitory on September 30, 2021 by guest. Protected copyright. concentrations (IC50 ) for A77 1726 were 0.09, 34 3.5, and 12.5 µmol/l, respectively. These IC50 values were similar for both receptor and non-receptor mediated (that is, phorbol ester Resting phase plus ionomycin) stimulation. 2+ Ca flux Although the immunoregulatory properties G0 IL2 transcription of A77 1726 have been well documented, we NFAT activation have only recently begun to understand the molecular targets and biochemical mecha- Mitosis nism(s) of action underlying these e ects. M V G1 De novo synthesis Using flow cytometric analysis of mitogen of stimulated T cells, Cherwinski et al showed pyrimidine ribonucleotides that A77 1726 prevented the cells from entering the DNA replication phase (S phase) of the cell cycle34 (see also Siemasko et al,13 Lang et 37 G2 al, and Herrmann M, Frangou CG, Kirsch- S baum B. Cell cycle control of the de novo pyrimidine synthesis inhibitor leflunomide through DNA the p53 and p21WAF-1 pathways. Paper presented replication at the Association of Rheumatology Health Figure 2 Cell cycle modulation. Some agents block signal transduction events in the Professionals 32nd National Scientific Meet- resting G0 phase. Other agents interfere with ribonucleotide biosynthesis in the G1 phase. In either case, transition into the DNA replication phase, or S phase, of the cell cycle is ing; 8–12 November 1997; Washington, DC). blocked. NFAT = nuclear factor of activated T cells. As illustrated in fig 2, A77 1726 may prevent

www.annrheumdis.com Leﬂunomide in the treatment of RA 843 Ann Rheum Dis: first published as 10.1136/ard.59.11.841 on 1 November 2000. Downloaded from cell progression into the S phase by acting at tions of A77 1726 which inhibit lymphocyte

several sites in the G0 (resting) or G1 (growth) proliferation are not suYcient to reduce IL2R phases, or both. Currently, two speciﬁc mecha- expression.93437 Furthermore, many of the nisms of action for A77 1726 have been identi- reported eVects of A77 1726 on signal trans- ﬁed: (a) inhibition of tyrosine kinases associ- duction might be secondary results of a ated with the initial stage of signal transduction primary mode of action (that is, dihydro-

in G0, and (b) inhibition of de novo pyrimidine orotate dehydrogenase inhibition).

nucleotide biosynthesis in late G1. As will be Recent evidence suggests that A77 1726 is a described below, the inhibition of de novo potent inhibitor of nuclear factor-êB (NF-êB) pyrimidine nucleotide biosynthesis occurs at activation.40 NF-êB is a transcriptional factor lower doses of A77 1726 than does the inhibi- critical to the function of cells in the immune tion of tyrosine kinase activity and is consid- system, and it plays a part in inﬂammation. ered the primary mechanism of action. Manna and Aggarwal showed that treatment of human Jurkat T cells with A77 1726, at a con- Inhibition of tyrosine kinase activity centration of 5–10 µmol/l, blocks TNF medi- Interference with the initial signal transduction ated activation of NF-êB.41 The eVect was not events which play a part in T cell activation constrained to TNF activation, as the stimulat- would prevent the transition of cells from the ing eVects of other inﬂammatory agents were

resting G0 phase to the G1 phase. Several proc- also blocked. In contrast, tyrosine kinase activ- esses involved in signal transduction, including ity was suppressed at a concentration of the inhibition of tyrosine kinase activity, are 150 µmol/l.12 A77 1726 targeted the degrada- aVected by high doses of A77 1726. In Jurkat tion of the inhibitory protein IêBá, which is T cells, the activities of the Src related tyrosine non-covalently associated with NF-êBinits kinases p56lck and p59fyn have been shown to inactive state in the cytoplasm. Inﬂammatory be inhibited by A77 1726.33 Tyrosine phospho- agents induce the phosphorylation dependent rylation of the chain of the CD3 complex, degradation of the inhibitory protein, which in mediated by p59fyn, is thought to be critical for turn unmasks the nuclear localisation signals successful T cell antigen receptor/CD3 com- on p65 and activates NF-êB. These observa- plex signalling. Likewise, p56lck mediated tions indicate an additional biochemical phosphorylation of the phospholipase C iso- mechanism for the immunomodulatory eVects zyme ã1 is important in the mobilisation of of A77 1726, as well as the observed anti- intracellular Ca2+. In another study A77 1726 inﬂammatory actions. was eVective in inhibiting the levels of tyrosine phosphorylated proteins in mouse leukaemia Inhibition of tyrosine kinase is not the cell line (LSTRA) cells, which overexpress principal target of A77 1726 p56lck.11 A77 1726 was also reported to Several observations are incompatible with suppress tyrosine phosphorylation in murine tyrosine kinase inhibition being the principal CTLL-4 cells stimulated by IL2.33 A similar mechanism underlying the immunoregulatory inhibition of IL2 receptor (IL2R) associated action of A77 1726. A77 1726 is equally eVec-

tyrosine kinase activity has been seen in a tive in inhibiting lymphocyte proliferation, http://ard.bmj.com/ staphylococcal nuclease-speciﬁc T cell line.38 whether it is stimulated through a cell surface More recently, Elder et al showed that receptor involving tyrosine kinases or by A77 1726 inhibited the phosphorylation of the mechanisms that bypass these initial signal Jak1 and Jak3 tyrosine kinases, which are nec- transduction events.13 34 It is also interesting to essary for IL2R signalling.14 note that the ability of A77 1726 to inhibit Other eVects of A77 1726 on signal trans- proliferation was the same, whether it was duction have also been observed, but the data added at the time of stimulation, eight hours are more equivocal. Reductions in IL2 produc- later,34 or even up to 24 hours later.13 The on September 30, 2021 by guest. Protected copyright. tion, possibly related to inhibition of p56lck, drug’s ability to aVect proliferation, independ- have been reported in A77 1726 treated ently of time, questions the role of tyrosine human T cells stimulated by anti-CD3 mono- kinase inhibition early in the signal transduc- clonal antibodies.33 Cao et al showed that tion.

A77 1726 augments the immunosuppressive Comparison of the IC50 values for the cytokine TGFâ1 (transforming growth factor regulation of lymphocyte proliferation versus â1), while simultaneously suppressing IL2.39 In inhibition of tyrosine kinase activity also contrast, another study failed to demonstrate suggests that early signalling events are not the an eVect of A77 1726 in IL2 production.37 This principal target of A77 1726. A discrepancy

latter observation is further supported by data between the IC50 values for A77 1726 mediated demonstrating that exogenous IL2 is unable to inhibition of proliferation and tyrosine kinase antagonise the immunoregulatory eVects of activity has been shown in the murine cytotoxic 39 A77 1726 (Herrmann M, Frangou CG, T cell line CTLL-4. The IC50 for inhibition of Kirschbaum B. Cell cycle control of the de proliferation was 2 µmol/l, whereas that for novo pyrimidine synthesis inhibitor leﬂuno- tyrosine kinase mediated phosphorylation of mide through the p53 and p21WAF-1 pathways. Jak1 and Jak3 kinases was 50 µmol/l.14 Paper presented at the Association of Rheuma- tology Health Professionals 32nd National Sci- Inhibition of de novo pyrimidine entiﬁc Meeting; 8–12 November 1997; Wash- synthesis ington, DC). Decreased IL2R expression has During proliferation the pyrimidine nucleotide also been invoked as a potential mechanism of pool within lymphocytes must expand by action of A77 1726.633 However, concentra- approximately eightfold.42 In contrast, the

www.annrheumdis.com 844 Breedveld, Dayer Ann Rheum Dis: first published as 10.1136/ard.59.11.841 on 1 November 2000. Downloaded from purine nucleotide pool increases by only selectively inhibits UTP and CTP synthesis with 42 twofold. Although salvage pathways for ob- an IC50 of 10 µmol/l, which is similar to the IC50 taining pyrimidines exist, hereditary disorders for its antiproliferative activity (10–30 µmol/l).11 of nucleotide metabolism suggest that highly These results indicate that A77 1726 inhibits proliferating cells such as lymphocytes need de novo pyrimidine synthesis. both de novo and salvage pathways to match the greatly increased demand for pyrimidine A77 1726 is a non-competitive inhibitor of nucleotides during proliferation.42 dihydroorotate dehydrogenase Three independent reports appeared in A77 1726 could inhibit de novo pyrimidine 1995 suggesting that A77 1726 is a reversible nucleotide biosynthesis at several sites. There inhibitor of pyrimidine nucleotide biosynthesis are six steps in the biosynthesis of uridine-5’- in vitro.6–8 Zielinski et al showed that exogenous monophosphate (UMP) from ATP and application of the pyrimidine nucleotide, urid- glutamine. The enzymes which catalyse these ine, could reverse the antiproliferative action of steps are carbamyl phosphate synthetase II A77 1726 in murine B and T cell lines studied (CPSII), L-aspartate transcarboxylase (ATC- 6 in vitro. In another study Cherwinski et al ase), L-dihydroorotase (DHOase), L-dihydro- tested the ability of both pyrimidine and purine orotate dehydrogenase (DHODH), orotate nucleotides to antagonise the antiproliferative phosphoribosyl transferase (OPRTase), and actions of A77 1726 in several cell lines, orotidine-5’-monophosphate decarboxylase including rat pheochromocytoma ACH2 cells, (OMPDC). The ﬁrst three enzymes in this human Jurkat T cells, mouse B cell lymphoma pathway form a cytosolic multienzyme com- A20–1.11 cells, and mouse B cell hybridoma plex, CPSII-ATCase-DHOase. Because of 3DO-18.3 cells.7 The antiproliferative eVect of their close association within this complex, 5 µM A77 1726 on all of these cell lines was enzymatic products are transferred eYciently completely blocked by 100 µM uridine. Cyti- from one active site to the next with minimum dine, which can be converted to uridine by losses due to diVusion into the adjacent intra- cytidine deaminase,43 also restored prolifera- cellular space. The fourth enzymatic step, cata- tion but with a reduced eYcacy. The purine lysed by DHODH, occurs on the outer face of nucleotides, adenosine and guanosine, had no the inner mitochondrial membrane. Finally, eVect on the antiproliferative actions of the last two enzymes in the pathway form A77 1726. Similar results were observed in another cytosolic multienzyme complex, mitogen stimulated normal rat, mouse, and OPRTase-OMPDC. human lymphocytes. The activities of all the enzymes in this path- Using human Jurkat T cells, Cao et al found way are allosterically controlled by the concen- that uridine and cytidine (at concentrations of trations of both precursors and products.45 Two up to 4 µmol/l) were equally successful in observations suggest, however, that the regula- reversing the immunoregulatory eVects of tion of pyrimidine synthesis may be particu- A77 1726.9 This was true for concentrations of larly sensitive to the catalytic step mediated by A77 1726 up to 24 µmol/l. At higher concen- DHODH. Firstly, lymphocytes have fewer 46 trations of A77 1726, uridine and cytidine were mitochondria than most cells. Secondly, both http://ard.bmj.com/ only partially successful in restoring prolifera- the precursor (dihydroorotate) and the product tion. Presumably, tyrosine kinase activity was (orotate) of the DHODH mediated step must being inhibited at these higher concentrations diVuse across the mitochondrial membrane. of A77 1726. It is interesting to note that urid- These two factors place DHODH at a strategic ine was found to be eVective in restoring position to act as a key step in regulating the de proliferation when added to cell cultures up to novo synthesis of UMP. Consistent with this 24 hours after the initial treatment with hypothesis, it has been shown that dihydro- A77 1726.9 orotate accumulates in human T lymphoblast- on September 30, 2021 by guest. Protected copyright. Subsequent studies have conﬁrmed and oid cells treated with A77 1726,8 while exog- extended the close association between the enous orotate antagonises the eVect of immunoregulatory actions of A77 1726 and A77 1726.44 altered pyrimidine nucleotide biosynthesis, Other studies have shown directly the selec- both in vitro8 10–15 44 and in vivo.10 16 17 44 tive inhibition of DHODH by A77 1726. The Changes in intracellular nucleotide pools eVects of A77 1726 on the six diVerent caused by exposure to A77 1726 have also enzymes in the de novo UMP synthetic been measured.7 In human T lymphoblastoma pathway were examined using a human T lym- CCRF.CEM cells, 10 µM A77 1726 decreased phoblastoma cell line.47 DHODH was shown

uridine triphosphate (UTP) and cytidine to be reversibly inhibited with a Ki value of triphosphate (CTP) levels by 58% and 51%, about 3 µmol/l. No other cytosolic enzymes in respectively. In normal rat spleen cells stimu- this biosynthetic pathway were aVected. In lated with concanavalin A (Con A), 1 µM another study the in vivo ability of a number of A77 1726 decreased UTP levels by 34% and synthetic A77 1726 analogues to inhibit CTP levels by 18%. At these doses of delayed-type hypersensitivity reactions in rats A77 1726 neither adenosine triphosphate and mice was found to be closely correlated (ATP) nor guanosine triphosphate (GTP) lev- with their in vitro ability to inhibit DHODH els were aVected. In IL2 stimulated murine activity.19 The inhibition of DHODH by T cells, 10 µM A77 1726 has been shown to A77 1726 has been conﬁrmed in a number decrease UTP levels by 95% and CTP levels by of subsequent in vitro studies.10 16 19 48 A 85% without aVecting either ATP or GTP lev- consistent ﬁnding in all of these studies was els.14 In the murine LSTRA cell line, A77 1726 that A77 1726 inhibited DHODH at drug

www.annrheumdis.com Leﬂunomide in the treatment of RA 845 Ann Rheum Dis: first published as 10.1136/ard.59.11.841 on 1 November 2000. Downloaded from Activation

De novo pathway

A77 1726 DHODH Salvage pathways

Uridine

Pyrimidine pools

CTP-, UTP-sugars Nucleotides CDP-lipids

Glycoproteins, glycolipids RNA, DNA Phospholipids

Cell-cell contact, adhesions, Lymphocyte Cellular membranes, diapedesis proliferation secondary messengers Figure 3 EVect of inhibition of de novo pyrimidine synthesis on various mechanisms of activated lymphocytes. (Adapted from Herrmann et al.49a) concentrations similar to those resulting in its “sensor” to detect low ribonucleotide levels immunoregulatory eVects, and at one to three (that is, reduced rUMP (ribonucleotide urid- orders of magnitude less than that needed for ine monophosphate) due to inhibition of the inhibition of tyrosine kinases. DHODH through A77 1726) and damaged

DHODH requires two substrates for cataly- DNA, as well as an “eVector” that inhibits the http://ard.bmj.com/ sis. In addition to dihydroorotate, which is cyclin D/cdk and E/cdk complexes when such converted to orotate, ubiquinone is used as an situations occur. The sensor function is accom- electron acceptor. In recombinant human plished by the tumour suppression protein, DHODH, it has been shown that A77 1726 p53, whose proto-oncogene is also upregulated inhibits the binding sites for both these after a mitogenic stimulus.50–52 substrates non-competitively.48 In a study ana- In dividing cells with adequate ribonucle- lysing the kinetics of DHODH inhibition, it otide levels, p53 is stabilised in the cytoplasm.50 was found that A77 1726 acted as a competi- In cells with low ribonucleotide levels, how- on September 30, 2021 by guest. Protected copyright. tive inhibitor of the ubiquinone binding site ever, p53 is activated and translocates into the and a non-competitive inhibitor of the dihy- nucleus. If damaged DNA is detected, p53 droorotate binding site.48 These actions of induces the transcription of yet another regula- A77 1726 distinguish it from another inhibitor tory gene, whose product is a 21 kilodalton of DHODH, brequinar sodium, which is a protein, p21.53 The p21 protein acts as the non-competitive inhibitor of both the ubiqui- eVector for the G1 checkpoint. It inhibits the none and dihydroorotate binding sites.49 Figure activity of the cyclin D/cdk and E/cdk com- 3 summarises the eVect of A77 1726 inhibition plexes, leading to the dephosphorylation of of DHODH on de novo synthesis of pyrimi- Rb.53 As a result, Rb can rebind to the dine and on the subsequent activities of transcription factor E2F/DP-1, and cause the activated lymphocytes. cell to become arrested in G1. Herrmann et al have shown in stimulated human peripheral T A77 1726 decreases levels of rUMP lymphocytes that both p53 and p21 become

leading to cell cycle arrest in late G1 upregulated after exposure to A77 1726, re- In regulating the phosphorylation state of sulting in the cells becoming arrested in late G1 retinoblastoma protein (Rb), the cyclin D/cdk (Herrmann M, Frangou CG, Kirschbaum B. (cyclin dependent kinase) and E/cdk com- Cell cycle control of the de novo pyrimidine

plexes serve as part of the G1 checkpoint, where synthesis inhibitor leﬂunomide through the cells with low ribonucleotide levels and/or p53 and p21WAF-1 pathways. Paper presented at damaged DNA are selectively prevented from the Association of Rheumatology Health Pro- progressing further into the cell cycle.50 Other fessionals 32nd National Scientiﬁc Meeting;

components of the G1 checkpoint include a 8–12 November 1997; Washington, DC).

www.annrheumdis.com 846 Breedveld, Dayer Ann Rheum Dis: first published as 10.1136/ard.59.11.841 on 1 November 2000. Downloaded from A77 1726 modulation of inflammation By reducing ATP dependent pools of UTP, and metalloproteinase induction UDP-Glu (uridine diphosphoglucose), and At the site of inflammation in chronic inflam- CTP (fig 3), the subsequent expression of matory diseases, inflammatory cells and cells of UDP sugars is inhibited, strongly influencing the native tissue are in close proximity, which the glycosylation of adhesion molecules. Simi- implies that a possible mechanism for cell larly, CTP lipids, which are essential for the communication is through direct cell-cell con- incorporation of mannose and fructose into tact in addition to soluble factors. In a series of glycoproteins, as well as dolichol-linked inter- studies54–59 of cell-cell contact between T mediates through GDP sugar precursors, are lymphocytes and monocytes, it has been shown suppressed (fig 3). Reduced GTP and GDP that cell-cell contact induces the production of sugars would aVect both lectin binding and cell both matrix metalloproteinases (MMPs) and surface topography, and as a result, would tissue inhibitor metalloproteinase-1 (TIMP-1). restrict mitogenic processes. These observa- In addition, direct contact causes the upregula- tions provide a mechanism for the anti- tion of proinflammatory cytokines (IL1 and inflammatory and immunomodulatory actions TNFá) and their inhibitors (IL1 receptor of A77 1726. antagonist and TNF soluble receptor).60 It is postulated that an imbalance between MMPs Clinical overview of leflunomide: eYcacy, and TIMPs and cytokines and their inhibitors safety, and combination therapy may lead to the matrix destruction characteris- The clinical eYcacy and safety of leflunomide tic of chronic inflammation and, in particular, has been assessed in a one dose ranging phase the matrix degradation associated with RA. II clinical trial,20 in two placebo controlled With these observations, Deage et al evalu- phase III clinical trials,62 63 and in one compara- ated the eVect of the anti-inflammatory agents, tive phase III trial.64a A77 1726 and dexamethasone, on the contact In both placebo controlled studies, clinical 60 activation of the monocytic cell line THP-1. improvement, as defined by the American Col- T lymphocytes were stimulated with phorbol lege of Rheumatology (ACR) 20% response myristate acetate and phytohaemagglutinin in criteria,64 was statistically significant for leflu- the presence of both drugs, and it was noted nomide (20 mg/day) compared with placebo. that A77 1726 and dexamethasone inhibited Leflunomide showed ACR 20% response rates the ability of stimulated T cells to activate the comparable with those of sulfasalazine (2 monocytic cells by 66%–97% and 43%–70%, g/day) and methotrexate (7.5–15 mg/week) in respectively. It was found that A77 1726 the placebo controlled clinical trials, and tended to favour the inhibition of proinflam- significant improvement in health related qual- matory and matrix destructive factors over that ity of life and functional ability compared with of anti-inflammatory factors and MMP inhibitors (fig 4), thus potentially limiting matrix 80 destruction. The exact mechanism for this A IL1β eVect remains to be determined. However, flow IL1Ra

cytometry indicates that surface molecules 60 http://ard.bmj.com/ (CD69 and CD11) of T lymphocytes that are, in part, involved in cell-cell contact signalling 40 with monocytes, were not modulated by either drug, indicating an alternative mechanism. Product (%) Preliminary clinical data on the expression of 20 cell adhesion molecules (CAMs) and MMPs in synovial tissue from 39 patients in a prospec- 0 on September 30, 2021 by guest. Protected copyright. tive, double blind trial comparing leflunomide DEX LEF and methotrexate, indicate that both treatments resulted in comparable reductions in synovial inflammation and decreases in pro- 80 duction of CAMs, and a decrease in the MMP/ B MMP-1 TIMP-1 ratio after four months of treatment TIMP-1 60 (Kraan MC, Reece RJ, Barg EC, et al.An explorative study of the changes in synovial tissue in patients with RA after treatment with 40 leflunomide or methotrexate. Paper presented

at the 14th European League Against Rheuma- Product (%) tism Congress; 6–11 June 1999; Glasgow, 20 Scotland). Results of the study indicate that both methotrexate and A77 1726 showed 0 comparable clinical eYcacy in reducing inflam- DEX LEF mation within the synovial tissue. Figure 4 (A) Leflunomide (LEF) diVerentially inhibits In addition to providing strong evidence the ability of stimulated T lymphocytes to activate THP-1 establishing that the primary mode of action of cells by direct cellular contact, favouring interleukin 1 receptor antagonist (IL1Ra) production, compared with A77 1726 is the inhibition of DHODH, Rück- dexamethasone (DEX). (B) Similarly, LEF diVerentially emann et al also found that A77 1726 (100 inhibits the ability of stimulated T lymphocytes to activate µmol/l) significantly depleted ATP and GTP THP-1 cells by direct cellular contact, favouring the 61 production of tissue inhibitor metalloproteinase-1. pools, thus strongly aVecting ATP dependent Concentrations of both LEF and DEX for figs 4A and B enzymes, which modulate the immune system. are 10−5 mol/l.60

www.annrheumdis.com Leflunomide in the treatment of RA 847 Ann Rheum Dis: first published as 10.1136/ard.59.11.841 on 1 November 2000. Downloaded from in the treatment of patients unresponsive to De novo pyrimidine synthesis De novo purine synthesis traditional monotherapy, and, in particular, in Glutamine Ribose-5-P the treatment of aggressive disease in early RA.68 69 Wilke and Clough highlight the need Leflunomide MTX for detailed information about the pharmacol- MTX ogy and modes of action for each of the Uridine UMP combined drugs, and emphasise that Xanthine XMP IMP AMP Adenine DMARDs used in combination should have UTP UDP dUMP diVerent modes of action.68 Furst concluded that eVective combination treatment requires a Cytidine MTX Guanine GMP ADP ATP thorough knowledge of the toxicity, modes of CTP dTMP action, and pharmacokinetics of the combined GTP GDP DMARDs.70 Because of its unique mechanism of action, the potential use of A77 1726 in combination treatment with DMARDs is dCTP dTTP dGTP dATP promising. DNA Methotrexate at the low doses used in the treatment of RA inhibits purine biosynthesis, Figure 5 Comparison of the eVect of leflunomide with that of methotrexate on nucleotide synthesis. MTX = methotrexate, dUMP = deoxyuridine monophosphate. and may have additional anti-inflammatory activity related to adenosine release. A77 1726, sulfasalazine and methotrexate.62 63 Lefluno- on the other hand, inhibits de novo pyrimidine mide was also shown to reduce the rate of biosynthesis and lymphocyte proliferation.71 radiographically assessed disease progression Figure 5 summarises the unique modes of significantly compared with the group receiv- action of both leflunomide (active metabolite ing placebo and to produce comparable rates of A77 1726) and methotrexate. The diVering progression compared with the groups receiv- modes of action present a rationale for the ing active treatment.62 63 combined treatment and may explain the syn- The comparative two year trial of lefluno- ergistic eVects of the two in combination.71 The mide versus methotrexate found both treat- combination of methotrexate and leflunomide ments comparable at two years in counts of has been proved to be eVective for the tender joints and patient global assessment of treatment of patients with active RA who were disease activity. Radiographic assessments of refractory to treatment with methotrexate disease progression at two years were also alone.64 comparable.64a The clinical eYcacy of combining A77 1726 The most common adverse events associated with cyclosporin A has not been thoroughly with leflunomide treatment were gastrointesti- investigated, but in vitro studies of the drug nal, consisting primarily of diarrhoea, raised combination in animal models exhibit interest- liver function tests, abdominal pain, and ing complementary interactions. The rationale nausea/vomiting.20 62 63 The frequency and se- for combining A77 1726 with drugs such as

verity of the gastrointestinal complications cyclosporin A is that these early acting drugs http://ard.bmj.com/ were highest during the ﬁrst two weeks of may not prevent all autoimmune lymphocytes

treatment with either leflunomide or metho- from entering G1. An additive interaction trexate and declined thereafter.64a In addition, between A77 1726 and cyclosporin A in leflunomide has been associated with rash/ preventing T cell proliferation has already been allergic reactions, and reversible alopecia. shown in vitro,12 and in a canine renal Interestingly, there were no significant prob- transplant model.72 A77 1726, in combination lems with anaemia, leucopenia, or thrombo- with cyclosporin, resulted in the longest mean cytopenia, nor was there any increase in the survival time, 68 days. It was also noted that a on September 30, 2021 by guest. Protected copyright. incidence of infection compared with placebo. combination of A77 1726 and cyclosporin, No opportunistic infections were noted. both at suboptimal doses, eVectively prolonged Treatment with leflunomide may have a mean survival time by three times that for con- cytostatic eVect, resulting from the inhibition trols, and that the animals had normal renal of pyrimidine biosynthesis in tissues with a function and weight. A similar eVect was high rate of cellular turnover, such as those of observed for antigen induced arthritis in rats, the gastrointestinal tract. This speculative where a suboptimal dose of both drugs signifi- mechanism may also explain the incidence of cantly inhibited chronic arthritis.73 reversible alopecia, as well as the lack of oppor- The current prescribing information defines tunistic infection, marrow toxicity, and mucosi- the recommended dose and administration of tis65 seen in clinical trials of leflunomide. leflunomide. Owing to the long half life in The gastrointestinal adverse eVects associ- patients with RA and the 24 hour dosing inter- ated with high dose leflunomide (35 mg/kg/ val, a loading dose of 100 mg/day for 3 days is day) in a Lewis rat model of transplantation recommended, followed by a maintenance were diarrhoea and pathological changes of the dose of 20 mg/day for the duration of small bowel and liver.66 These adverse eVects treatment.74 If dosing at 20 mg/day is not well were significantly reduced by uridine co- tolerated, the dose may be decreased to 10 administration, which implies the mechanism mg/day. Liver enzyme levels should be leading to these side eVects was related to monitored, and dose adjustments made, if DHODH dependent cell proliferation. necessary. Because of the long half life for Early discussions of combination DMARD the active metabolite, it is advised that during treatment67–69 recognised the potential benefit dose adjustment patients should be carefully

www.annrheumdis.com 848 Breedveld, Dayer Ann Rheum Dis: first published as 10.1136/ard.59.11.841 on 1 November 2000. Downloaded from monitored, as the levels of metabolite may take 15 Siemasko K, Chong ASF, Jack H-M, Gong H, Williams JW, 74 Finne A. Inhibition of JAK3 and STAT6 tyrosine phospho- several weeks to reduce. rylation by the immunosuppressive drug leflunomide leads to a block in IgG1 production. J Immunol 1998;160: 1581–8. Summary and conclusions 16 Spinella-Jaegle S, Williamson RA, Yea CM, Robson PA, Curnock AP, Adher S, et al. Dihydroorotate dehydrogenase Leflunomide is a promising new antirheumatic is a target for the biological eVects of leflunomide. drug with novel properties and mild side Transplant Proc 1996;28:3088–91. 17 Silva HT, Cao W, Shorthouse R, Morris RE. Mechanism of eVects. 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