Targeting DMARD resistance in Rheumatoid Arthritis Rheumatoid in resistance DMARD Targeting Targeting DMARD resistance in Rheumatoid Arthritis

Joost van der Heijden - - Joost van der Heijden der van Joost

Targeting DMARD resistance in Rheumatoid Arthritis

Joost van der Heijden The research described in this thesis was performed at the department of VRIJE UNIVERSITEIT Rheumatology (head prof. dr. B.A.C. Dijkmans) in collaboration with the department of Pathology (head prof. dr. C.J.L.M. Meijer), VU University Medical Center, Amsterdam, the Netherlands. Joost van der Heijden was supported by the Dutch Arthritis Foundation (Grant NRF-03-I-40) and ZonMW (The Netherlands Organization for Health Research and Development; Grant 920-03-362). Targeting DMARD resistance in

Publication of this thesis was financially supported by: Abbott B.V., Amgen B.V., Rheumatoid Arthritis AstraZeneca B.V., Janssen-Cilag B.V., Merck Sharp & Dohme B.V., Pfizer B.V., Roche B.V., sanofi-aventis B.V. and Schering-Plough.

ACADEMISCH PROEFSCHRIFT

ter verkrijging van de graad Doctor aan de Vrije Universiteit Amsterdam, op gezag van de rector magnificus prof.dr. L.M. Bouter, in het openbaar te verdedigen ten overstaan van de promotiecommissie van de faculteit der Geneeskunde op vrijdag 12 december 2008 om 10.45 uur in de aula van de universiteit, De Boelelaan 1105

ISBN nummer: 978 90 8659 253 1 door

Cover design and lay-out: Esther Beekman, www.estherontwerpt.nl Photo cover: ‘Schiphol 2006’, by Maarten van Haaff, www.maartenvanhaaff.nl Johan Willem van der Heijden Printed by: PrintPartners Ipskamp, Enschede © Copyright: Joost van der Heijden 2008 geboren te Gouda promotoren: prof. dr. B.A.C. Dijkmans prof. dr. R.J. Scheper copromotoren: dr. G. Jansen prof. dr. W.F. Lems

‘I, a physician, am delighted to stand here with two distinguished chemists, Drs. Reichstein and Kendall. Perhaps the ratio of one physician to two chemists is symbolic, since medicine is so firmly linked to chemistry by a double bond. For medicine, especially during the past twenty-five years, has been receiving its finest weapons from the hands of the chemists, and the chemist finds his richest reward as the fruits of his labor rescue countless thousands from the long shadows of the sickroom’.

Philip S. Hench, rheumatologist*

*Nobel Prize Laureate (together with prof. dr. Tadeus Reichstein and prof. dr. Edward C. Kendall) in Physiology and Medicine in 1950 for their discoveries relating to the hormones of the adrenal cortex, their structure and biological effects. Contents

Chapter 1 General introduction and introduction into the chapters 10

Chapter 2 Drug insight: resistance to methotrexate and other disease-modifying antirheumatic drugs - from bench to bedside. Nature Clinical Practice Rheumatology 2007; 3: 26-34. 28

Chapter 3 Development of sulfasalazine resistance in human T cells induces expression of the mulltidrug resistance transporter ABCG2 (BCRP) and augmented production of TNFα. Annals of the Rheumatic Diseases 2004; 63: 138-143. 46

Chapter 4 Acquired resistance of human T cells to sulfasalazine: stability of the resistant phenotype and sensitivity to non-related DMARDs. Annals of the Rheumatic Diseases 2004; 63: 131-137. 62

Chapter 5 Sulfasalazine is a potent inhibitor of the reduced folate carrier: implications for combination therapies with methotrexate in rheumatoid arthritis. Arthritis & Rheumatism 2004; 50: 2130-2139. 80

Chapter 6 Involvement of Breast Cancer Resistance Protein expression on RA synovial tissue macrophages in resistance to methotrexate and leflunomide. Submitted for publication 98

Chapter 7 Anti-folate drug combinations for inflammatory diseases. In: Chemistry and Biology of Pteridines and Folates 2007; 365-377. 114

Chapter 8 Enhanced capacity of selected methotrexate analogues to inhibit TNF-α production in whole blood from RA patients. Submitted for publication 128

Chapter 9 Folate receptor-β as potential delivery route for novel folate antagonists to macrophages in synovial tissue of rheumatoid arthritis patients. Accepted for publication in Arthritis & Rheumatism 2008 148

Chapter 10 The proteasome inhibitor bortezomib inhibits the release of NFκB-inducible cytokines and induces apoptosis of activated T-cells from rheumatoid arthritis patients. Accepted for publication in Clinical and Experimental Rheumatology 2008 168

Chapter 11 General discussion and future perspectives 182

Chapter 12 Summary 198 Dutch Summary – Nederlandse samenvatting 208 Acknowledgements – Dankwoord 214 Curriculum Vitae 218 List of publications 220 List of abbreviations List of abbreviations

ABC ATP-binding cassette AICAR 5-aminoimidazole-4-carboxamide ribonucleotide AICARTF 5-aminoimidazole-4-carboxamide ribonucleotide formyltransferase APRTF Amidophosphoribosyltransferase ATRA All-trans retinoic acid BCRP Breast cancer resistance protein CCP Cyclic citrunillated peptide CHQ Chloroquine DAS Disease activity score DC Dendritic cell DHFR Dihydrofolate reductase DMARD Disease modifying antirheumatic drug ELISA Enzyme-Linked Immuno Sorbent Assay FA Folic acid FACS Fluorescent-activated cell sorting FPGH Folylpolyglutamate hydrolase FPGS Folylpolyglutamate synthetase FR Folate receptor GARTFase Glycinamide ribonucleotide transformylase HDAC Histone deacetylase HLA Human leukocyte antigen

IC-50 Drug concentration required to inhibit cell growth or cytokine production by 50% compared to non-treated cells IκB Inhibitor-κB IκK Inhibitor-κB kinase IL Interleukin LV Leucovorin MDR Multi-drug resistance MHC Major histocompatibility complex MMP Matrix metalloproteinase MRP Multi-drug resistance protein MTHFR Methylene tetrahydrofolate reductase MTX Methotrexate NFκB Nuclear factor κB NSAID Non steroidal anti-inflammatory drug PBL Peripheral blood lymphocyte PBMC Peripheral blood mononuclear cell PCR Polymerase chain reaction Pgp P-glycoprotein RA Rheumatoid arthritis RF Rheumatoid factor RFC Reduced folate carrier SNP Single nucleotide polymorphism SSZ Sulfasalazine TNF Tumor necrosis factor TS Thymidylate synthase chapter 1 General introduction and introduction into the chapters chapter 1 General introduction and introduction into the chapters

General introduction Figure 1

A B Rheumatoid Arthritis (RA) is a chronic inflammatory and joint destructive disease that affects approximately 1% of the Dutch population and commonly leads to significant disability and consequent reduction in the quality of life of RA patients. RA can start at any age, with a peak incidence between the fourth to sixth decades of life, and is three times more frequent in women than in men. RA is associated with high costs and, if not treated appropriately, with a reduction in life expectancy (1, 2).

Pathogenesis of RA The exact pathogenesis of RA is not fully elucidated, but it is a disease where the immune and inflammatory systems are intimately linked to the destruction of cartilage and bone. Given the detection of auto-antibodies, with IgM-rheumatoid factor (RF) as the classical and prototypical serum marker, RA is classified as an auto-immune disorder. During the Figure 1: Schematic view of a normal joint and its changes in RA. last decade, auto-antibodies against citrullinated peptides (anti-CCP antibodies) have (A) The normal joint. The synovial joint is composed of two adjacent bony ends, each covered with a layer been identified in two third of RA patients, which turned out to be more specific for this of cartilage, separated by a joint space and surrounded by the synovial membrane and joint capsule. The synovial membrane consists of a thin (1-3 cells) layer of synoviocytes without a basement membrane. disease (3). These antibodies reflect an abnormal humoral response against citrullinated (B) The arthritic joint. RA is characterized by an inflammatory response of the synovial membrane (referred self-proteins (4). Interestingly, these antibodies can be detected years before the first to as synovitis) that is conveyed by a transendothelial influx and local activation of a variety of mononuclear onset of clinical symptoms and antibody levels are correlated with RA disease activity (5, 6). cells such as T-cells, B-cells, plasma cells, mast cells, dendritic cells and macrophages, as well as by angiogenesis. The lymphoid infiltrate can be diffuse or, commonly, form lymphoid-follicle-like structures. The question remains whether the occurrence of anti-CCP antibodies are of pathogenetic The lining layer becomes hyperplastic and the synovial membrane expands and forms villi; the destructive ‚ relevance in RA. portion of the synovial membrane is termed ‘pannus . Enzymes produced by macrophages, synovial fibroblasts and neutrophils induce cartilage destruction; activation of present osteoclasts eventually leads to bone destruction. Bone repair by osteoblasts usually does not occur in active RA. From: Smolen JS et al. Conceptually, the onset of joint inflammation in RA is induced by the following events, Lancet 2007; 370:1861-1874. depicted in figure 1. Influx of dendritic cells (DCs) into the synovial compartment occurs early in disease pathology, and it is thought that DCs play a role in the initiation and Therapeutic strategies perpetuation of disease by presentation of (unknown) arthritogenic (auto) antigens. For decades therapy of RA patients consisted of non-steroidal anti-inflammatory drugs Several genetic loci are known to be associated with the susceptibility for and the severity (NSAIDs), reducing the pain and inflammation but not modifying the course of the of RA; the most well established being a specific amino-acid sequence in the HLA-DR4 disease. The first Disease Modifying Anti-Rheumatic Drugs (DMARDs) were intramuscular allele, the so called shared epitope, which is involved in antigen presentation (7). Upon gold (J. Forestier, 1928) and sulfasalazine (N. Svartz, 1938), interfering with the course presentation of auto-antigens by appropriate HLA molecules, invading (pre-activated) and progression of the disease (1,10). The modifying effect of DMARDs is reflected in T-cells become (further) activated by cell-cell contact or cytokine signalling and start improvement of the Disease Activity Score (DAS) (a composite index of the patients to produce cytokines. These cytokines in turn stimulate on one hand B-cells to produce opinion of disease activity, a count of tender and swollen joints by the physician and the auto-antibodies (e.g. RF and anti-CCP) and on the other hand macrophages and synovial erythrocyte sedimentation rate) (11) and parameters of joint damage as seen on X-rays of fibroblasts to produce pro-inflammatory cytokines (like TNFα and IL1-β) and matrix hands and feet. metalloproteinase’s (MMPs) which provoke cartilage destruction. Activation of joint Corticoids are used since the 1940’s with very good clinical response, but there are osteoclasts eventually leads to bone destruction. Tissue infiltration by inflammatory limitations to treatment duration because of the occurrence of side effects upon cells results in thickening of the synovial membrane (referred to as synovitis) and is prolonged use, like osteoporosis, hypertension and diabetes. Methotrexate (MTX) was first accompanied by neovascularisation (1, 8, 9). introduced around 1950, but it was not given the credits it deserves at that time. Three developments can be considered as real breakthroughs in the treatment of RA patients:

12 13 chapter 1 General introduction and introduction into the chapters

(a) the rediscovery of MTX in the 1980’s (b) the introduction of combination therapies and DMARD-resistance (c) the introduction of biologic agents in the 1990’s. The term biologics refers to a group Observational studies and meta-analyses of treatment efficacy, average treatment of therapeutic agents that specifically target a particular cell or cytokine involved in the duration and adverse effects for RA-patients consistently demonstrate variable responses pathogenesis of RA. These highly effective anti-inflammatory agents include infliximab for individual DMARDs and DMARD combinations (26, 26-30). Unfortunately, many and adalimumab (both antibodies against TNFα), etanercept (soluble TNFα receptors), RA patients experience loss of efficacy upon chronic treatment with DMARDs, which rituximab (antibodies to CD20/B-cells) and abatacept (antibodies to the co-stimulatory could point to the onset of acquired drug resistance. Galindo-Rodrigues et al showed in molecule B7 on antigen presenting cells to prevent T-cell activation) (1, 8, 10, 12-14). These a retrospective practice-based study between 1985 and 1994, including 2296 DMARD drugs are usually combined with MTX. In the Netherlands physicians agreed with the therapies, that after 16 months 50% of treatments had been discontinued because of government to prescribe biologics only to those patients that sequentially failed on two inefficacy and/or toxicity and that after 4.5 years 75% of therapies had been discontinued. regular DMARDs, mainly to reduce drug related costs. MTX appeared to be the best drug in the first 5 years of disease; approximately 50% of RA patients were still receiving MTX after 3 years of treatment, compared to 33% for Today, many studies have proven that early and aggressive therapy with (combinations of) antimalarials and 25% for sulfasalazine (31) (figure 2). At the time this study was performed DMARDs and/or biologic agents is beneficial in terms of preservation of functional ability however, TNFα antagonists were not yet available. Despite the initial good response to and slowing down of joint damage progression (12). After remission induction, DMARDs MTX for many patients, there is room for improvement of DMARD therapy and a need for and/or biologic agents can then be tapered and in some cases even stopped (15). studies that unravel possible mechanisms of DMARD resistance and studies that define Despite the current success of biologic therapies, DMARDs have an established place in the parameters for predicting DMARD efficacy and toxicity. treatment of RA because of their efficacy, convenience, safety and low related drug costs The issue of drug resistance as a cause of therapy failure and the search for new therapeutic (16). Currently, the most commonly applied DMARDs are MTX, sulfasalazine, leflunomide, approaches to circumvent drug-resistance has received much attention in cancer prednisone and antimalarials like hydroxychloroquine. Their therapeutic efficacy can be treatment (32); however it just starts to be appreciated in RA treatment (33-36). Human enhanced by combining these DMARDs, however a rationale behind these combinations beings have intrinsic and inducible mechanisms against daily exposure to hundreds or is often lacking (17, 18). Sulfasalazine plus MTX is a frequently used combination; however, even thousands of xenobiotic substances present in our environment and food (32, 37, this combination does not display additive or synergistic effects over monotherapy (19, 38). Most chemically designed therapeutic drugs, including DMARDs, are recognized 20), unless a third DMARD (hydroxychloroquine or prednisone) is added (21-25). by target cells as foreign substances. Given this notion, sooner or later immunologic or cellular defence mechanisms will become operative and inactivate the drugs at various molecular levels. Cellular drug resistance can be categorized under two main headings: Figure 2 (1) inherited or primary resistance (referring to cells that are already resistant before receiving therapy) and (2) acquired drug resistance (which indicates that cells were initially sensitive to the drug, but developed resistance during the course of treatment). General mechanisms of cellular drug resistance are: diminished drug delivery, impaired drug uptake, energy dependent cellular drug extrusion via MDR transporters, decreased drug activation, drug sequestration or enhanced drug detoxification, alterations in the target of the drug or enhanced repair of damage/ impaired capacity for cells to go into apoptosis (33) (figure 3). These and other mechanisms of drug resistance are extensively described in chapter two.

Figure 2: Reasons for discontinuation of traditional DMARDs in patients with RA. Black bars: inefficacy; grey bars: toxicity; light grey bars: both inefficacy and toxicity; white bars: total Resistance to biologic agents discontinuations. IM: intramuscular; *study completed in 1994 – no other choices available at that time for While treatment with biologic agents provides great benefit to the majority of RA those with inadequate effect requiring alternative therapy after MTX. Source: Retrospective audit of records of patients with onset of RA between January 1985 and June 1994. References: Galindo-Rodriguez G et al. J patients, some patients experience persistent active disease or loss of efficacy upon Rheumatol 1999; 26(11):2337-2343 and Fleischmann RM et al. J Rheumatol Suppl 2005; 73:3-7. prolonged treatment. Biologic agents are proteins and therefore antibodies can be

14 15 chapter 1 General introduction and introduction into the chapters

produced against these agents, diminishing clinical efficacy. Wolbink et al showed that Figure 3 in serum of 43% (22/51) of consecutive RA patients treated with infliximab, anti-infliximab antibodies were detectable. A negative correlation was found for the titer of anti- infliximab antibodies and the clinical response (39). The same observation was made in a group of Ankylosis Spondylitis (AS) patients treated with infliximab (40). Adalimumab, a fully human anti-TNFα antibody, was thought to be less immunogenic than the chimeric infliximab. Nevertheless Bartelds et al showed that in serum of 17% (21/121) of consecutive RA patients anti-adalimumab antibodies were detectable and that the antibody-titer was associated with diminished adalimumab concentrations and subsequently with an impaired clinical response (41). There is evidence that MTX administration prolongs the duration of response to biologic agents, probably due to the inhibition of the production of anti-biologic antibodies (42); in several trials, the efficacy of the combination of MTX Figure 3: Molecular mechanisms of cellular drug resistance. (1) Diminished drug delivery (2) impaired drug uptake, (3) energy dependent cellular drug extrusion via MDR plus a biologic agent appeared to be superior over monotherapy with MTX or the biologic transporters, (4) decreased drug activation, (5) drug sequestration or (6) enhanced drug detoxification (7) agent (43-48). For this reason, it is recommended to combine a biologic agent with MTX. alterations in the target of the drug or (8) enhanced repair of damage/ impaired capacity for cells to go into apoptosis

Aim and outline of the thesis This thesis focuses on the mechanisms involving DMARD resistance in the treatment of of MDR proteins on inflammatory cells (T-cells, macrophages) in synovial tissue of RA RA patients and alternative ways to target RA inflammatory cells in case of resistance to patients and assessed whether expression of these proteins correlated with clinical currently available DMARDs. outcome after treatment with MTX or leflunomide.

From the field of oncology, increased drug efflux has been recognized as one important Besides MTX efflux by MDR transporters (33, 37, 38, 50-52), cellular resistance to MTX mechanism of drug resistance. Cell-membrane proteins responsible for drug efflux belong might be conferred by diminished uptake via its cell membrane carrier, the reduced to the family of ATP-binding cassette (ABC) transporters of which 49 different proteins folate carrier (RFC). Also impaired polyglutamylation by the enzyme folylpolyglutamate were identified from the human genome project (http://nutrigene.4t.com/humanabc. synthethase (FPGS) or alterations in the target enzymes of MTX (a.o. dihydrofolate htm). Tissue distribution studies showed that these proteins are expressed in tissues reductase) may contribute to a resistant phenotype (53-59). that are heavily exposed to toxic agents, microbial and exogenous compounds, eg. liver, The second section of this thesis focuses on novel experimental therapeutic strategies to kidney and intestine (49). Consistently, one of the primary functions of ABC-transporters overcome MTX resistance. In this context, we utilized activated T-cells from RA patients is extrusion of toxic substances. Some of these transporters are extensively characterized to test potential anti-inflammatory effects of novel generation antifolate drugs that were while the functional properties of others are still unknown (37, 38, 50). A wide range of rationally designed to overcome known mechanisms of MTX resistance in cancer patients structurally and functionally different drugs can be pumped out of cells by these ABC- (60). Furthermore, we evaluated whether the Folate Receptor β (FRβ), expressed on transporter proteins, thereby conferring a so-called multiple-drug resistant (MDR) synovial tissue macrophages, could be selectively targeted by novel generation antifolate phenotype. Therapeutic drugs are frequently extruded in co-transport with gluthatione, drugs that display a higher affinity for this receptor than MTX (61, 62). or after conversion to gluthatione-, glucuronide- or sulphate conjugates (figure 4). Several DMARDs seem to be among the substrates of MDR proteins (33). This notion led to our Finally, in the last section of this thesis we focussed on a novel class of experimental drugs hypothesis that these proteins might also provide a cellular defence mechanism against that may retain therapeutic activity against resistant cells, due to the fact that they are no DMARDs, leading to DMARD-resistance. In the first section of this thesis we evaluated substrates for MDR transporters. Such class of compounds include proteasome inhibitors, the potential role of ABC-transporters in conferring resistance to DMARDs. To this end, which interfere in intracellular protein degradation (63). Bortezomib, a boronic acid we first investigated whether MDR proteins become upregulated on inflammatory model dipeptide, is the clinically used representative of this class of drugs (64-66). Bortezomib cells upon chronic exposure to DMARDs in vitro. In addition, we evaluated the expression has recently been approved for the treatment of therapy refractory multiple myeloma.

16 17 chapter 1 General introduction and introduction into the chapters

Figure 5 Figure 4

Figure 4: Cellular drug extrusion via MDR transporters. Following entry of drugs (1-2), drugs can be subjected to extrusion (3-5) via an energy (ATP)-dependent process involving an ABC transporter (4). Drugs can be extruded in co-transport with glutathione, or after conversion to glutathione, glucuronide or sulphate conjugates. Figure 5: Bortezomib and NFκB. Upon activation, the transcription factor NFκB is translocated to the nucleus of the cell which results in transcription of genes encoding for pro-inflammatory cytokines and anti-apoptotic factors. By inhibiting the Conceptually, bortezomib may elicit potential anti-inflammatory effects by inhibiting the proteasome (red cross) and therefore the activation of NFκB, that is dependent on the proteasome-mediated breakdown of its inhibitor (IκB) (orange crosses), bortezomib abrogates the production of pro-inflammatory degradation of the natural inhibitor of the transcription factor NFκB , i.e. IκBα, thereby cytokines, anti-apoptotic factors and adhesion molecules. Reference: Paramore A and Frantz S. Nat Rev inhibiting nuclear translocation of NFκB and transcription of several pro-inflammatory Drug Discov 2003; 2(8):611-612. cytokines such as TNFα (67, 68) (figure 5). We therefore tested the anti-inflammatory effects of this drug, using TNFα release from ex-vivo activated T-cells of RA patients as a read out. (MRP1) and breast cancer resistance protein (BCRP). In addition, we assessed the impact of sulfasalazine resistance on the ability of T-cells to secrete TNFα. Chapter four describes the dynamics of sulfasalazine resistance in human T-cells after withdrawal of sulfasalazine Introduction into the chapters and after rechallenging these cells again with this drug, by measuring the expression of MDR transporters under these conditions. Finally, we evaluated the impact of sulfasalazine In chapter two, a literature review is given that outlines molecular mechanisms that could resistance on responsiveness to other, non-related DMARDs. be involved in the onset of resistance to DMARDs in RA patients, including methotrexate, sulfasalazine, hydroxychloroquine, azathioprine and leflunomide. The mechanisms Since we observed (chapter four) that sulfasalazine resistant T-cells in vitro were cross- suggested are based on findings from experimental laboratory studies of specific drug- resistant to MTX when co-incubated with sulfasalazine, along with the clinical notion uptake and drug-efflux transporters, alterations in intracellular drug metabolism and that combination therapy of sulfasalazine and MTX does not show improvement over genetic polymorphisms of drug transporters and metabolic enzymes. In this chapter we monotherapy with MTX or sulfasalazine, we investigated whether exposure of cells also discuss strategies to overcome resistance and the current clinical studies aiming to to sulfasalazine provokes intervention with the cellular pharmacology of MTX. For this predict the response to treatments and risk of toxic effects. purpose in chapter five we studied the effect of sulfasalazine treatment on the functional activity and expression of the cell membrane transporter responsible for the uptake of In chapter three we determined whether overexpression of MDR proteins contribute to MTX, the Reduced Folate Carrier (RFC). a diminished efficacy of sulfasalazine after prolonged exposure of human T-cells to this DMARD. For this purpose, a sulfasalazine resistant human T-cell line was characterized In chapter six we described an inventory study of expression of MDR-transporters for expression of the MDR-proteins P-glycoprotein (Pgp), Multidrug resistance protein 1 on inflammatory cells in RA synovial tissue before and after treatment with MTX or

18 19 chapter 1 General introduction and introduction into the chapters

leflunomide. With immunohistochemical techniques we assessed the expression of Pgp, MRP1-5, MRP8, MRP9 and BCRP. Since the latter two groups of MDR transporters have MTX and leflunomide among their substrates, we examined whether expression of these drug efflux transporters correlated with the clinical response to these DMARDs.

Chapter seven reviews the leading manuscripts involving RA treatment with the antifolate drug MTX and leflunomide as monotherapy and in combination regimes with other DMARDs or biologic agents. This chapter serves as a clinical introduction to chapter 8-10, where we describe: (1) possible alternative targets in the folate pathway in inflammatory cells making use of novel, rationally designed, antifolate drugs; (2) selective targeting of synovial tissue macrophages by folate antagonists and (3) experimental drugs possessing novel mechanisms of action.

From the field of oncology, where MTX is used against childhood leukemia, novel generation of antifolate drugs are available that were designed to circumvent known mechanisms of resistance against MTX. In chapter eight we describe the potential anti- inflammatory properties of these drugs by measuring the inhibition of TNFα release from activated T-cells in whole blood of RA patients. In chapter nine we focus on the folate receptor β (FRβ) as a potential target for RA treatment with novel antifolate drugs. Since it has been described that this receptor is selectively expressed on activated macrophages in inflamed synovial fluid of RA patients, and may be involved in MTX transport in these cells, we assessed the expression of FRβ on inflammatory cells in intact RA synovial tissue by immunohistochemistry and PCR analysis. Beyond this, we determined FRβ binding affinities for several novel antifolate drugs, in search for drugs that may be more selective than MTX in targeting activated synovial tissue macrophages via the FRβ.

Chapter ten describes a study of the anti-inflammatory properties of the proteasome inhibitor bortezomib, a drug currently used in the treatment of therapy refractory multiple myeloma. Since activation of the nuclear transcription factor NFκB, that is dependent on the proteasome-mediated breakdown of its inhibitor (IκB), is thought to play a central role in the onset and progression of inflammation in RA, selective inhibition of the activity of this transcription factor by low-dose bortezomib treatment might be a very efficient therapeutic option that warrants further investigation. In this study, we measured the inhibitory effects of bortezomib on production of TNFα by activated T-cells from RA patients, along with the induction of apoptosis in these cells.

In chapter eleven the general discussion is provided. Chapter twelve summarizes the highlights of this thesis and is followed by a summary in Dutch.

20 21 References References

References methotrexate and sulphasalazine in patients with rheumatoid arthritis: pharmacokinetic analysis and relationship to clinical response, Br.J.Clin.Pharmacol., 42: 195-200, 1996. 1 Smolen,J.S. and Steiner,G. Therapeutic strategies for rheumatoid arthritis, Nat.Rev.Drug Discov., 20 Haagsma,C.J., van Riel,P.L., de Jong,A.J. and van de Putte,L.B. Combination of sulphasalazine 2: 473-488, 2003. and methotrexate versus the single components in early rheumatoid arthritis: a randomized, 2 Smolen,J.S., Aletaha,D., Koeller,M., Weisman,M.H. and Emery,P. New therapies for treatment of controlled, double-blind, 52 week clinical trial, Br.J.Rheumatol., 36: 1082-1088, 1997. rheumatoid arthritis, Lancet, 370: 1861-1874, 2007. 21 Boers,M., Verhoeven,A.C., Markusse,H.M., van de Laar,M.A., Westhovens,R., van Denderen,J.C., 3 Schellekens,G.A., Visser,H., de Jong,B.A.C., van den Hoogen,F.H., Hazes,J.M., Breedveld,F.C. and van Zeben,D., Dijkmans,B.A.C., Peeters,A.J., Jacobs,P., van den Brink,H.R., Schouten,H.J., van der van Venrooij,W.J. The diagnostic properties of rheumatoid arthritis antibodies recognizing a Heijde,D.M., Boonen,A. and Van der Linden,S. Randomised comparison of combined step-down cyclic citrullinated peptide, Arthritis Rheum., 43: 155-163, 2000. prednisolone, methotrexate and sulphasalazine with sulphasalazine alone in early rheumatoid 4 Vossenaar,E.R. and van Venrooij,W.J. Citrullinated proteins: sparks that may ignite the fire in arthritis, Lancet, 350: 309-318, 1997. rheumatoid arthritis, Arthritis Res.Ther., 6: 107-111, 2004. 22 Barrera,P., Haagsma,C.J., Boerbooms,A.M., van Riel,P.L., Borm,G.F., van de Putte,L.B. and Van der 5 Nielen,M.M., van Schaardenburg, D., Reesink,H.W., van de Stadt,R.J., van der Horst-Bruinsma IE, Meer,J.W. Effect of methotrexate alone or in combination with sulphasalazine on the production de Koning,M.H., Habibuw,M.R., Vandenbroucke,J.P. and Dijkmans,B.A.C. Specific autoantibodies and circulating concentrations of cytokines and their antagonists. Longitudinal evaluation in precede the symptoms of rheumatoid arthritis: a study of serial measurements in blood donors, patients with rheumatoid arthritis, Br.J.Rheumatol., 34: 747-755, 1995. Arthritis Rheum., 50: 380-386, 2004. 23 Landewe,R.B., Boers,M., Verhoeven,A.C., Westhovens,R., van de Laar,M.A., Markusse,H.M., 6 Rantapaa-Dahlqvist,S., de Jong,B.A.C., Berglin,E., Hallmans,G., Wadell,G., Stenlund,H., Sundin,U. van Denderen,J.C., Westedt,M.L., Peeters,A.J., Dijkmans,B.A.C., Jacobs,P., Boonen,A., van der and van Venrooij,W.J. Antibodies against cyclic citrullinated peptide and IgA rheumatoid factor Heijde,D.M. and Van der Linden,S. COBRA combination therapy in patients with early rheumatoid predict the development of rheumatoid arthritis, Arthritis Rheum., 48: 2741-2749, 2003. arthritis: Long-term structural benefits of a brief intervention, Arthritis Rheum., 46: 347-356, 7 Bowes,J. and Barton,A. Recent advances in the genetics of RA susceptibility, Rheumatology. 2002. (Oxford), 47: 399-402, 2008. 24 O’Dell,J.R., Haire,C.E., Erikson,N., Drymalski,W., Palmer,W., Eckhoff,P.J., Garwood,V., 8 Smolen,J.S., Aletaha,D., Koeller,M., Weisman,M.H. and Emery,P. New therapies for treatment of Maloley,P., Klassen,L.W., Wees,S., Klein,H. and Moore,G.F. Treatment of rheumatoid arthritis rheumatoid arthritis, Lancet, 2007. with methotrexate alone, sulfasalazine and hydroxychloroquine, or a combination of all three 9 McInnes,I.B. and Schett,G. Cytokines in the pathogenesis of rheumatoid arthritis, Nat.Rev. medications, N.Engl.J.Med., 334: 1287-1291, 1996. Immunol., 7: 429-442, 2007. 25 O’Dell,J.R., Leff,R., Paulsen,G., Haire,C., Mallek,J., Eckhoff,P.J., Fernandez,A., Blakely,K., 10 O’Dell,J.R. Therapeutic strategies for rheumatoid arthritis, N.Engl.J.Med., 350: 2591-2602, 2004. Wees,S., Stoner,J., Hadley,S., Felt,J., Palmer,W., Waytz,P., Churchill,M., Klassen,L. and Moore,G. 11 van der Heijde,D.M., van ‘t Hof,M., van Riel,P.L. and van de Putte,L.B. Validity of single variables Treatment of rheumatoid arthritis with methotrexate and hydroxychloroquine, methotrexate and indices to measure disease activity in rheumatoid arthritis, J.Rheumatol., 20: 538-541, 1993. and sulfasalazine, or a combination of the three medications: Results of a two-year, randomized, 12 Goekoop-Ruiterman,Y.P., De Vries-Bouwstra,J.K., Allaart,C.F., van Zeben,D., Kerstens,P.J., Hazes,J. double-blind, placebo-controlled trial, Arthritis Rheum., 46: 1164-1170, 2002. M., Zwinderman,A.H., Ronday,H.K., Han,K.H., Westedt,M.L., Gerards,A.H., van Groenendael,J. 26 Fleischmann,R.M. Is there a need for new therapies for rheumatoid arthritis?, J.Rheumatol.Suppl, H., Lems,W.F., van Krugten,M.V., Breedveld,F.C. and Dijkmans,B.A.C. Clinical and radiographic 73: 3-7, 2005. outcomes of four different treatment strategies in patients with early rheumatoid arthritis (the 27 Wolfe,F. The epidemiology of drug treatment failure in rheumatoid arthritis, Baillieres Clin. BeSt study): a randomized, controlled trial, Arthritis Rheum., 52: 3381-3390, 2005. Rheumatol., 9: 619-632, 1995. 13 Elliott,M.J., Maini,R.N., Feldmann,M., Kalden,J.R., Antoni,C., Smolen,J.S., Leeb,B., Breedveld,F. 28 de la Mata,J., Blanco,F.J. and Gomez-Reino,J.J. Survival analysis of disease modifying antirheumatic C., Macfarlane,J.D., Bijl,H. et al. Randomised double-blind comparison of chimeric monoclonal drugs in Spanish rheumatoid arthritis patients, Ann.Rheum.Dis., 54: 881-885, 1995. antibody to tumour necrosis factor alpha (cA2) versus placebo in rheumatoid arthritis, Lancet, 29 Maetzel,A., Wong,A., Strand,V., Tugwell,P., Wells,G. and Bombardier,C. Meta-analysis of 344: 1105-1110, 1994. treatment termination rates among rheumatoid arthritis patients receiving disease-modifying 14 Ruderman,E.M. and Pope,R.M. Drug Insight: abatacept for the treatment of rheumatoid arthritis, anti-rheumatic drugs, Rheumatology.(Oxford), 39: 975-981, 2000. Nat.Clin.Pract.Rheumatol., 2: 654-660, 2006. 30 Aletaha,D. and Smolen,J.S. Effectiveness profiles and dose dependent retention of traditional 15 van der Kooij,S.M., Goekoop-Ruiterman,Y.P., De Vries-Bouwstra,J.K., Peeters,A.J., van Krugten,M. disease modifying antirheumatic drugs for rheumatoid arthritis. An observational study, V., Breedveld,F.C., Dijkmans,B.A.C. and Allaart,C.F. Probability of continued low disease activity J.Rheumatol., 29: 1631-1638, 2002. in patients with recent onset rheumatoid arthritis treated according to the disease activity score, 31 Galindo-Rodriguez,G., vina-Zubieta,J.A., Russell,A.S. and Suarez-Almazor,M.E. Disappointing Ann.Rheum.Dis., 67: 266-269, 2008. longterm results with disease modifying antirheumatic drugs. A practice based study, 16 Cannella,A.C. and O’Dell,J.R. Is there still a role for traditional disease-modifying antirheumatic J.Rheumatol., 26: 2337-2343, 1999. drugs (DMARDs) in rheumatoid arthritis?, Curr.Opin.Rheumatol., 15: 185-192, 2003. 32 Szakacs,G., Paterson,J.K., Ludwig,J.A., Booth-Genthe,C. and Gottesman,M.M. Targeting 17 Fathy,N. and Furst,D.E. Combination therapy for autoimmune diseases: the rheumatoid arthritis multidrug resistance in cancer, Nat.Rev.Drug Discov., 5: 219-234, 2006. model, Springer Semin.Immunopathol., 23: 5-26, 2001. 33 Jansen,G., Scheper,R.J. and Dijkmans,B.A.C. Multidrug resistance proteins in rheumatoid arthritis, 18 Smolen,J.S., Aletaha,D. and Keystone,E. Superior efficacy of combination therapy for rheumatoid role in disease-modifying antirheumatic drug efficacy and inflammatory processes: an overview, arthritis: fact or fiction?, Arthritis Rheum., 52: 2975-2983, 2005. Scand.J.Rheumatol., 32: 325-336, 2003. 19 Haagsma,C.J., Russel,F.G., Vree,T.B., van Riel,P.L. and van de Putte,L.B. Combination of 34 Wollheim,F.A. Drug resistance in rheumatology: an area in search of investigators, Curr.

22 23 References References

Rheumatol.Rep., 5: 333-335, 2003. 48 Donahue,K.E., Gartlehner,G., Jonas,D.E., Lux,L.J., Thieda,P., Jonas,B.L., Hansen,R.A., Morgan,L. 35 Morgan,C., Lunt,M., Brightwell,H., Bradburn,P., Fallow,W., Lay,M., Silman,A. and Bruce,I.N. C. and Lohr,K.N. Systematic review: comparative effectiveness and harms of disease-modifying Contribution of patient related differences to multidrug resistance in rheumatoid arthritis, Ann. medications for rheumatoid arthritis, Ann.Intern.Med., 148: 124-134, 2008. Rheum.Dis., 62: 15-19, 2003. 49 Scheffer,G.L., Kool,M., Heijn,M., de Haas,M., Pijnenborg,A.C., Wijnholds,J., van Helvoort,A., de 36 Tsujimura,S., Saito,K., Nawata,M., Nakayamada,S. and Tanaka,Y. Overcoming drug resistance Jong,M.C., Hooijberg,J.H., Mol,C.A., van der Linden,M., de Vree,J.M., van der Valk,P., Elferink,R.P., induced by P-glycoprotein on lymphocytes in patients with refractory rheumatoid arthritis, Ann. Borst,P. and Scheper,R.J. Specific detection of multidrug resistance proteins MRP1, MRP2, MRP3, Rheum.Dis., 67: 380-388, 2008. MRP5, and MDR3 P-glycoprotein with a panel of monoclonal antibodies, Cancer Res., 60: 5269- 37 Deeley,R.G., Westlake,C. and Cole,S.P. Transmembrane transport of endo- and xenobiotics by 5277, 2000. mammalian ATP-binding cassette multidrug resistance proteins, Physiol Rev., 86: 849-899, 50 Borst,P. and Oude Elferink,R.P. Mammalian ABC transporters in health and disease, Annu.Rev. 2006. Biochem., 71: 537-592, 2002. 38 Sarkadi,B., Homolya,L., Szakacs,G. and Varadi,A. Human multidrug resistance ABCB and ABCG 51 Wielinga,P., Hooijberg,J.H., Gunnarsdottir,S., Kathmann,I., Reid,G., Zelcer,N., van der Born,K., de transporters: participation in a chemoimmunity defense system, Physiol Rev., 86: 1179-1236, Haas,M., van der Heijden,I., Kaspers,G., Wijnholds,J., Jansen,G., Peters,G. and Borst,P. The human 2006. multidrug resistance protein MRP5 transports folates and can mediate cellular resistance against 39 Wolbink,G.J., Vis,M., Lems,W., Voskuyl,A.E., de,G.E., Nurmohamed,M.T., Stapel,S., Tak,P.P., antifolates, Cancer Res., 65: 4425-4430, 2005. Aarden,L. and Dijkmans,B.A.C. Development of antiinfliximab antibodies and relationship to 52 Hooijberg,J.H., Broxterman,H.J., Kool,M., Assaraf,Y.G., Peters,G.J., Noordhuis,P., Scheper,R.J., clinical response in patients with rheumatoid arthritis, Arthritis Rheum., 54: 711-715, 2006. Borst,P., Pinedo,H.M. and Jansen,G. Antifolate resistance mediated by the multidrug resistance 40 de Vries,M.K., Wolbink,G.J., Stapel,S.O., de,V.H., van Denderen,J.C., Dijkmans,B.A.C., Aarden,L.A. proteins MRP1 and MRP2, Cancer Res., 59: 2532-2535, 1999. and van der Horst-Bruinsma IE. Decreased clinical response to infliximab in ankylosing spondylitis 53 Rots,M.G., Pieters,R., Peters,G.J., Noordhuis,P., Van Zantwijk,C.H., Henze,G., Janka-Schaub,G.E., is correlated with anti-infliximab formation, Ann.Rheum.Dis., 66: 1252-1254, 2007. Veerman,A.J. and Jansen,G. Methotrexate resistance in relapsed childhood acute lymphoblastic 41 Bartelds,G.M., Wijbrandts,C.A., Nurmohamed,M.T., Stapel,S., Lems,W.F., Aarden,L., Dijkmans,B. leukaemia, Br.J.Haematol., 109: 629-634, 2000. A.C., Tak,P.P. and Wolbink,G.J. Clinical response to adalimumab: relationship to anti-adalimumab 54 Kager,L., Cheok,M., Yang,W., Zaza,G., Cheng,Q., Panetta,J.C., Pui,C.H., Downing,J.R., Relling,M. antibodies and serum adalimumab concentrations in rheumatoid arthritis, Ann.Rheum.Dis., 66: V. and Evans,W.E. Folate pathway gene expression differs in subtypes of acute lymphoblastic 921-926, 2007. leukemia and influences methotrexate pharmacodynamics, J.Clin.Invest, 115: 110-117, 2005. 42 Maini,R.N., Breedveld,F.C., Kalden,J.R., Smolen,J.S., Davis,D., Macfarlane,J.D., Antoni,C., 55 Kremer,J.M. Toward a better understanding of methotrexate, Arthritis Rheum., 50: 1370-1382, Leeb,B., Elliott,M.J., Woody,J.N., Schaible,T.F. and Feldmann,M. Therapeutic efficacy of multiple 2004. intravenous infusions of anti-tumor necrosis factor alpha monoclonal antibody combined with 56 Dervieux,T., Furst,D., Lein,D.O., Capps,R., Smith,K., Walsh,M. and Kremer,J. Polyglutamation low-dose weekly methotrexate in rheumatoid arthritis, Arthritis Rheum., 41: 1552-1563, 1998. of methotrexate with common polymorphisms in reduced folate carrier, aminoimidazole 43 Maini,R., St Clair,E.W., Breedveld,F., Furst,D., Kalden,J., Weisman,M., Smolen,J., Emery,P., carboxamide ribonucleotide transformylase, and thymidylate synthase are associated with Harriman,G., Feldmann,M. and Lipsky,P. Infliximab (chimeric anti-tumour necrosis factor alpha methotrexate effects in rheumatoid arthritis, Arthritis Rheum., 50: 2766-2774, 2004. monoclonal antibody) versus placebo in rheumatoid arthritis patients receiving concomitant 57 Ranganathan,P., Eisen,S., Yokoyama,W.M. and McLeod,H.L. Will pharmacogenetics allow better methotrexate: a randomised phase III trial. ATTRACT Study Group, Lancet, 354: 1932-1939, 1999. prediction of methotrexate toxicity and efficacy in patients with rheumatoid arthritis?, Ann. 44 Weinblatt,M.E., Kremer,J.M., Bankhurst,A.D., Bulpitt,K.J., Fleischmann,R.M., Fox,R.I., Jackson,C. Rheum.Dis., 62: 4-9, 2003. G., Lange,M. and Burge,D.J. A trial of etanercept, a recombinant tumor necrosis factor receptor:Fc 58 Wessels,J.A., De Vries-Bouwstra,J.K., Heijmans,B.T., Slagboom,P.E., Goekoop-Ruiterman,Y. fusion protein, in patients with rheumatoid arthritis receiving methotrexate, N.Engl.J.Med., 340: P., Allaart,C.F., Kerstens,P.J., van Zeben,D., Breedveld,F.C., Dijkmans,B.A.C., Huizinga,T.W. and 253-259, 1999. Guchelaar,H.J. Efficacy and toxicity of methotrexate in early rheumatoid arthritis are associated 45 Weinblatt,M.E., Keystone,E.C., Furst,D.E., Moreland,L.W., Weisman,M.H., Birbara,C.A., Teoh,L. with single-nucleotide polymorphisms in genes coding for folate pathway enzymes, Arthritis A., Fischkoff,S.A. and Chartash,E.K. Adalimumab, a fully human anti-tumor necrosis factor alpha Rheum., 54: 1087-1095, 2006. monoclonal antibody, for the treatment of rheumatoid arthritis in patients taking concomitant 59 Wessels,J.A., van der Kooij,S.M., le Cessie S., Kievit,W., Barerra,P., Allaart,C.F., Huizinga,T.W. methotrexate: the ARMADA trial, Arthritis Rheum., 48: 35-45, 2003. and Guchelaar,H.J. A clinical pharmacogenetic model to predict the efficacy of methotrexate 46 Klareskog,L., van der Heijde,D., de Jager,J.P., Gough,A., Kalden,J., Malaise,M., Martin,M.E., monotherapy in recent-onset rheumatoid arthritis, Arthritis Rheum., 56: 1765-1775, 2007. Pavelka,K., Sany,J., Settas,L., Wajdula,J., Pedersen,R., Fatenejad,S. and Sanda,M. Therapeutic 60 Rots,M.G., Pieters,R., Peters,G.J., Noordhuis,P., Van Zantwijk,C.H., Kaspers,G.J., Hahlen,K., effect of the combination of etanercept and methotrexate compared with each treatment alone Creutzig,U., Veerman,A.J. and Jansen,G. Role of folylpolyglutamate synthetase and in patients with rheumatoid arthritis: double-blind randomised controlled trial, Lancet, 363: 675- folylpolyglutamate hydrolase in methotrexate accumulation and polyglutamylation in childhood 681, 2004. leukemia, Blood, 93: 1677-1683, 1999. 47 Breedveld,F.C., Weisman,M.H., Kavanaugh,A.F., Cohen,S.B., Pavelka,K., van Vollenhoven, R., 61 Nakashima-Matsushita,N., Homma,T., Yu,S., Matsuda,T., Sunahara,N., Nakamura,T., Tsukano,M., Sharp,J., Perez,J.L. and Spencer-Green,G.T. The PREMIER study: A multicenter, randomized, Ratnam,M. and Matsuyama,T. Selective expression of folate receptor beta and its possible role double-blind clinical trial of combination therapy with adalimumab plus methotrexate versus in methotrexate transport in synovial macrophages from patients with rheumatoid arthritis, methotrexate alone or adalimumab alone in patients with early, aggressive rheumatoid arthritis Arthritis Rheum., 42: 1609-1616, 1999. who had not had previous methotrexate treatment, Arthritis Rheum., 54: 26-37, 2006. 62 Gibbs,D.D., Theti,D.S., Wood,N., Green,M., Raynaud,F., Valenti,M., Forster,M.D., Mitchell,F.,

24 25 References References

Bavetsias,V., Henderson,E. and Jackman,A.L. BGC 945, a novel tumor-selective thymidylate synthase inhibitor targeted to alpha-folate receptor-overexpressing tumors, Cancer Res., 65: 11721-11728, 2005. 63 Glickman,M.H. and Ciechanover,A. The ubiquitin-proteasome proteolytic pathway: destruction for the sake of construction, Physiol Rev., 82: 373-428, 2002. 64 Rajkumar,S.V., Richardson,P.G., Hideshima,T. and Anderson,K.C. Proteasome inhibition as a novel therapeutic target in human cancer, J.Clin.Oncol., 23: 630-639, 2005. 65 Elliott,P.J., Zollner,T.M. and Boehncke,W.H. Proteasome inhibition: a new anti-inflammatory strategy, J.Mol.Med., 81: 235-245, 2003. 66 Paramore,A. and Frantz,S. Bortezomib, Nat.Rev.Drug Discov., 2: 611-612, 2003. 67 Tak,P.P. and Firestein,G.S. NF-kappaB: a key role in inflammatory diseases, J.Clin.Invest, 107: 7-11, 2001. 68 Firestein,G.S. NF-kappaB: Holy Grail for rheumatoid arthritis?, Arthritis Rheum., 50: 2381-2386, 2004.

26 27 chapter 2 Resistance to methotrexate and other disease-modifying antirheumatic drugs – from bench to bedside

Joost W. van der Heijden1, Ben A.C. Dijkmans1, Rik J. Scheper2 and Gerrit Jansen1

Departments of 1Rheumatology and 2Pathology, VU University Medical Center, Amsterdam, The Netherlands

Nature Clinical Practice Rheumatology 2007; 3: 26-34 chapter 2 DMARD resistance: a review

Summary risk of adverse effects and could ultimately lead to discontinuation of treatment. The issue of drug resistance as a cause for therapy failure has received considerable attention in the The chronic nature of rheumatoid arthritis (RA) means that patients require drug therapy treatment of cancer and infectious diseases (4), but has just started to be appreciated in for many years. Many RA patients however, have to discontinue treatment due to drug- RA treatment (5, 6). Whereas endpoint evaluations for anticancer drug resistance usually related toxic effects, loss of efficacy, or both. The underlying molecular cause for loss refer to loss of antiproliferative effects of target cells, DMARD resistance might also be of efficacy of antirheumatic drugs is not fully understood, but it might be mediated, defined as loss of ability to block the release of proinflammatory cytokines, in addition to at least in part, by mechanisms shared with resistance to anticancer drugs. This review a loss of antiproliferative effects. outlines molecular mechanisms that could be involved in the onset of resistance to, or This review describes the current knowledge on the molecular mechanisms of cellular the loss of efficacy of, disease-modifying antirheumatic drugs in RA patients, including resistance to DMARDs (methotrexate, sulfasalazine, chloroquine, hydroxychloroquine, methotrexate, sulfasalazine, chloroquine, hydroxychloroquine, azathioprine and azathioprine, auranofin, aurothiomalate, ciclosporin, gold and leflunomide). The leflunomide. The mechanisms suggested are based on findings from experimental suggested mechanisms are mainly based on in vitro laboratory studies and theories about laboratory studies of specific drug-uptake and drug-efflux transporters belonging to the emergence of DMARD resistance in RA. We also discuss potential strategies to deal the superfamily of multidrug-resistance transporters, alterations in intracellular drug with drug resistance. metabolism, and genetic polymorphisms of drug transporters and metabolic enzymes. We also discuss strategies to overcome resistance and the current clinical studies aiming General features of drug resistance to predict response and risk of toxic effects. More in-depth knowledge of the mechanisms Human beings have defense mechanisms against daily exposure to hundreds or even behind these features could help facilitate a more efficient use of disease-modifying thousands of xenobiotic substances present in our environment and food. For this reason, anrtirheumatic drugs. most chemically designed therapeutic drugs, including DMARDs, are recognized by target cells as foreign substances. Sooner or later, immunological or cellular defense mechanisms will become operative and inactivate the drugs. In a clinical setting, such Review criteria a process manifests as reduced drug efficacy, for which, at a molecular level, the onset We searched PubMed using the keywords “DMARDs”, “resistance”, “multidrug resistance”, of drug resistance might be an underlying cause (7-12). Cellular drug resistance can be “methotrexate”, “sulfasalazine” and “rheumatoid arthritis”. Searches included full text categorized under two main headings: inherent or primary resistance (referring to cells papers and abstracts from annual American College of Rheumatology (ACR) meetings and that are already resistant before receiving therapy) and acquired drug resistance (which European League against Rheumatology (EULAR) meetings from 1995–2006. We have also indicates that cells were initially sensitive to drugs but developed resistance during the included experimental data from our own research aimed at identifying the molecular course of treatment). In RA treatment, a study by Morgan et al (13) showed that 5–10% mechanisms of resistance to DMARDs in experimental model systems and patients. of RA patients had inherent or primary resistance to DMARDs, which might account for variable responses to DMARDs seen among RA patients during initial therapy. Similarly, some animal strains have displayed an inherent genetic basis for treatment failure of methotrexate in collagen-induced arthritis (14). The majority of cases of DMARD treatment Introduction failure are, however, considered to be the result of various underlying mechanisms, some of which could be specific to the type of DMARD used. Despite the current success of biological therapies (inhibitors of tumor necrosis factor [TNF], interleukin-1β-receptor antagonists and antibodies to CD20) (1-3), disease- Experimental studies of drug resistance mechanisms modifying antirheumatic drugs (DMARDs) have an established place in the treatment of The mechanisms of resistance to DMARDs might be similar to those of resistance to rheumatoid arthritis (RA) because of their convenience, safety and low related costs. One anticancer and antimicrobial drugs. Box 1 outlines several mechanisms of drug resistance common phenomenon associated with chronic DMARD treatment, however, is a gradual for disease-specific target cells; in RA, the causes of resistance could involve cells reduction in drug efficacy, which could point to the onset of drug resistance. Increases in of the immune system (T lymphocytes and macrophages) implicated in the disease drug doses might partly regain therapeutic efficacy, but this approach also increases the pathophysiology. Possible mechanisms of drug resistance include impaired drug

30 31 chapter 2 DMARD resistance: a review

Box 1 Multiple mechanisms of cellular resistance to different DMARDs. delivery to target cells, defective cellular uptake, increased drug extrusion, alterations in intracellular drug activation, target inhibition, processes downstream of target inhibition, or a combination of these features. For methotrexate, an example of impaired Methotrexate drug delivery is its metabolism to the less active metabolite 7-hydroxymethotrexate. For • Defective transport via reduced folate carrier (decreased protein levels, altered kinetics) sulfasalazine, the activity of intestinal drug-efflux transporters is a determining factor in • Slow transport via folate receptor (lower affinity for methotrexate than folic acid) variability of plasma drug concentrations (15). • Increased efflux via ATP-binding cassette (ABC) subfamily transporters ABCC1-ABCC5 Increased drug efflux has been recognized as an important mechanism of drug resistance. and ABCG2 (polymorphic variations) Cell membrane proteins responsible for drug efflux belong to the family of ATP-binding • Impaired polyglutamylation (decreased expression or activity of folylpolyglutamate synthetase protein; increased expression or activity of folylpolyglutamate hydrolase cassette (ABC) transporters (4, 16). A wide range of structurally and functionally different protein, polymorphic variants) drugs can be pumped out of the cell by these proteins, leading to multidrug resistance. • Altered target enzymes (increased dihydrofolate reductase activity or expression, Several DMARDs seem to be among the substrates of ABC transporters. kinetically altered dihydrofolate reductase, increased 5-aminoimidazole-4- A summary of reported mechanisms of resistance to various DMARDs is shown in Box 1. On carboxamide ribonucleotide formyltransferase activity, polymorphic variants) the basis of laboratory and preclinical data from all the DMARDs, resistance mechanisms • Increased salvage (exogenous/endogenous folates, purines) to methotrexate have been best characterized (17, 18) and are shown in Figure 1. A first • Increased metabolism of methotrexate to 7-OH-methotrexate in the liver limiting step in the mechanism of action of methotrexate is cellular uptake via the reduced folate carrier (RFC, also designated SLC19A1), which is constitutively expressed Sulfasalazine in almost all immune effector cells (19), or by folate receptor-β, which has a restricted • Increased efflux via ABCG2 (polymorphic variants) expression on activated macrophages, such as in the synovium (20). Reduced expression • Increased metabolism to 5-aminosalacylic acid and sulfapyridine in colon of, or alterations in, the transport kinetics of these proteins will diminish cellular uptake of Chloroquine and hydroxychloroquine methotrexate, resulting in lower intracellular concentrations of the drug, which will thus • Increased efflux via ABCB1, ABCC1 (polymorphic variants) not reach thresholds for therapeutic effects. Within the cell, methotrexate is converted • Sequestration in intracellular compartments (lysosomes) to polyanionic polyglutamated forms via the action of the enzyme folylpolyglutamate synthetase, which improves drug retention because the polyglutamated forms are Auranofin/aurothiomalate/gold poorer substrates for efflux transporters such as ABCC1–5 or ABCG2 (21, 22). Thus, • Increased expression of metallothioneins (metal-binding proteins) cells with a low capacity to retain polyglutamated forms of methotrexate (because of decreased folylpolyglutamate synthetase activity or increased activity of the Leflunomide breakdown enzyme folylpolyglutamate hydrolase) will be more prone to reduced activity • Altered target enzyme (increased activity or expression of dihydro-orotate dehydrogenase, kinetically altered protein) because of drug efflux mechanisms mediated by drug-transporters. Polyglutamated forms of methotrexate inhibit several key enzymes in folate metabolism (dihydrofolate Azathioprine reductase and thymidylate synthase) and purine metabolism (aminoimidazole • Increased efflux via ABCC4, ABCC5 carboxamide ribonucleotide transformylase). Elevated levels of these enzymes or • Increased activity of thiopurine methyltransferase linked to genetic polymorphism kinetically altered enzymes with lower binding affinities will necessitate higher doses of methotrexate to achieve optimum inhibition. Glucocorticoids For the DMARD sulfasalazine, increased drug efflux via ABCG2 has been reported as a • Decreased levels of glucocorticoid receptor α potential mechanism of cellular resistance (23, 24). Molecular mechanisms of resistance for • Increased ratio of glucocorticoid receptor β isoforms to glucocorticoid receptor α isoform the antimalarial DMARDs chloroquine and hydroxychloroquine include drug sequestration • Diminished phosphorylation of glucocorticoid receptors, leading to shorter half-life in lysosomal compartments or drug efflux via ABC transporters ABCB1 (also called Pgp) • Diminished crosstalk with transcription factors and ABCC1 (25). With respect to the DMARDs auranofin and aurothiomalate, studies by • Enhanced efflux via ABCB1 Glennas et al (26) revealed that upregulated expression of the metal-binding protein

32 33 chapter 2 DMARD resistance: a review

Figure 1 mechanisms of resistance to leflunomide in B cells revealed a marked upregulation of its target enzyme dihydro-orotate dehydrogenase (29), which therefore requires higher drug doses to be effectively inhibited. Finally, two reviews have summarized molecular mechanisms of resistance to glucocorticoids in RA. These mechanisms might involve reduced levels of the α isoform of the glucocorticoid receptor and an increased ratio of the β isoform to the α isoform, (the β isoform lacks the high affinity glucocorticoid- binding capacity and acts as a dominant-negative regulator of the α isoform), impaired crosstalk with transcription factors, or increased drug efflux via the multidrug resistance protein ABCB1 (30, 31). Clinical laboratory studies have identified several molecular mechanisms that could contribute to a reduction in efficacy of various DMARDs. To test whether these mechanisms, alone or in combination, are actually operative in a clinical setting will require further evaluation of specific markers for DMARD resistance in prospective clinical studies. One additional point of consideration is that the onset of resistance to a specific DMARD in immune effector cells might be accompanied by other genotypic Figure 1: Mechanisms of pharmacokinetic and cellular resistance to methotrexate. Metabolism of methotrexate (MTX) in the liver to partly inactive 7-hydroxy-methotrexate reduces and phenotypic changes that collaterally influence the efficacy of other DMARDs, the plasma concentrations of methotrexate and creates competition for cellular uptake via the RFC, which release of proinflammatory cytokines, or both. Findings from in vitro studies showed that also internalizes reduced folates and folic acid, although with low affinity for the latter. Folate receptors (FR) mediate low affinity endocytosis of methotrexate. Quantitative or qualitative alterations in RFC or acquired resistance to chloroquine in T cells is accompanied by a markedly reduced release folate-receptor expression confer transport-related resistance to methotrexate, as do high levels of of TNF and interleukin-8, along with a marked loss of sensitivity for glucocorticoids (25). exogenous folates competing for cell entry. Intracellularly, methotrexate is metabolized to polyglutamated Conversely, acquired resistance to sulfasalazine in the same T cells is accompanied by a forms by FPGS, and these are broken down by FPGH. The monoglutamate forms and, to some extend, polyglutamated forms are expelled via the transporters ABCC1–ABCC5 and ABCG2. ABCB1 does not markedly increased sensitivity for glucocorticoids (23). Hence, upon chronic exposure, transport anionic drugs such as methotrexate. Polyglutamated forms of methotrexate can inhibit several DMARDs might exert both beneficial and adverse pleiotropic effects. Such effects might key enzymes in folate metabolism, such as DHFR and TS, preventing de novo purine biosynthesis by APRTFase and AICARTFase. Enzyme inhibition, folate depletion and direct or indirect effects on cytokine have been unrecognized when the anti-inflammatory effects of specific DMARDs were release signaling pathways (such as those mediated by NFκB and IKK) all create routes via which methotrexate tested over a short time frame in a laboratory setting.

could suppress RA (51, 57, 58). Abbreviations: 5-CH3-THF, 5-methytetrahydrofolate; 5,10-CH2-THF,5,10- methylenetetrahydrofolate; 7-OH-MTX, 7-hydroxymethotrexaat; ABC, ATP-binding cassette (subfamily specific); AICARTFase, 5-aminoimidazole-4-carboxamide ribonucleotide formyltransferase; APRTFase, DMARD resistance or inefficacy in RA

amidophosphoribosyltransferase; DHFR, dihydrofolate reductase; FA, folic acid; FH2, dihydrofolate; FH4, Observational studies and meta-analyses of treatment efficacy, average treatment tetrahydrofolate; FPGH, folylpolyglutamate hydrolase; FPGS, folylpolyglutamate synthetase; FR, folate duration and adverse effects for RA patients consistently demonstrate variable responses receptor; IKK, inhibitor of IκB kinase; LV, leucovorin; MAPK, mitogen-activated protein kinase; MTHFR, methylenetetrahydrofolate reductase; MTX, methotrexate; MTX-PG, polyglutamated forms of methotrexate; for individual DMARDs and DMARD combinations (7, 8, 10-12, 32, 33). For example, in a 10- NFκB, nuclear factor κB; RFC, reduced folate carrier; TS, thymidylate synthase; TNF, tumor necrosis factor. year follow-up study (1985-1994), that included 2,296 RA patients, 25% of patients had to discontinue DMARD treatment owing to inefficacy and 20% as a result of adverse effects metallothionein was the dominant mechanism of acquired resistance to this DMARD. The (7, 33). The percentage of RA patients who had to discontinue DMARD monotherapy for DMARD azathioprine is a prodrug of the nucleoside analogue 6-mercaptopurine. At least inefficacy was highest for auranofin (37%), sulfasalazine (36%), and the antimalarials two mechanisms could be implicated in a diminished azathioprine activity: one relates to chloroquine and hydroxychloroquine (25%), and lowest for methotrexate (9%) (7). variations in metabolism (and toxicity) associated with specific gene polymorphisms of the Unfortunately, except for clinical endpoints, the molecular basis for DMARD inefficacy enzyme thiopurine methyltransferase (27); and in the other, two drug efflux transporters, was not further unraveled in these studies. This lack of findings was probably the results of ABCC4 and ABCC5, mediate cellular extrusion of 6-mercaptopurine to confer resistance an insufficient number of clinical samples and sample sizes of peripheral blood or synovial (28). Cellular resistance to ciclosporin has been reported in two laboratory studies but tissue, which made biochemical and molecular analyses of potential DMARD resistance- the molecular basis for resistance was not disclosed (5). Preliminary reports investigating related parameters impossible.

34 35 chapter 2 DMARD resistance: a review

Multidrug resistance and RA inflammatory responses can be modulated (5). Furthermore, evidence increasingly shows Over the past decade, attempts have been made to associate or correlate loss of DMARD that the transcriptional regulation of these transporters involves cytokine mediation (42). efficacy with one or more of the resistance parameters that have been identified in Hence, basal expression of individual drug efflux transporters on immune effector cells of laboratory studies. In particular, studies have assessed the potential role of multidrug RA patients could be a reflection of disease activity at the time therapy is started. Hider resistance proteins and aimed to establish parameters that could predict the response et al. (43) showed that ABCC1 expression on peripheral blood cells of RA patients declined to methotrexate. Early studies by Salmon and Dalton (34) suggested that the drug efflux compared with baseline after 6 months of therapy with methotrexate. Consistent with transporter ABCB1 might mediate the cellular release of TNF, but this hypothesis could the notion of cytokine mediation, expression of this drug efflux transporter might be not be confirmed experimentally. Maillefert et al. (35) observed ABCB1 expression on upregulated again when the efficacy of methotrexate treatment is reduced over time. peripheral blood cells in higher percentages of prednisone-treated RA patients (10.7%) than controls (3.3%). This finding might be consistent with the notion that prednisone Methotrexate efficacy or resistance in RA is a substrate for ABCB1. In a small group of eight RA patients, Llorente et al. (36) noted The discovery of different mechanisms of resistance to methotrexate from in vitro studies a higher percentage of peripheral blood lymphocytes with high ABCB1 activity among has led to the identification of common mechansisms for sensitivity or resistance in patients who did not respond to therapy than among those who did (34.4% vs. 3.3%) RA patients. Stranzl et al. (44) observed in a cross-sectional study that messenger RNA Finally, Yudoh et al. (37) noted higher percentages of T-helper-1 cells expressing ABCB1 in a levels of folylpolyglutamate synthetase were negatively correlated with response to group of RA patients who did not respond to therapy with bucillamine and sulfasalazine; methotrexate therapy. In 55% of 141 RA patients, no folylpolyglutamate synthetase mRNA 9.6% at baseline and 15.4% after 3 months of therapy versus 3.3% in responding patients was measurable; the response rate to methotrexate therapy was significantly higher at both baseline and 3 months of therapy. Commenting on this study, Rahman et al. than among patients with folylpolyglutamate synthetase mRNA expression (57% vs. (38) suggested that increased ABCB1 expression might also contribute to resistance to 33%; P = 0.005). A positive correlation would have been expected between expression methotrexate and referred to the findings of Norris et al. (39). In the study by Norris et and response, as proficient folylpolyglutamate synthetase activity should allow better al., however, human lymphoblastic cell lines were used that already had an extremely intracellular retention of methotrexate; however, as cellular activation or proliferation high level of resistance to methotrexate (four orders of magnitude higher than their can upregulate folylpolyglutamate synthetase mRNA levels and activity, the reduced methotrexate-sensitive counterparts) as measured from the defective cellular uptake via response rates might reflect augmented disease activity. the reduced folate carrier. In addition to this finding, ABCB1 contributed only to a minor In a study of 163 RA patients, Wolf et al. (45) analyzed parameters of cellular uptake extent to the methotrexate resistant phenotype. Given the general substrate preference transporters (RFC mRNA) and efflux transporters (functional activity of ABCC1–4). of ABCB1 (neutral or cationic compounds) a general role in transporting an anionic drug Patients with a high RFC status and low ABCC activity might be expected to respond such as methotrexate seems unlikely. Studies of ABCB1 in reconstituted vesicles produced well to methotrexate treatment. Irrespective of RFC status, however, more patients similar findings. The involvement of other drug transporters in methotrexate efflux has with ABCC activity on peripheral blood cells had better response rates, according to the been demonstrated (Figure 1) and could contribute to resistance to this drug. Preliminary European League Against Rheumatism criteria, than those with a low ABCC activity (53– data by Oerlemans et al. (40) show that the transporters ABCC1, ABCC5 and ABCG2 could 60% versus 29%, respectively). Whether low levels of RFC mRNA were actually translated be identified on monocyte-derived macrophages of RA patients. Detailed studies on the into negligible RFC protein levels was not established, and it is unclear how defective presence of drug efflux transporters in RA synovial tissue are lacking. Preliminary data methotrexate uptake could confer a good response. These parameters should be further from our laboratory revealed the presence of ABCG2 on macrophages in the synovial evaluated in prospective studies. sublining of a small group of RA patients (41). Studies are required to further elaborate on Several groups have investigated the contribution of common polymorphisms of genes or confirm possible correlations with DMARD efficacy. coding for folate or methotrexate transporters, such as RFC, and key enzymes in folate or purine metabolism (Figure 1) (46-51). They have been searching for associations with Multidrug resistance and inflammation toxic effects and response to methotrexate in RA patients. Dervieux et al. (47) reported As well as their possible role in DMARD resistance, drug efflux transporters have other that RA patients with homozygous variant genotypes, such as 80AA in the RFC region, physiologic functions. The substrates of ABCB1, ABCC1–ABCC5 and ABCG2, for example, 347GG in the aminoimidazolecarboxamide ribonucleotide transformylase region and include leukotrienes, prostaglandins, cyclic AMP, cyclic GMP and steroids, through which two 28 bp tandem repeats in the thymidylate synthase enhancer region (*2/*2), had a 3.7-

36 37 chapter 2 DMARD resistance: a review

fold increased likelihood of a good response after at least 3 months’ therapy . In addition, Box 2 General mechanisms of cellular drug resistance. patients with red blood cell concentrations of methotrexate long-chain polyglutamates of higher than 60 nmol/l were 14-fold more likely to have a good response. Impaired drug delivery to cells In another cross-sectional study, Dervieux et al. (48) showed that RA patients harboring Pharmacokinetic resistance or bioavailability (impaired intestinal absorption, raised urinary the 401TT genotype in the promoter region of folylpolyglutamate hydrolase, a lysosomal secretion, extensive protein binding) folate/methotrexate polyglutamate breakdown enzyme, were 4.8-fold more likely to have long-chain methotrexate polyglutamates in their red blood cells than patients Impaired cellular uptake with the CC or CT genotypes. A composite of cumulative homozygous genotypes Quantitative or qualitative defects of specific drug-uptake transporters associated with toxic side effects included methylenetetrahydrofolate reductase Increased cellular efflux 677TT, thymidylate synthase enhancer region *2/*2, aminoimidazolecarboxamide ribonucleotide transformylase 347GG and serine hydroxymethyltransferase 1420CC Impaired drug activation or increased intracellular drug inactivation (49). For methotrexate treatment in early RA, Wessels et al. (50) observed that patients Defective activation (phosphorylation, polyglutamylation) of drugs harboring the methylenetetrahydrofolate reductase 1298AA and 677CC genotypes Raised metabolism to non-active drugs showed a greater clinical improvement than those with the other genotype combinations, Sequestration in intracellular compartments (e.g. lysosomes) and patients carrying the methylenetetrahydrofolate reductase 1298C allele were more prone to methotrexate-related toxic effects. For all these polymorphic variants, the extent Alterations in drug target levels to which they translate into functionally altered proteins in terms of kinetics, stability or Increase in drug target (enzyme) levels (e.g. by transcriptional activation or gene amplification) other properties needs to be established. Whether these features are causative in relation Altered target (modified drug-binding properties) to methotrexate inefficacy or toxic effects also requires clarification. Ultimately, these polymorphic variants should prove their value in predicting responsiveness and toxicity Altered events downstream of target in prospective studies. Improved repair of drug-induced damage Defective drug-induced apoptosis Strategies to overcome DMARD resistance Improved bypassing of drug-induced effect Since resistance to anticancer drugs has various causes, several strategies have been proposed to overcome resistance (4), some of which might also apply to DMARD resistance in RA. In a clinical setting, when loss of efficacy for a particular DMARD is observed, making controlled, incremental increases in dose is a logical option until toxic effects manifest or, if drugs are used in combination, drug interactions become apparent (52, 53). In general, or taking advantage of the mode of resistance. Engagement, for example in the case of alternating drugs, either in a step-up approach (therapy is started as monotherapy and drug-efflux transporters, could involve the use of inhibitors specific to these proteins. In drug(s) are added in cases of insufficient response) or a step-down approach (therapy is cancer chemotherapy, however, this approach had, for various reasons, limited success started with multiple drugs but one is stopped at a time), is thought to slow the onset of (4). The lower drug doses given to RA patients might make an engagement strategy more drug resistance seen with monotherapy. In cases where combination DMARD therapy has efficacious, but the likelihood of this outcome has not yet been established. proved unsuccessful, addition of a third DMARD has proved to be highly efficacious in Blocking of drug efflux transporters could raise plasma and intracellular drug certain combinations (e.g. for methotrexate plus sulfasalazine plus hydroxychloroquine concentrations. Zaher et al. (15) showed that ABCG2-knockout mice had markedly altered (54) or methotrexate plus sulfasalazine plus prednisolone (3). Whether the superiority of pharmacokinetics for sulfasalazine; plasma levels were more than 100-fold higher than in these triple therapy schedules is explained by minimizing the contribution of resistance- control mice. These same raised plasma levels of sulfasalazine could be achieved in normal related parameters is not fully clear (25). mice by co-administering gefitinib, an ABCG2 inhibitor. This example suggests that when When a specific mechanism of resistance has been identified for a type of drug, including DMARDs are substrates for drug efflux transporters, therapy with transporter blockers DMARDs, strategies to overcome resistance might include engagement with, bypassing, could modulate plasma levels, reducing the risk of adverse effects.

38 39 chapter 2 DMARD resistance: a review

Accumulated knowledge on resistance mechanisms for methotrexate has led to the Key points rational design of second-generation folate antagonists. Use of these drugs might • Disease-modifying antirheumatic drugs (DMARDs) and anticancer drugs share common overcome the issue of methotrexate resistance, because they are more efficiently taken molecular mechanisms of resistance up by RFC and are better substrates for folylpolyglutamate synthetase, which prevents • Resistance to DMARDs can be acquired by upregulation of drug-efflux proteins efflux by drug exporters (55). Preliminary results in ex vivo experiments indicate that some belonging to the family of multidrug-resistance transporters of these second-generation folate antagonists are potent inhibitors of TNF production • Multidrug-resistance proteins exert primary physiological functions in the cellular in activated T cells of RA patients. Some second-generation folate antagonists might export of inflammatory mediators also target folate receptors (see Figure 1). By following this route of entry, they could • The current knowledge of molecular mechanisms of resistance to methotrexate bypass efflux routes encountered after cell entry via the RFC route. The current status facilitates the prediction of patient response to methotrexate by target-directed of this research is not, however, advanced enough to predict whether any of the second- genetic and biochemical screening of blood cells generation therapies will be able to replace methotrexate. • Identification of the molecular mechanisms of resistance to various DMARDs opens up new strategies for circumvention of drug resistance

Conclusions

Given the long duration of drug treatment that most RA patients face, maintaining the efficacy of DMARDs and biological agents for as long as possible is a challenge (3, 56). When efficacy is insufficient or lost, rheumatologists switch to alternative regimens, the long-lasting effects of which might be unpredictable. Interdisciplinary research into drug resistance has provided extensive knowledge of mechanisms associated with resistance and toxic effects. Some of the findings might apply to DMARD treatment in RA. Laboratory studies have revealed various factors that can contribute to diminished activity of specific DMARDs (Box 2), some of which are currently being evaluated in clinical practice. Of particular interest are studies aiming to predict response to methotrexate as it is used as the main drug in RA treatment. The most useful studies will assess response in relation to specific resistance-related parameters during the course of therapy that may precede clinical signs of loss of efficacy. This approach will allow the extrapolation of data into predictive criteria. Thus, pharmacogenetically guided prospective clinical studies in early RA are warranted. The investigations of ways in which to make more efficient use of the currently available DMARDs would also be useful. For example, modulation of pharmacokinetics by blocking intestinal drug-efflux transporters and ways in which second-generation of drugs might overcome resistance deserve further attention. Finally, beyond the level of immune effector cells in peripheral blood, more insight into factors of DMARD resistance related to inflamed synovial tissue would be helpful to assist therapeutic interventions. Any extension of DMARD efficacy over time in monotherapy or in combination with other DMARDs or biological agents will be beneficial from therapeutic and socioeconomic perspectives.

40 41 References References

References 2003. 20 Nakashima-Matsushita,N., Homma,T., Yu,S., Matsuda,T., Sunahara,N., Nakamura,T., Tsukano,M., 1 Smolen,J.S. and Steiner,G. Therapeutic strategies for rheumatoid arthritis, Nat.Rev.Drug Discov., Ratnam,M. and Matsuyama,T. Selective expression of folate receptor beta and its possible role 2: 473-488, 2003. in methotrexate transport in synovial macrophages from patients with rheumatoid arthritis, 2 O’Dell,J.R. Therapeutic strategies for rheumatoid arthritis, N.Engl.J.Med., 350: 2591-2602, 2004. Arthritis Rheum., 42: 1609-1616, 1999. 3 Goekoop-Ruiterman,Y.P., De Vries-Bouwstra,J.K., Allaart,C.F., van Zeben,D., Kerstens,P.J., Hazes,J. 21 Hooijberg,J.H., Broxterman,H.J., Kool,M., Assaraf,Y.G., Peters,G.J., Noordhuis,P., Scheper,R.J., M., Zwinderman,A.H., Ronday,H.K., Han,K.H., Westedt,M.L., Gerards,A.H., van Groenendael,J. Borst,P., Pinedo,H.M. and Jansen,G. Antifolate resistance mediated by the multidrug resistance H., Lems,W.F., van Krugten,M.V., Breedveld,F.C. and Dijkmans,B.A.C. Clinical and radiographic proteins MRP1 and MRP2, Cancer Res., 59: 2532-2535, 1999. outcomes of four different treatment strategies in patients with early rheumatoid arthritis (the 22 Shafran,A., Ifergan,I., Bram,E., Jansen,G., Kathmann,I., Peters,G.J., Robey,R.W., Bates,S.E. and BeSt study): a randomized, controlled trial, Arthritis Rheum., 52: 3381-3390, 2005. Assaraf,Y.G. ABCG2 harboring the Gly482 mutation confers high-level resistance to various 4 Szakacs,G., Paterson,J.K., Ludwig,J.A., Booth-Genthe,C. and Gottesman,M.M. Targeting hydrophilic antifolates, Cancer Res., 65: 8414-8422, 2005. multidrug resistance in cancer, Nat.Rev.Drug Discov., 5: 219-234, 2006. 23 Van der Heijden,J., de Jong,M.C., Dijkmans,B.A.C., Lems,W.F., Oerlemans,R., Kathmann,G.A., 5 Jansen,G., Scheper,R.J. and Dijkmans,B.A.C. Multidrug resistance proteins in rheumatoid Scheffer,G.L., Scheper,R.J., Assaraf,Y.G. and Jansen,G. Acquired resistance of human T cells to arthritis, role in disease-modifying antirheumatic drug efficacy and inflammatory processes: an sulphasalazine: stability of the resistant phenotype and sensitivity to non-related DMARDs, Ann. overview, Scand.J.Rheumatol., 32: 325-336, 2003. Rheum.Dis., 63: 131-137, 2004. 6 Wollheim,F.A. Drug resistance in rheumatology: an area in search of investigators, Curr. 24 Van der Heijden,J., de Jong,M.C., Dijkmans,B.A.C., Lems,W.F., Oerlemans,R., Kathmann,G. Rheumatol.Rep., 5: 333-335, 2003. A., Schalkwijk,C.G., Scheffer,G.L., Scheper,R.J. and Jansen,G. Development of sulphasalazine 7 Fleischmann,R.M. Is there a need for new therapies for rheumatoid arthritis?, J.Rheumatol.Suppl, resistance in human T cells induces expression of the multidrug resistance transporter ABCG2 73: 3-7, 2005. (BCRP) and augmented production of TNFalpha, Ann.Rheum.Dis., 63: 138-143, 2004. 8 Choy,E.H., Smith,C., Dore,C.J. and Scott,D.L. A meta-analysis of the efficacy and toxicity of 25 Oerlemans,R., van der Heijden J., Vink,J., Dijkmans,B.A.C., Kaspers,G.J., Lems,W.F., Scheffer,G.L., combining disease-modifying anti-rheumatic drugs in rheumatoid arthritis based on patient Ifergan,I., Scheper,R.J., Cloos,J., Assaraf,Y.G. and Jansen,G. Acquired resistance to chloroquine in withdrawal, Rheumatology.(Oxford), 44: 1414-1421, 2005. human CEM T cells is mediated by multidrug resistance-associated protein 1 and provokes high 9 Nurmohamed,M.T. and Dijkmans,B.A.C. Efficacy, tolerability and cost effectiveness of disease- levels of cross-resistance to glucocorticoids, Arthritis Rheum., 54: 557-568, 2006. modifying antirheumatic drugs and biologic agents in rheumatoid arthritis, Drugs, 65: 661-694, 26 Glennas,A. and Rugstad,H.E. Cultured human cells with high levels of gold-binding cytosolic 2005. metallothionein are not resistant to the growth inhibitory effect of sodium aurothiomalate, Ann. 10 Aletaha,D. and Smolen,J.S. Effectiveness profiles and dose dependent retention of traditional Rheum.Dis., 45: 101-109, 1986. disease modifying antirheumatic drugs for rheumatoid arthritis. An observational study, 27 Clunie,G.P. and Lennard,L. Relevance of thiopurine methyltransferase status in rheumatology J.Rheumatol., 29: 1631-1638, 2002. patients receiving azathioprine, Rheumatology.(Oxford), 43: 13-18, 2004. 11 Bingham,S. and Emery,P. Resistant rheumatoid arthritis clinics- a necessary development?, 28 Wielinga,P.R., Reid,G., Challa,E.E., van der Heijden,I., van Deemter,L., de Haas,M., Mol,C., Kuil,A. Rheumatology.(Oxford), 39: 2-5, 2000. J., Groeneveld,E., Schuetz,J.D., Brouwer,C., De Abreu,R.A., Wijnholds,J., Beijnen,J.H. and Borst,P. 12 Wolfe,F. The epidemiology of drug treatment failure in rheumatoid arthritis, Baillieres Clin. Thiopurine metabolism and identification of the thiopurine metabolites transported by MRP4 Rheumatol., 9: 619-632, 1995. and MRP5 overexpressed in human embryonic kidney cells, Mol.Pharmacol., 62: 1321-1331, 2002. 13 Morgan,C., Lunt,M., Brightwell,H., Bradburn,P., Fallow,W., Lay,M., Silman,A. and Bruce,I.N. 29 Loffler,M., Klein,A., Hayek-Ouassini,M., Knecht,W. and Konrad,L. Dihydroorotate dehydroge- Contribution of patient related differences to multidrug resistance in rheumatoid arthritis, Ann. nase mRNA and protein expression analysis in normal and drug-resistant cells, Nucleosides Rheum.Dis., 62: 15-19, 2003. Nucleotides Nucleic Acids, 23: 1281-1285, 2004. 14 Delano,D.L., Montesinos,M.C., Desai,A., Wilder,T., Fernandez,P., D’Eustachio,P., Wiltshire,T. and 30 Chikanza,I.C. and Kozaci,D.L. Corticosteroid resistance in rheumatoid arthritis: molecular and Cronstein,B.N. Genetically based resistance to the antiinflammatory effects of methotrexate in cellular perspectives, Rheumatology.(Oxford), 43: 1337-1345, 2004. the air-pouch model of acute inflammation, Arthritis Rheum., 52: 2567-2575, 2005. 31 Buttgereit,F., Straub,R.H., Wehling,M. and Burmester,G.R. Glucocorticoids in the treatment of 15 Zaher,H., Khan,A.A., Palandra,J., Brayman,T.G., Yu,L. and Ware,J.A. Breast Cancer Resistance rheumatic diseases: an update on the mechanisms of action, Arthritis Rheum., 50: 3408-3417, Protein (Bcrp/abcg2) is a major determinant of sulfasalazine absorption and elimination in 2004. mouse, Molecular Pharmaceutics, 3: 55-61, 2006. 32 Maetzel,A., Wong,A., Strand,V., Tugwell,P., Wells,G. and Bombardier,C. Meta-analysis of 16 Borst,P. and Oude Elferink,R.P. Mammalian ABC transporters in health and disease, Annu.Rev. treatment termination rates among rheumatoid arthritis patients receiving disease-modifying Biochem., 71: 537-592, 2002. anti-rheumatic drugs, Rheumatology.(Oxford), 39: 975-981, 2000. 17 Gorlick,R., Goker,E., Trippett,T., Waltham,M., Banerjee,D. and Bertino,J.R. Intrinsic and acquired 33 Galindo-Rodriguez,G., vina-Zubieta,J.A., Russell,A.S. and Suarez-Almazor,M.E. Disappointing resistance to methotrexate in acute leukemia, N.Engl.J.Med., 335: 1041-1048, 1996. longterm results with disease modifying antirheumatic drugs. A practice based study, 18 Rots,M.G., Pieters,R., Kaspers,G.J., Veerman,A.J., Peters,G.J. and Jansen,G. Classification of ex J.Rheumatol., 26: 2337-2343, 1999. vivo methotrexate resistance In acute lymphoblastic and myeloid leukaemia, Br.J.Haematol., 110: 34 Salmon,S.E. and Dalton,W.S. Relevance of multidrug resistance to rheumatoid arthritis: 791-800, 2000. development of a new therapeutic hypothesis, J.Rheumatol.Suppl, 44: 97-101, 1996. 19 Matherly,L.H. and Goldman,D.I. Membrane transport of folates, Vitam.Horm., 66: 403-456, 35 Maillefert,J.F., Maynadie,M., Tebib,J.G., Aho,S., Walker,P., Chatard,C., Dulieu,V., Bouvier,M.,

42 43 References References

Carli,P.M. and Tavernier,C. Expression of the multidrug resistance glycoprotein 170 in the 50 Wessels,J.A., De Vries-Bouwstra,J.K., Heijmans,B.T., Slagboom,P.E., Goekoop-Ruiterman,Y. peripheral blood lymphocytes of rheumatoid arthritis patients. The percentage of lymphocytes P., Allaart,C.F., Kerstens,P.J., van Zeben,D., Breedveld,F.C., Dijkmans,B.A.C., Huizinga,T.W. and expressing glycoprotein 170 is increased in patients treated with prednisolone, Br.J.Rheumatol., Guchelaar,H.J. Efficacy and toxicity of methotrexate in early rheumatoid arthritis are associated 35: 430-435, 1996. with single-nucleotide polymorphisms in genes coding for folate pathway enzymes, Arthritis 36 Llorente,L., Richaud-Patin,Y., Diaz-Borjon,A., Alvarado,D.L.B., Jakez-Ocampo,J., De La Fuente Rheum., 54: 1087-1095, 2006. H., Gonzalez-Amaro,R. and Diaz-Jouanen,E. Multidrug resistance-1 (MDR-1) in rheumatic 51 Kremer,J.M. Toward a better understanding of methotrexate, Arthritis Rheum., 50: 1370-1382, autoimmune disorders. Part I: Increased P-glycoprotein activity in lymphocytes from rheumatoid 2004. arthritis patients might influence disease outcome, Joint Bone Spine, 67: 30-39, 2000. 52 Smolen,J.S., Aletaha,D. and Keystone,E. Superior efficacy of combination therapy for rheumatoid 37 Yudoh,K., Matsuno,H., Nakazawa,F., Yonezawa,T. and Kimura,T. Increased expression of arthritis: fact or fiction?, Arthritis Rheum., 52: 2975-2983, 2005. multidrug resistance of P-glycoprotein on Th1 cells correlates with drug resistance in rheumatoid 53 Jansen,G., Van der Heijden,J.W., Oerlemans,R., Lems,W.F., Ifergan,I., Scheper,R.J., Assaraf,Y.G. arthritis, Arthritis Rheum., 42: 2014-2015, 1999. and Dijkmans,B.A.C. Sulfasalazine is a potent inhibitor of the reduced folate carrier: implications 38 Rahman,P., Hefferton,D. and Robb,D. Increased MDR1 P-glycoprotein expression in methotrexate for combination therapies with methotrexate in rheumatoid arthritis, Arthritis Rheum., 50: 2130- resistance: comment on the article by Yudoh et al, Arthritis Rheum., 43: 1661-1662, 2000. 2139, 2004. 39 Norris,M.D., De Graaf,D., Haber,M., Kavallaris,M., Madafiglio,J., Gilbert,J., Kwan,E., Stewart,B. 54 O’Dell,J.R., Leff,R., Paulsen,G., Haire,C., Mallek,J., Eckhoff,P.J., Fernandez,A., Blakely,K., W., Mechetner,E.B., Gudkov,A.V. and Roninson,I.B. Involvement of MDR1 P-glycoprotein in Wees,S., Stoner,J., Hadley,S., Felt,J., Palmer,W., Waytz,P., Churchill,M., Klassen,L. and Moore,G. multifactorial resistance to methotrexate, Int.J.Cancer, 65: 613-619, 1996. Treatment of rheumatoid arthritis with methotrexate and hydroxychloroquine, methotrexate 40 Oerlemans,R., Dijkmans,B.A.C., Van der Heijden,J.W., Lems,W.F., Reurs,A.W., Scheffer,G.L., and sulfasalazine, or a combination of the three medications: Results of a two-year, randomized, Scheper,R.J. and Jansen,G. Differential expression of multidrug resistance-related proteins double-blind, placebo-controlled trial, Arthritis Rheum., 46: 1164-1170, 2002. on monocyte-derived macrophages from rheumatoid arthritis patients, Arthritis Research & 55 Rots,M.G., Pieters,R., Peters,G.J., Van Zantwijk,C.H., Mauritz,R., Noordhuis,P., Willey,J.C., Therapy, 7: S32, 2005. Hahlen,K., Creutzig,U., Janka-Schaub,G., Kaspers,G.J., Veerman,A.J. and Jansen,G. Circumvention 41 Van der Heijden,J.W., Oerlemans,R., Tak,P.P., Smeets,T.J.M., Scheper,R.J., Scheffer,G.L., Assaraf,Y. of methotrexate resistance in childhood leukemia subtypes by rationally designed antifolates, G., Lems,W.F., Dijkmans,B.A.C. and Jansen,G. Expression of the multidrug resistance protein Blood, 94: 3121-3128, 1999. BCRP in synovial tissue of RA patients - a marker for inflammation or resistance to MTX?, Arthritis 56 Wolbink,G.J., Vis,M., Lems,W., Voskuyl,A.E., de Groot,E., Nurmohamed,M.T., Stapel,S., Tak,P. Rheum., 52: S540, 2005. P., Aarden,L. and Dijkmans,B.A.C. Development of antiinfliximab antibodies and relationship to 42 Scotto,K.W. Transcriptional regulation of ABC drug transporters, Oncogene, 22: 7496-7511, clinical response in patients with rheumatoid arthritis, Arthritis Rheum., 54: 711-715, 2006. 2003. 57 Cronstein,B.N. Molecular therapeutics. Methotrexate and its mechanism of action, Arthritis 43 Hider,S.L., Owen,A., Hartkoorn,R., Khoo,S., Back,D.J., Silman,A. and Bruce,I.N. Down-Regulation Rheum., 39: 1951-1960, 1996. Of MRP1 Expression In Early RA Patients Exposed To Methotrexate As A First DMARD, Ann Rheum. 58 Cutolo,M., Sulli,A., Pizzorni,C., Seriolo,B. and Straub,R.H. Anti-inflammatory mechanisms of Dis., 65: 1390-1393, 2006. methotrexate in rheumatoid arthritis, Ann.Rheum.Dis., 60: 729-735, 2001. 44 Stranzl,T., Wolf,J., Leeb,B.F., Smolen,J.S., Pirker,R. and Filipits,M. Expression of folylpolyglutamyl synthetase predicts poor response to methotrexate therapy in patients with rheumatoid arthritis, Clin.Exp.Rheumatol., 21: 27-32, 2003. 45 Wolf,J., Stranzl,T., Filipits,M., Pohl,G., Pirker,R., Leeb,B.F. and Smolen,J.S. Expression of resistance markers to methotrexate predicts clinical improvement in patients with rheumatoid arthritis, Ann.Rheum.Dis., 64: 564-568, 2004. 46 Ranganathan,P., Eisen,S., Yokoyama,W.M. and McLeod,H.L. Will pharmacogenetics allow better prediction of methotrexate toxicity and efficacy in patients with rheumatoid arthritis?, Ann. Rheum.Dis., 62: 4-9, 2003. 47 Dervieux,T., Furst,D., Lein,D.O., Capps,R., Smith,K., Walsh,M. and Kremer,J. Polyglutamation of methotrexate with common polymorphisms in reduced folate carrier, aminoimidazole carboxamide ribonucleotide transformylase, and thymidylate synthase are associated with methotrexate effects in rheumatoid arthritis, Arthritis Rheum., 50: 2766-2774, 2004. 48 Dervieux,T., Kremer,J., Lein,D.O., Capps,R., Barham,R., Meyer,G., Smith,K., Caldwell,J. and Furst,D.E. Contribution of common polymorphisms in reduced folate carrier and gamma- glutamylhydrolase to methotrexate polyglutamate levels in patients with rheumatoid arthritis, Pharmacogenetics, 14: 733-739, 2004. 49 Weisman,M.H., Furst,D.E., Park,G.S., Kremer,J.M., Smith,K.M., Wallace,D.J., Caldwell,J. R. and Dervieux,T. Risk genotypes in folate-dependent enzymes and their association with methotrexate-related side effects in rheumatoid arthritis, Arthritis Rheum., 54: 607-612, 2006.

44 45 chapter 3 Development of sulfasalazine resistance in human T cells induces expression of the multidrug resistance transporter ABCG2 (BCRP) and augmented production of TNFα

Joost W. van der Heijden1, Mariska C. de Jong2, Ben A.C. Dijkmans1, Willem F. Lems1, Ruud Oerlemans1, Ietje Kathmann3, Casper G. Schalkwijk4, George L. Scheffer2, Rik J. Scheper2 and Gerrit Jansen1

Departments of 1Rheumatology, 2Pathology, 3Medical Oncology and 4Clinical Chemistry, VU University Medical Center, Amsterdam, The Netherlands

Annals of the Rheumatic diseases 2004; 63: 138-143 chapter 3 Sulfasalazine resistance in T-cells is mediated by BCRP

Abstract activity, most withdrawals of SSZ resulted from the lack of efficacy rather than toxicity (13). The underlying mechanism(s) for this lack of efficacy has not been established. Given Objective: To determine whether overexpression of cell membrane associated drug the long term DMARD treatment RA patients receive, we rationalized that the onset of efflux pumps belonging to the family of ATP binding cassette (ABC) proteins contributes acquired drug resistance to SSZ, analogous to resistance to anticancer drugs or anti- to a diminished efficacy of sulfasalazine (SSZ) after prolonged cellular exposure to this infectious drugs (14-16), might contribute to its lack of efficacy. From an anti-inflammatory disease modifying antirheumatic drug. perspective, drug resistance may refer to a diminished ability of DMARDs to inhibit Methods: A T-cell model system of human CEM (T) cells was used to expose cells in secretion of pro-inflammatory cytokines by inflammatory cells. Mechanistically, this can vitro to increasing concentrations of SSZ for a period up to six months. Cells were then be provoked by either augmented basal levels of cytokine secretion or, indirectly, through characterized for the expression of drug efflux pumps: P-glycoprotein (Pgp, ABCB1), attenuated antiproliferative/apoptotic effects of DMARDs. Diminished antiproliferative multidrug resistance protein 1 (MRP1, ABCC1) and Breast Cancer Resistance Protein (BCRP, effects mediated by overexpression of specific energy (ATP) dependent drug efflux pumps ABCG2). are a common and established mechanism of resistance to anticancer drugs. These drug Results: Prolonged exposure of CEM (T) cells to SSZ provoked resistance to SSZ as efflux pumps belong to a superfamily of ATP-Binding Cassette (ABC) transporters (17- manifested by a 6.4-fold diminished antiproliferative effect of SSZ compared with 19). ABC transporters with an established role in drug resistance include: P-glycoprotein parental CEM (T) cells. CEM cells resistant to SSZ (CEM/SSZ) showed a marked induction (Pgp/ABCB1) (20), Multidrug Resistance associated Proteins 1-5 (MRP1-5/ABCC1-5) (21, of ABCG2/BCRP, Pgp expression was not detectable, while MRP1 expression was even 22) and Breast Cancer Resistance Protein (BCRP/ABCG2) (15, 23). Currently, very little is down regulated. A functional role of ABCG2/BCRP in SSZ resistance was demonstrated known about SSZ as a potential substrate for one or more of the different MDR pumps or by a 60% reversal of SSZ resistance by the ABCG2/BCRP blocker Ko143. Release of the other transporters (24). On the basis of its chemical structure as organic anion, SSZ might proinflammatory cytokine tumour necrosis factor α (TNFα) was threefold higher in CEM/ belong to a class of compounds transported by the drug efflux pump MRP1 that has a SSZ cells than in CEM cells. Moreover, twofold higher concentrations of SSZ were required preferred substrate affinity of anionic (glutathione-conjugated) compounds (21, 22). to inhibit TNFα production from CEM/SSZ cells compared with CEM cells. To gain insight from a rheumatological perspective into possible mechanism(s) of Conclusion: Collectively, ABCG2 induction, augmented TNFα release and less efficient resistance to the anti-proliferative/anti-inflammatory effects of SSZ, we provoked inhibition of TNFα production by SSZ may contribute to diminished efficacy after acquired resistance to SSZ in an in vitro model system for human T cells (CEM) by stepwise prolonged exposure to SSZ. These results warrant further clinical studies to verify whether exposure of CEM (T) cells to gradually increasing concentrations of SSZ. drug efflux pumps, originally identified for their roles in cytostatic drug resistance, can In this study we observed that SSZ resistance in CEM (T) cells was conferred by also be induced by SSZ or other disease-modifying antirheumatic drugs. overexpression of a drug efflux pump, the multidrug resistance transporter BCRP/ ABCG2. Consistent with reduced drug uptake, inhibition of nuclear NFκB activity in SSZ- resistant cells, as reflected by TNFα production, required at least two-fold higher SSZ Introduction concentrations as compared with CEM (T) cells.

Sulfasalazine (SSZ) is a widely applied disease modifying anti-rheumatic drug (DMARD), either as a single agent or in combination with other DMARDs (1-5). The anti-inflammatory Materials and methods properties of SSZ have been attributed to diminished production of pro-inflammatory cytokines such as Tumor Necrosis Factor α (TNFα) trough multiple mechanisms, including Materials (a) inhibition of the activation of nuclear factor kappa B (NFκB) by inhibition of Inhibitor Sulfasalazine, 5’-aminosalicylic acid, sulfapyridine, phorbol 12-myristate 13-acetate (PMA), kappa B Kinase (IKK) (6, 7), (b) through inhibition of the purine biosynthesis de novo ionomycine, verapamil and phenylmethylsulfonyl fluoride (PMSF) were purchased from enzyme 5-aminoimidazole-4-carboxamideribonucleotide (AICAR) transformylase (8, 9) Sigma Chem Co. (St. Louis, MO, U.S.A.). Protease Inhibitor Cocktail (PIC) and Triton X-100 and (c) by induction of apoptosis in lymphocytes and macrophages (10, 11). were from Boehringer Mannheim (Ingelheim, Germany). The ABCG2 inhibitor Ko143 (25) In clinical practice the median duration of use of SSZ is 1-2 years (12). A meta-analysis was kindly provided by Dr A. Schinkel (Netherlands Cancer Institute, Amsterdam, the of DMARD treatment termination rates based on 159-studies showed that after initial Netherlands). The MRP1 blocker MK571 was provided by Merck Frosst, Quebec, Canada.

48 49 chapter 3 Sulfasalazine resistance in T-cells is mediated by BCRP

RPMI-1640 tissue culture medium and fetal calf serum (FCS) were obtained from Gibco Western blotting Chemical Co, Grand Island, NY, USA. For analysis of expression of Pgp, MRP1 and BCRP/ABCG2 cells were harvested in the mid-log phase of growth and washed three times with ice cold Hepes buffered saline, Selection of SSZ resistance in CEM (T) cells pH 7.4. Total cell lysates of 107 cells were prepared by suspending them in 500 µl of lysis

In this study human CEM T-lineage lymphocytic cells were used as a model system for buffer containing 50 mM Tris-HCl (pH 7.6), 5 mM DTT, 20 µl PIC (1 tablet/ml H2O), 20% human T cells. CEM (T) cells were originally isolated by Foley et al. (26) and characterized by glycerol and 0.5% NP-40. The suspension was sonicated (MSE sonicator, amplitude 6, several T cell marker molecules, including CD3, CD4, CD8 and CD25 (27). CEM (T) cells have for 3x5 seconds with 30 second time intervals at 4oC) and centrifuged in an Eppendorf been employed as a representative model to study resistance mechanisms for DMARDs microcentrifuge (10 minutes, 14000 rpm, 4oC). Protein content of the supernatant was like methotrexate (28-30). CEM (T) cells were cultured at an initial density of 3 x 105 cells/ determined by Biorad protein assay. Fifty microgram of total cell lysates was fractionated ml in RPMI-1640 medium supplemented with 10% FCS, 2 mM L-glutamine, and 100 µg/ml on a 7.5% polyacrylamide gel and transferred onto a nitrocellulose membrane. The

o o penicillin and streptomycin in a 5% CO2 incubator at 37 C as described previously (31).Cell nitrocellulose membranes were pre-incubated overnight at 4 C in blocking buffer (5% cultures were refreshed twice weekly. SSZ was added to the suspension culture of CEM (T) Biorad Blocker in TBS-T (10 mM Tris-HCl, pH 8.0, 0.15 M NaCl, 0.1% Tween-20) to prevent cells at an initial concentration of 0.4 mM (IC50 concentration). When cell growth in SSZ- aspecific antibody binding. After blocking, the membranes were incubated for 1 hour containing medium approximated growth of control CEM (T) cells, SSZ concentrations in at room temperature with the primary antibodies for Pgp (JSB1, 1:500), MRP1 (MRPr1, the medium were step wise increased by 0.1-0.2 mM. After a period of 4-6 months SSZ- 1:500) or ABCG2/BCRP (BXP21, 1:400) as described by Scheffer et al. (32, 33). As control resistant CEM (T) cells selected at 1.5 mM SSZ (CEM/SSZ1.5) and 2.5 mM SSZ (CEM/SSZ2.5) for loading, β-actin was used (MAB1501R, 1:3000, Chemicon International, Ca, U.S.A.). were used for further characterization. After three washing steps with TBS-T, the membrane was incubated for one hour with Antiproliferative effects of SSZ were analyzed by plating 1.25 x 105 cells/ml in individual horseradish peroxidise labelled anti-rat/mouse (1:2000, Dako) as secondary antibody. wells of a 24 well plate containing up to 50 µl of SSZ solutions. Inhibition of cell growth Detection of the antibody binding was measured by enhanced chemoluminescence (ECL) was determined after 72 hours’ incubation by counting viable cells on the basis of trypan according to the manufacturers’ instructions (Amersham International, Buckinghamshire, blue exclusion. The SSZ concentration required to inhibit cell growth by 50% compared to UK). Protein levels were determined by densitometric scanning (GelDoc and Molecular control growth is defined as IC50 concentration. Analyst, Biorad Laboratories) of the X-ray films (Hyperfim ECL, Amersham International, Buckinghamshire, UK). Nuclear fractionation For analysis of the presence of the p65 subunit of NFkB in the nucleus, 30 µg of nuclear Ten million CEM (T) and CEM/SSZ1.5 cells were harvested and washed three times with protein was fractionated on a 7.5% polyacrylamide gel, transferred to nitrocellulose 25 ml ice cold Hepes buffered saline (pH 7.4). All further experiments were carried out at membranes, and probed with NFκB p65 antibody (Santa Cruz, sc8008, 1: 2000) before ECL o 4 C. The final cell pellet was suspended in 0.5 ml buffer A (10 mM Hepes, 1.5 mM MgCl2, detection as described above. 10 mM KCl, 1 mM dithiotreitol (DTT), 1 mM phenylmethylsulfonyl fluoride (PMSF, pH 7.9) and centrifuged (5 minutes; 1200 rpm). The pellet was resuspended in 80 µl of buffer A TNFα ELISA supplemented with 0.1% Triton X-100 and 3 µl Protease Inhibitor Cocktail (PIC; 1 tablet/ml TNFα production was analyzed after stimulation of CEM (T) and CEM/SSZ cells (3x105/ o H2O). After 10 min at 4 C the suspension was centrifuged (5 minutes; 1200 rpm) and the ml) for 24 hours with PMA (10 ng/ml)/ionomycin (1 µM). After this period, supernatants pellet was washed once with 0.5 ml buffer A and centrifuged (5 minutes; 1200 rpm). The were collected by centrifugation (5 minutes; 3000 rpm) and analyzed for TNFα by ELISA pellet was carefully resuspended in 70 µl buffer B (20 mM Hepes, 25% (v/v) glycerol, 0.42 M (Central Laboratory of Blood Transfusion, Amsterdam) according to the manufacturer’s

NaCl, 1.5 mM MgCl2, 0.2 mM EDTA, 1 mM DTT, 1 mM PMSF and 3 µl PIC, pH 7.9) to allow lysis instruction. The detection limit of the TNFα ELISA was 5 pg/ml. of the nuclear membrane. After 30 minutes’ incubation the extract was centrifuged in an Eppendorf microcentrifuge (20 min, 14,000 rpm, 4oC). Aliquots of the nuclear extracts Statistical analysis were sampled for protein content (Biorad assay), the remainder was stored at –80oC for Statistical significance of differences was analyzed by Student’s t-test. A p-value < 0.05 NFκB p65 Western blotting. was considered to be statistically different.

50 51 chapter 3 Figuur_2B.pzf:Data 1 graph - Mon Aug 18 13:26:56 2008Sulfasalazine resistance in T-cells is mediated by BCRP

Results Figure 2 A Onset of SSZ resistance in CEM (T) cells To investigate whether CEM (T) cells developed over time less sensitivity to SSZ, CEM (T) cells were continuously exposed in vitro to stepwise increasing concentrations of SSZ. After a period of 4-6 months’ culture, CEM (T) cells could be maintained in SSZ concentrations that exceeded 3.5-6 fold the initial SSZ selection concentration of 0.4 mM SSZ (Figure 1A). Cells selected at an intermediate level of resistance (1.5 mM SSZ) and a final level of 2.5 mM SSZ were used for further characterization. Figure 1B shows the antiproliferative effect of SSZ against CEM (T) cells, CEM/SSZ1.5 and CEM/SSZ2.5. Concentrations of SSZ required for 50% growth inhibition were 4.3-fold (p<0.001) and 6.4-fold (p<0.001) higher for CEM/ SSZ1.5 (IC50: 1.72 ±0.17 mM) and CEM/SSZ2.5 cells (IC50: 2.55 ±0.21 mM), respectively, as Figure 2 B

compared to CEM (T) cells (IC50: 0.40 ± 0.03 mM). 120 CEM (T) cells Figuur_1A.pzf:Data 1 graph - Mon Aug 18 13:25:21 2008 Because in vivo SSZ can be catabolised into two components, that is 5’-aminosalicylic 100 CEM/SSZ1.5 Figuur_1B.pzf:Data 1 graph - Mon Aug 18 13:26:32 2008 ABCG2 blocker Ko143 acid (5’-ASA) and sulfapyridine (SP) (34), we analyzed whether one or both of these 80 Pgp blocker Verapamil components might contribute to the SSZ resistant phenotype. It was noted that neither 5’- 60 MRP1 blocker MK571 ASA nor SP, either separate or combined, displayed an antiproliferative effect at equimolar 40 concentrations of intact SSZ (results not shown). 20 These results indicate that human CEM (T) cells can develop resistance to SSZ upon long Cell growth (% of control) 0 term exposure to this DMARD. Resistance to SSZ does not involve its in vivo catabolites 0.0 0.5 1.0 1.5 2.0 2.5 [SSZ] (mmol/l) 5’-ASA and SP.

Expression of multidrug resistance transporters in CEM (T) and CEM/SSZ cells Figure 2: (A) Expression of MDR transporters Pgp, MRP1 and ABCG2/BCRP in CEM (T) and CEM/SSZ cells. Because SSZ is an organic anion that might be a potential substrate for ATP-driven drug Western blots were performed using total cell lysates of CEM (T) and CEM/SSZ1.5 cells. Cell lysates of CEM/ VBL100 (64), 2008/MRP1 (32) and MCF7/MR (40) served as positive control for Pgp, MRP1 and ABCG2/BCRP, efflux pumps, we investigated whether SSZ resistance in CEM (T) cells was mediated by respectively. For CEM (T) and CEM/SSZ1.5 cells, 50 µg of cell lysate was applied on the SDS-PAGE gel and for the controls 10 µg protein. Pgp was detected by the monoclonal antibody JSB1, MRP1 was detected by the monoclonal antibody MRPr1 and ABCG2/BCRP was detected by the monoclonal antibody BXP21 (32, 33). (B) Reversal of SSZ resistance in CEM/SSZ1.5 cells by ABCG2/BCRP blocker Ko143, but not by Pgp Figure 1 A Figure 1 B blocker Verapamil or MRP1 blocker MK571. CEM/SSZ1.5 cells were incubated in medium containing non-toxic 3.0 120 concentrations of the ABCG2/BCRP blocker Ko143 (0.5 µM), MRP1 blocker MK571 (20 µM) or Pgp blocker CEM (T) 2.5 verapamil (10 µM) and evaluated for SSZ sensitivity compared to parental CEM (T) cells as described in Figure 100 CEM/SSZ1.5 1B. Results are presented as the mean of 3 experiments (SD <20%). 2.0 CEM/SSZ2.5 80 1.5 60 1.0 [SSZ] (mmol/l) 40 upregulated expression of one or more of the major multidrug resistance transporters

0.5 20 Pgp, MRP1 or ABCG2/BCRP. CEM/SSZ1.5 cells and CEM/SSZ2.5 cells displayed a marked Cell growth (% of control)

0.0 0 upregulation of ABCG2/BCRP compared with CEM (T) cells, in which ABCG2 expression 0 50 100 150 200 0.0 0.5 1.0 1.5 2.0 2.5 was negligible (Figure 2A). In contrast, control levels of MRP1 expression in CEM (T) cells Time (days) [SSZ] (mmol/l) were down regulated in CEM/SSZ1.5 and CEM/SSZ2.5 cells. Along with Pgp, also other MDR Figure 1: (A) Onset of SSZ resistance in human CEM (T) cells. SSZ resistance was provoked by culturing CEM pumps (MRP2, MRP3 and LRP (35)) were undetectable in both CEM (T), CEM/SSZ1.5 and (T) cells in stepwise increasing concentrations SSZ over time. (B) SSZ sensitivity for CEM (T) cells, CEM/SSZ1.5 and CEM/SSZ2.5 cells. Antiproliferative effects were assessed after 72 hours’ exposure to SSZ. Results are the CEM/SSZ2.5 cells (not shown). These results demonstrate that the drug resistance efflux mean of 3-5 separate experiments (SD <20%). pump ABCG2 is markedly upregulated during acquired resistance of CEM (T) cells to SSZ.

52 53 Figuur_3A.pzf:Data 1 graph - Mon Aug 18 13:27:22 2008

chapter 3 Sulfasalazine resistance in T-cells is mediated by BCRP

Reversal of ABCG2/BCRP mediated SSZ resistance in CEM/SSZ cells Figure 3 A

To establish whether ABCG2/BCRP is functionally active in conferring resistance to SSZ, 1500 CEM (T) ABCG2/BCRP was blocked by a specific inhibitor, Ko143 (25), to verify reversal of resistance. CEM/SSZ1.5 CEM/SSZ2.5 Blocking of ABCG2/BCRP reverted SSZ resistance by 50-60%, but not completely to CEM 1000 (T) cell sensitivity (Figure 2B). Blockers of MRP1 (MK571) or Pgp (verapamil) did not reverse (pg/ml) SSZ resistance. These results demonstrate that ABCG2/BCRP plays a functional role in α

TNF 500 conferring resistance to SSZ.

0 TNFα production and nuclear NFκB p65 expression in CEM (T) and CEM/SSZ cells 0.0 0.5 1.0 1.5 2.0 2.5 [SSZ] (mmol/l) SSZ is a potent inhibitor of the production of the pro-inflammatory cytokine TNFα by inhibiting the nuclear activation of the transcription factor NFκB (6, 36). To assess whether SSZ resistance affected production of TNFα, secretion of this cytokine was determined for Figure 3 B CEM (T) cells and CEM/SSZ cells after 24 hours’ PMA/ionomycin stimulation in the absence or presence of a concentration range of SSZ (Figure 3A). The basal level of TNFα production by CEM/SSZ1.5 and CEM/SS2.5 cells was markedly increased: 2.0-fold (p<0.001) and 2.9- fold (p<0.001), respectively compared with CEM (T) cells. Beyond this, twofold higher concentrations of SSZ were required to inhibit TNFα production by 50% in CEM/SSZ1.5 cells (IC50: 0.95 mM) and CEM/SSZ2.5 cells (IC50: 1.1 mM) compared with CEM (T) cells (IC50: 0.55 mM). Consequently, the area under the curve of TNFα production measured over the range of SSZ concentrations (Figure 3A) was 2.5-fold and 4.6-fold greater for CEM/SSZ1.5 and CEM/SSZ2.5 cells, respectively, compared with CEM (T) cells. Figure 3: (A) TNFα production by CEM (T), CEM/SSZ1.5 and CEM/SSZ2.5 cells (3x105/ml) after 24 hours’ stimulation with PMA/ionomycin in the absence or presence of a concentration range of SSZ. TNF levels were determined by ELISA. Results are mean (SD) of 5-7 separate experiments. Because SSZ inhibits TNFα production by preventing the cytosolic to nuclear translocation α (B) NFkB p65 expression in nuclear extracts of CEM (T) cells and CEM/SSZ1.5 cells after 24 hours’ stimulation of the nuclear transcription factor NFκB (6, 36), we analyzed whether increased nuclear with PMA/ionomycin in the absence or presence of SSZ (1.0 mM). NFκB levels themselves and/or an impaired potency of SSZ to reduce nuclear NFκB expression could explain the observed differences in absolute TNFα production and the diminished inhibitory effect of SSZ on TNFα production for CEM/SSZ2.5 cells compared to Discussion CEM (T) cells. At the time of TNFα analysis, after 24 hours’ PMA/ionomycine stimulation, expression of the nuclear NFκB p65 subunit was determined in CEM (T) cells and CEM/ As far as we know, this study is the first report to show that long term exposure of human SSZ1.5 cells in the absence or presence of SSZ. Unstimulated CEM (T) cells and CEM/SSZ1.5 T cells to the DMARD sulfasalazine can lead to development of resistance by induction of cells did not appear to differ in their basal level of nuclear NFκB p65 expression (Figure 3B, a drug efflux pump, the MDR transporter BCRP/ABCG2. Reduced intracellular drug levels lanes A vs. E). SSZ exposure to CEM (T) cells resulted in a markedly decreased nuclear NFκB not only led to impaired antiproliferative effects, but also to a less effective inhibition p65 expression, both in unstimulated cells (lanes A vs. B) or after PMA/ionomycin exposure of nuclear NFκB activation by SSZ in the resistant cells. Thus, SSZ-resistant cells required (lanes C vs. D). In contrast, SSZ exposure had little effect on nuclear NFκB p65 expression more SSZ to inhibit TNFα secretion. in either unstimulated CEM/SSZ1.5 cells (lanes E vs. F) or PMA/ionomycin stimulated CEM/ SSZ1.5 cells (lanes G vs. H). Similar results were observed for CEM/SSZ2.5 cells (results not Development of drug resistance is a common cause of treatment failure for anticancer shown). Together, these results suggest that, owing to ABCG2/BCRP mediated efflux, SSZ drugs or antimalarial drugs (16, 37). Drug resistance to DMARDs has received little attention is less effective in inhibiting nuclear NFκB p65 activation in CEM/SSZ cells than in CEM (T) in rheumatology even though loss of efficacy for several DMARDs, including SSZ, is well cells. established upon long term treatment (12, 13). In this study we mimicked the onset of SSZ

54 55 chapter 3 Sulfasalazine resistance in T-cells is mediated by BCRP

resistance by exposing human CEM (T) cells in vitro to stepwise increasing concentrations associated with alterations in the NFκB signalling pathway that controls the transcription of SSZ. Interestingly, SSZ resistance was associated with an upregulated expression of of anti-apoptotic and pro-inflammatory cytokine/chemokine genes (53-55). a recently discovered MDR transporter, ABCG2/BCRP, originally discovered in breast cancer cells selected for resistance (loss of antiproliferative effects) to the anticancer In contrast with the up regulated expression of ABCG2/BCRP in CEM/SSZ cells, we noted agents mitoxantrone and doxorubicin (38-41). Tissue distribution studies demonstrated a down regulated expression of another drug efflux pump MRP1 (Figure 2A). MRP1 has that ABCG2/BCRP expression is not restricted to breast cancer tissues. ABCG2/BCRP is an established role in exporting hydrophobic and hydrophilic drugs, the latter often as expressed in a limited number of normal tissues (liver, colon epithelium, kidney, mammary glutathione conjugates (21, 22). Immunologically, MRP1 has an important function in gland and blood vessels) (33). Expression of ABCG2/BCRP in synovial cells and tissues has dendritic cells by exporting the cysteinyl leucotriene LTC4, which mediates the signalling not been explored. Very recently, ABCG2/BCRP expression was described in blood cells, for chemokine CCL19 chemotaxis and migration of dendritic cells to the lymph nodes notably CD34+ hematopoietic progenitor cells (42) and in acute myeloid leukaemia cells (56). Because SSZ has been reported to inhibit LTC synthetase (57), this may suggest that (43, 44). The physiological function of BCRP/ABCG2 is as yet unknown, although recent a diminished LTC production coincides with a lowered expression of the LTC4 transporter studies with BCRP-/- knock out mice indicated a crucial role in the cellular extrusion and MRP1. detoxification of dietary phototoxins (45). Collectively, this in vitro study illustrates that long term exposure of human T cells to In our study, evidence for a functionally active ABCG2/BCRP drug efflux pump in CEM/ SSZ can result in up regulated expression of the MDR pump ABCG2/BCRP and increased SSZ cells was demonstrated by the fact that Ko143 (25), a blocker of BCRP/ABCG2, could production of TNFα, a key component of progressive disease in RA (58, 59). In this respect, reverse resistance to SSZ. In addition, CEM/SSZ displayed cross resistance and reversal by we also noted up regulation of ABCG2/BCRP expression in human monocytic THP1 and Ko143 to mitoxantrone (39), a prototypical substrate for ABCG2. U937 cells after in vitro exposure to SSZ (Oerlemans R et al, unpublished observation) The observation that the antirheumatic drug SSZ, like distinct anticancer drugs, can suggesting that this phenomenon is also of relevance for other inflammatory-related induce upregulation of ABCG2/BCRP expression may imply that these drugs share a cell types. Given the accumulating evidence for involvement of MDR pumps in conferring common target that triggers a response of ABCG2/BCRP upregulation. Unlike the ABC resistance to at least three DMARDs; SSZ (ABCG2/BCRP) (this study), chloroquine (MRP1) transporter Pgp (46), there is no evidence for a direct transcriptional regulation of ABCG2 (60), and MTX (MRP1, ABCG2/BCRP) (61, 62) further research is warranted to examine via an NFκB binding site on the ABCG2/BCRP promoter (47). However, most drugs involved whether MDR pumps also have a role in the reduced (preclinical and clinical) efficacy of in ABCG2 upregulation, mitoxantrone (48), doxorubicin (49), daunorubicin (50), CPT-11/ other DMARDs after long term treatment of patients with RA. Thus studies evaluating SN38 (49, 51) and flavopiridol (52) all have been documented to target the NFκB signalling inhibitors of ABCG2/BCRP (23, 25, 63) as possible chemosensitizers for SSZ activity deserve pathway by activating NFκB and stimulating IKBα degradation. Together, these results further attention. suggest that sustained (danger) signals mediated through the NFκB signalling pathway may provoke upregulation of drug efflux pumps like ABCG2/BCRP.

Along with the loss of antiproliferative effects of SSZ against CEM/SSZ cells (Figure 1B), a diminished anti-inflammatory effect was also noted as markedly higher concentrations of SSZ were required to inhibit TNFα production in CEM/SSZ cells than in CEM (T) cells (Figure 3A). Mechanistically this might be consistent with the fact that after PMA/ ionomycin stimulation the activation and nuclear translocation of NFκB in CEM/SSZ cells is less effectively inhibited by SSZ (6, 7, 36) because of drug efflux via ABCG2/BCRP. Consequently, TNFα production is less inhibited than in CEM (T) cells. Besides differences in the potency of SSZ to inhibit TNFα production, it is important to recognize that after PMA/ionomycine stimulation, basal levels of TNFα production were 2-3 fold higher in CEM/SSZ cells than in CEM (T) cells. This may indicate that induction of SSZ resistance is

56 References References

References 16 Djimde,A., Doumbo,O.K., Cortese,J.F., Kayentao,K., Doumbo,S., Diourte,Y., Dicko,A., Su,X. Z., Nomura,T., Fidock,D.A., Wellems,T.E., Plowe,C.V. and Coulibaly,D. A molecular marker for 1 Boers,M., Verhoeven,A.C., Markusse,H.M., van de Laar,M.A., Westhovens,R., van Denderen,J.C., chloroquine-resistant falciparum malaria, N.Engl.J.Med., 344: 257-263, 2001. van Zeben,D., Dijkmans,B.A.C., Peeters,A.J., Jacobs,P., van den Brink,H.R., Schouten,H.J., van der 17 Dean,M. and Allikmets,R. Complete characterization of the human ABC gene family, J.Bioenerg. Heijde,D.M., Boonen,A. and van der Linden,S. Randomised comparison of combined step-down Biomembr., 33: 475-479, 2001. prednisolone, methotrexate and sulphasalazine with sulphasalazine alone in early rheumatoid 18 Klein,I., Sarkadi,B. and Varadi,A. An inventory of the human ABC proteins, Biochim.Biophys. arthritis, Lancet, 350: 309-318, 1997. Acta, 1461: 237-262, 1999. 2 Barrera,P., Haagsma,C.J., Boerbooms,A.M., van Riel,P.L., Borm,G.F., van de Putte,L.B. and van der 19 Borst,P. and Oude Elferink,R.P. Mammalian ABC transporters in health and disease, Annu.Rev. Meer,J.W. Effect of methotrexate alone or in combination with sulphasalazine on the production Biochem., 71: 537-592, 2002. and circulating concentrations of cytokines and their antagonists. Longitudinal evaluation in 20 Ambudkar,S.V., Dey,S., Hrycyna,C.A., Ramachandra,M., Pastan,I. and Gottesman,M.M. patients with rheumatoid arthritis, Br.J.Rheumatol., 34: 747-755, 1995. Biochemical, cellular, and pharmacological aspects of the multidrug transporter, Annu.Rev. 3 Goekoop,Y.P., Allaart,C.F., Breedveld,F.C. and Dijkmans,B.A.C. Combination therapy in Pharmacol.Toxicol., 39: 361-398, 1999. rheumatoid arthritis, Curr.Opin.Rheumatol., 13: 177-183, 2001. 21 Borst,P., Evers,R., Kool,M. and Wijnholds,J. A family of drug transporters: the multidrug 4 Landewe,R.B., Boers,M., Verhoeven,A.C., Westhovens,R., van de Laar,M.A., Markusse,H.M., resistance-associated proteins, J.Natl.Cancer Inst., 92: 1295-1302, 2000. van Denderen,J.C., Westedt,M.L., Peeters,A.J., Dijkmans,B.A.C., Jacobs,P., Boonen,A., van der 22 Leslie,E.M., Deeley,R.G. and Cole,S.P. Toxicological relevance of the multidrug resistance protein Heijde,D.M. and van der Linden,S. COBRA combination therapy in patients with early rheumatoid 1, MRP1 (ABCC1) and related transporters, Toxicology, 167: 3-23, 2001. arthritis: Long-term structural benefits of a brief intervention, Arthritis Rheum., 46: 347-356, 23 Allen,J.D. and Schinkel,A.H. Multidrug resistance and pharmacological protection mediated by 2002. the breast cancer resistance protein (BCRP/ABCG2), Molecular Cancer Therapeutics, 1: 427-434, 5 O’Dell,J.R., Leff,R., Paulsen,G., Haire,C., Mallek,J., Eckhoff,P.J., Fernandez,A., Blakely,K., 2002. Wees,S., Stoner,J., Hadley,S., Felt,J., Palmer,W., Waytz,P., Churchill,M., Klassen,L. and Moore,G. 24 Liang,E., Proudfoot,J. and Yazdanian,M. Mechanisms of transport and structure-permeability Treatment of rheumatoid arthritis with methotrexate and hydroxychloroquine, methotrexate relationship of sulfasalazine and its analogs in Caco-2 cell monolayers, Pharm.Res., 17: 1168-1174, and sulfasalazine, or a combination of the three medications: Results of a two-year, randomized, 2000. double-blind, placebo-controlled trial, Arthritis Rheum., 46: 1164-1170, 2002. 25 Allen,J.D., van Loevezijn,A., Lakhai,J.M., van der Valk,M., van Tellingen,O., Reid,G., Schellens,J. 6 Wahl,C., Liptay,S., Adler,G. and Schmid,R.M. Sulfasalazine: a potent and specific inhibitor of H.M., Koomen,G.J. and Schinkel,A.H. Potent and specific inhibition of the breast cancer nuclear factor kappa B, J.Clin.Invest, 101: 1163-1174, 1998. resistance protein multidrug transporter in vitro and in mouse intestine by a novel analogue of 7 Weber,C.K., Liptay,S., Wirth,T., Adler,G. and Schmid,R.M. Suppression of NF-kappaB activity by fumitremorgin C, Molecular Cancer Therapeutics, 1: 417-425, 2002. sulfasalazine is mediated by direct inhibition of IkappaB kinases alpha and beta, Gastroenterology, 26 Foley,G.E., Lazarus,H., Farber,S., Uzman,B.G., Boone,B.A. and McCarthy,R.E. Continuous culture 119: 1209-1218, 2000. of human lymphoblasts from peripheral blood of a child with acute leukemia, Cancer, 4: 522- 8 Gadangi,P., Longaker,M., Naime,D., Levin,R.I., Recht,P.A., Montesinos,M.C., Buckley,M.T., 529, 1965. Carlin,G. and Cronstein,B.N. The anti-inflammatory mechanism of sulfasalazine is related to 27 Baz,A., Henry,L., Caravano,R., Scherrer,K. and Bureau,J.P. Changes in the subunit distribution of adenosine release at inflamed sites, J.Immunol., 156: 1937-1941, 1996. prosomes (MCP-proteasomes) during the differentiation of human leukemic cells, Int.J.Cancer, 9 Cutolo,M., Sulli,A., Pizzorni,C., Seriolo,B. and Straub,R.H. Anti-inflammatory mechanisms of 72: 467-476, 1997. methotrexate in rheumatoid arthritis, Ann.Rheum.Dis., 60: 729-735, 2001. 28 Galpin,A.J., Schuetz,J.D., Masson,E., Yanishevski,Y., Synold,T.W., Barredo,J.C., Pui,C.H., Relling,M. 10 Liptay,S., Bachem,M., Hacker,G., Adler,G., Debatin,K.M. and Schmid,R.M. Inhibition of nuclear V. and Evans,W.E. Differences in folylpolyglutamate synthetase and dihydrofolate reductase factor kappa B and induction of apoptosis in T- lymphocytes by sulfasalazine, Br.J.Pharmacol., expression in human B-lineage versus T-lineage leukemic lymphoblasts: mechanisms for lineage 128: 1361-1369, 1999. differences in methotrexate polyglutamylation and cytotoxicity, Mol.Pharmacol., 52: 155-163, 11 Rodenburg,R.J., Ganga,A., van Lent,P.L., van de Putte,L.B. and van Venrooij,W.J. The 1997. antiinflammatory drug sulfasalazine inhibits tumor necrosis factor alpha expression in 29 Mauritz,R., Bekkenk,M.W., Rots,M.G., Pieters,R., Mini,E., Van Zantwijk,C.H., Veerman,A.J., macrophages by inducing apoptosis, Arthritis Rheum., 43: 1941-1950, 2000. Peters,G.J. and Jansen,G. Ex vivo activity of methotrexate versus novel antifolate inhibitors of 12 Wolfe,F. The epidemiology of drug treatment failure in rheumatoid arthritis, Baillieres Clin. dihydrofolate reductase and thymidylate synthase against childhood leukemia cells, Clin.Cancer Rheumatol., 9: 619-632, 1995. Res., 4: 2399-2410, 1998. 13 Maetzel,A., Wong,A., Strand,V., Tugwell,P., Wells,G. and Bombardier,C. Meta-analysis of 30 Rots,M.G., Pieters,R., Kaspers,G.J., Veerman,A.J., Peters,G.J. and Jansen,G. Classification of ex treatment termination rates among rheumatoid arthritis patients receiving disease-modifying vivo methotrexate resistance In acute lymphoblastic and myeloid leukaemia, Br.J.Haematol., anti-rheumatic drugs, Rheumatology.(Oxford), 39: 975-981, 2000. 110: 791-800, 2000. 14 Bradshaw,D.M. and Arceci,R.J. Clinical relevance of transmembrane drug efflux as a mechanism 31 Jansen,G., Mauritz,R., Drori,S., Sprecher,H., Kathmann,I., Bunni,M., Priest,D.G., Noordhuis,P., of multidrug resistance, J.Clin.Oncol., 16: 3674-3690, 1998. Schornagel,J.H., Pinedo,H.M., Peters,G.J. and Assaraf,Y.G. A structurally altered human reduced 15 Litman,T., Druley,T.E., Stein,W.D. and Bates,S.E. From MDR to MXR: new understanding of folate carrier with increased folic acid transport mediates a novel mechanism of antifolate multidrug resistance systems, their properties and clinical significance, Cell Mol.Life Sci., 58: resistance, J.Biol.Chem., 273: 30189-30198, 1998. 931-959, 2001. 32 Scheffer,G.L., Kool,M., Heijn,M., de Haas,M., Pijnenborg,A.C., Wijnholds,J., van Helvoort,A., de

58 59 References References

Jong,M.C., Hooijberg,J.H., Mol,C.A., van der Linden,M., de Vree,J.M., van der Valk,P., Elferink,R. G2) gene, Biochim.Biophys.Acta, 1520: 234-241, 2001. P., Borst,P. and Scheper,R.J. Specific detection of multidrug resistance proteins MRP1, MRP2, 48 Boland,M.P., Fitzgerald,K.A. and O’Neill,L.A. Topoisomerase II is required for mitoxantrone to MRP3, MRP5, and MDR3 P-glycoprotein with a panel of monoclonal antibodies, Cancer Res., 60: signal nuclear factor kappa B activation in HL60 cells, J.Biol.Chem., 275: 25231-25238, 2000. 5269-5277, 2000. 49 Bottero,V., Busuttil,V., Loubat,A., Magne,N., Fischel,J.L., Milano,G. and Peyron,J.F. Activation 33 Maliepaard,M., Scheffer,G.L., Faneyte,I.F., van Gastelen,M.A., Pijnenborg,A.C., Schinkel,A.H., of nuclear factor kappaB through the IKK complex by the topoisomerase poisons SN38 and van De Vijver,M.J., Scheper,R.J. and Schellens,J.H. Subcellular localization and distribution of the doxorubicin: a brake to apoptosis in HeLa human carcinoma cells, Cancer Res., 61: 7785-7791, breast cancer resistance protein transporter in normal human tissues, Cancer Res., 61: 3458- 2001. 3464, 2001. 50 Boland,M.P., Foster,S.J. and O’Neill,L.A. Daunorubicin activates NFkappaB and induces kappaB- 34 Smedegard,G. and Bjork,J. Sulphasalazine: mechanism of action in rheumatoid arthritis, dependent gene expression in HL-60 promyelocytic and Jurkat T lymphoma cells, J.Biol.Chem., Br.J.Rheumatol., 34 Suppl 2: 7-15, 1995. 272: 12952-12960, 1997. 35 Scheffer,G.L., Schroeijers,A.B., Izquierdo,M.A., Wiemer,E.A. and Scheper,R.J. Lung resistance- 51 Huang,T.T., Wuerzberger-Davis,S.M., Seufzer,B.J., Shumway,S.D., Kurama,T., Boothman,D.A. and related protein/major vault protein and vaults in multidrug-resistant cancer, Curr.Opin.Oncol., Miyamoto,S. NF-kappaB activation by camptothecin. A linkage between nuclear DNA damage 12: 550-556, 2000. and cytoplasmic signaling events, J.Biol.Chem., 275: 9501-9509, 2000. 36 Tak,P.P. and Firestein,G.S. NF-kappaB: a key role in inflammatory diseases, J.Clin.Invest, 107: 7-11, 52 Tsai,S.H., Liang,Y.C., Lin-Shiau,S.Y. and Lin,J.K. Suppression of TNFalpha-mediated NFkappaB 2001. activity by myricetin and other flavonoids through downregulating the activity of IKK in ECV304 37 Gottesman,M.M., Fojo,T. and Bates,S.E. Multidrug resistance in cancer: role of ATP-dependent cells, J.Cell Biochem., 74: 606-615, 1999. transporters, Nat.Rev.Cancer, 2: 48-58, 2002. 53 Yamamoto,Y. and Gaynor,R.B. Therapeutic potential of inhibition of the NF-kappaB pathway in 38 Ross,D.D., Yang,W., Abruzzo,L.V., Dalton,W.S., Schneider,E., Lage,H., Dietel,M., Greenberger,L., the treatment of inflammation and cancer, J.Clin.Invest, 107: 135-142, 2001. Cole,S.P. and Doyle,L.A. Atypical multidrug resistance: breast cancer resistance protein 54 Baldwin,A.S. Control of oncogenesis and cancer therapy resistance by the transcription factor messenger RNA expression in mitoxantrone-selected cell lines, J.Natl.Cancer Inst., 91: 429-433, NF-kappaB, J.Clin.Invest, 107: 241-246, 2001. 1999. 55 Chen,F., Castranova,V. and Shi,X. New insights into the role of nuclear factor-kappaB in cell 39 Doyle,L.A., Yang,W., Abruzzo,L.V., Krogmann,T., Gao,Y., Rishi,A.K. and Ross,D.D. A multidrug growth regulation, Am.J.Pathol., 159: 387-397, 2001. resistance transporter from human MCF-7 breast cancer cells, Proc.Natl.Acad.Sci.U.S.A, 95: 56 Robbiani,D.F., Finch,R.A., Jager,D., Muller,W.A., Sartorelli,A.C. and Randolph,G.J. The leukotriene 15665-15670, 1998. C(4) transporter MRP1 regulates CCL19 (MIP-3beta, ELC)- dependent mobilization of dendritic 40 Taylor,C.W., Dalton,W.S., Parrish,P.R., Gleason,M.C., Bellamy,W.T., Thompson,F.H., Roe,D. cells to lymph nodes, Cell, 103: 757-768, 2000. J. and Trent,J.M. Different mechanisms of decreased drug accumulation in doxorubicin and 57 Bach,M.K., Brashler,J.R. and Johnson,M.A. Inhibition by sulfasalazine of LTC synthetase and of rat mitoxantrone resistant variants of the MCF7 human breast cancer cell line, Br.J.Cancer, 63: 923- liver glutathione S-transferases, Biochem.Pharmacol., 34: 2695-2704, 1985. 929, 1991. 58 Ulfgren,A.K., Andersson,U., Engstrom,M., Klareskog,L., Maini,R.N. and Taylor,P.C. Systemic 41 Scheffer,G.L., Maliepaard,M., Pijnenborg,A.C., van Gastelen,M.A., de Jong,M.C., Schroeijers,A.B., anti-tumor necrosis factor alpha therapy in rheumatoid arthritis down-regulates synovial tumor van der Kolk,D.M., Allen,J.D., Ross,D.D., van der Valk,P., Dalton,W.S., Schellens,J.H. and Scheper,R. necrosis factor alpha synthesis, Arthritis Rheum., 43: 2391-2396, 2000. J. Breast cancer resistance protein is localized at the plasma membrane in mitoxantrone- and 59 Lipsky,P.E., van der Heijde,D.M., St Clair,E.W., Furst,D.E., Breedveld,F.C., Kalden,J.R., Smolen,J.S., topotecan-resistant cell lines, Cancer Res., 60: 2589-2593, 2000. Weisman,M., Emery,P., Feldmann,M., Harriman,G.R. and Maini,R.N. Infliximab and methotrexate 42 Zhou,S., Schuetz,J.D., Bunting,K.D., Colapietro,A.M., Sampath,J., Morris,J.J., Lagutina,I., in the treatment of rheumatoid arthritis. Anti-Tumor Necrosis Factor Trial in Rheumatoid Arthritis Grosveld,G.C., Osawa,M., Nakauchi,H. and Sorrentino,B.P. The ABC transporter Bcrp1/ABCG2 is with Concomitant Therapy Study Group, N.Engl.J.Med., 343: 1594-1602, 2000. expressed in a wide variety of stem cells and is a molecular determinant of the side-population 60 Jansen,G., Scheper,R.J. and Dijkmans,B.A.C. Chloroquine resitance in human CEM (T) cells is phenotype, Nat.Med., 7: 1028-1034, 2001. mediated by multidrug resistance protein 1, Annals of the Rheumatic Diseases, 60: 89, 2001. 43 Ross,D.D., Karp,J.E., Chen,T.T. and Doyle,L.A. Expression of breast cancer resistance protein in 61 Hooijberg,J.H., Broxterman,H.J., Kool,M., Assaraf,Y.G., Peters,G.J., Noordhuis,P., Scheper,R.J., blast cells from patients with acute leukemia, Blood, 96: 365-368, 2000. Borst,P., Pinedo,H.M. and Jansen,G. Antifolate resistance mediated by the multidrug resistance 44 van der Kolk,D.M., Vellenga,E., Scheffer,G.L., Muller,M., Bates,S.E., Scheper,R.J. and de Vries,E.G. proteins MRP1 and MRP2, Cancer Res., 59: 2532-2535, 1999. Expression and activity of breast cancer resistance protein (BCRP) in de novo and relapsed acute 62 Volk,E.L., Rohde,K., Rhee,M., McGuire,J.J., Doyle,L.A., Ross,D.D. and Schneider,E. Methotrexate myeloid leukemia, Blood, 99: 3763-3770, 2002. cross-resistance in a mitoxantrone-selected multidrug- resistant MCF7 breast cancer cell line is 45 Jonker,J.W., Buitelaar,M., Wagenaar,E., van der Valk,M.A., Scheffer,G.L., Scheper,R.J., Plosch,T., attributable to enhanced energy-dependent drug efflux, Cancer Res., 60: 3514-3521, 2000. Kuipers,F., Elferink,R.P., Rosing,H., Beijnen,J.H. and Schinkel,A.H. The breast cancer resistance 63 de Bruin,M., Miyake,K., Litman,T., Robey,R. and Bates,S.E. Reversal of resistance by GF120918 in protein protects against a major chlorophyll-derived dietary phototoxin and protoporphyria, cell lines expressing the ABC half-transporter, MXR, Cancer Lett., 146: 117-126, 1999. Proc.Natl.Acad.Sci.U.S.A, 99: 15649-15654, 2002. 64 Beck,W.T., Cirtain,M.C., Look,A.T. and Ashmun,R.A. Reversal of Vinca alkaloid resistance but not 46 Zhou,G. and Kuo,M.T. NF-kappaB-mediated induction of mdr1b expression by insulin in rat multiple drug resistance in human leukemic cells by verapamil, Cancer Res., 46: 778-784, 1986. hepatoma cells, J.Biol.Chem., 272: 15174-15183, 1997. 47 Bailey-Dell,K.J., Hassel,B., Doyle,L.A. and Ross,D.D. Promoter characterization and genomic organization of the human breast cancer resistance protein (ATP-binding cassette transporter

60 61 chapter 4 Acquired resistance of human T cells to sulfasalazine: stability of the resistant phenotype and sensitivity to non-related DMARDs

Joost W. van der Heijden1, Mariska C. de Jong2, Ben A.C. Dijkmans1, Willem F. Lems1, Ruud Oerlemans1, Ietje Kathmann3, George L. Scheffer2, Rik J. Scheper2, Yehuda G. Assaraf4 and Gerrit Jansen1

Departments of 1Rheumatology, 2Pathology and 3Medical Oncology, VU University Medical Center, Amsterdam, The Netherlands. 4The Fred Wyszkowski Cancer Research Laboratory, department of Biology, The Technion, Israel Institute of Technology, Haifa, Israel

Annals of the Rheumatic Diseases 2004; 63: 131-137 chapter 4 Characterization of sulfasalazine resistant T-cells

Abstract In clinical rheumatology, loss of drug efficacy is also noted for patients with rheumatoid arthritis (RA) receiving long term treatment with disease modifying anti-rheumatic Background: A recent study from our laboratory showed that induction of the multidrug drugs (DMARDs) (14-17). Whether this loss of DMARD efficacy is related to the onset of resistance related drug efflux pump ABCG2/BCRP contributed to acquired resistance of acquired resistance to DMARDs, with mechanisms similar to anticancer drug resistance, human T cells to the disease-modifying antirheumatic drug (DMARD) sulfasalazine (SSZ). is not known (18). Meta-analyses of DMARD treatment termination rates showed that Objective: To investigate the duration of SSZ resistance and ABCG2/BCRP expression after the DMARD sulfasalazine (SSZ) (19, 20) had a relatively shorter lasting efficacy than other withdrawal of SSZ and rechallenging with SSZ, and to assess the impact of SSZ resistance DMARDs, in particular the ‘golden standard’ MTX (14, 16-18). In general, lack of efficacy on responsiveness to other DMARDs. may be related to diminished anti-inflammatory and/or antiproliferative effects of Methods: Human CEM (T) cells with acquired resistance to SSZ (CEM/SSZ) were DMARDs against pro-inflammatory cytokine releasing cells at inflammatory sites (21). characterized for (a) SSZ sensitivity and ABCG2/BCRP expression during withdrawal and rechallenge of SSZ, and (b) antiproliferative efficacy of other DMARDs. To gain insight into the possible mechanism(s) of resistance to the antiproliferative/anti- Results: ABCG2/BCRP protein expression was stable for at least four weeks when CEM/SSZ inflammatory effects of the DMARD SSZ, we previously provoked acquired resistance to cells were grown in the absence of SSZ, but gradually declined, along with SSZ resistance SSZ in an in vitro model system of human T-lymphocytes by stepwise exposure of CEM levels, to non-detectable levels after withdrawal of SSZ for six months. Rechallenging (T) cells to gradually increasing concentrations of SSZ (22). This study showed that SSZ with SSZ led to a rapid (<2.5 weeks) resumption of SSZ resistance and ABCG2/BCRP resistance in CEM (T) cells was conferred by overexpression of a drug efflux pump - notably expression as in the original CEM/SSZ cells. CEM/SSZ cells displayed diminished sensitivity the multidrug resistance transporter ABCG2/BCRP. Owing to enhanced drug efflux in to the DMARDs leflunomide (5.1-fold) and methotrexate (1.8-fold), were moderately more SSZ resistant cells (CEM/SSZ), higher concentrations of SSZ were required to inhibit the sensitive (1.6-2.0 fold) to cyclosporin A and chloroquine, and markedly more sensitive (13- secretion of the proinflammatory cytokine tumour necrosis factor α (TNFα) (22). fold) to the glucocorticoid dexamethasone as compared to parental CEM cells. In the present study, we investigated the stability of the resistant phenotype of CEM/ Conclusion: The drug efflux pump ABCG2/BCRP has a major role in conferring resistance SSZ cells after withdrawal of SSZ in order to establish whether SSZ resistance is slowly/ to SSZ. The collateral sensitivity of SSZ resistant cells for some other (non-related) rapidly lost over time and reinducible after SSZ rechallenge. Moreover, we analyzed to DMARDs may provide a further rationale for sequential mono- or combination therapies what extent SSZ resistance affected antiproliferative effects of other DMARDs and anti- with distinct DMARDs upon decreased efficacy of SSZ. inflammatory drugs. The results demonstrate that SSZ resistance, along with ABCG2/ BCRP expression, is gradually lost over a period of six months after withdrawal of SSZ, but rapidly re-emerged, within 2 weeks upon rechallenge with SSZ. Drug sensitivity was as Introduction follows: CEM/SSZ cells displayed lower sensitivity (fivefold) to the DMARD leflunomide, a slightly diminished sensitivity to MTX (twofold), moderately increased sensitivities for Acquired drug resistance is recognized as a common cause of treatment failure for cyclosporin A and chloroquine (up to twofold), but a markedly enhanced sensitivity for patients with cancer after sustained treatment with cytostatic drugs (1, 2). An important dexamethasone (13-fold). mechanism of acquired cellular drug resistance to anticancer drugs is the overexpression of drug efflux pumps belonging to the superfamily of adenosine triphosphate (ATP) dependent binding cassette (ABC) transporters (2-6). ABC transporters with an established Materials and methods role in drug efflux and drug resistance include: (a) P-glycoprotein (Pgp, ABCB1), which confers resistance preferentially to lipophilic drugs (7); (b) members of the multidrug Materials: Sulfasalazine, leflunomide, chloroquine, phenylmethylsulfonyl fluoride resistance associated protein subfamily (MRP1-6, ABCC1-6), which confer resistance (PMSF) and dexamethasone were purchased from Sigma Chem Co. (St. Louis, MO, U.S.A.). to various anionic charged/glutathione conjugated drugs such as methotrexate (MTX) Protease Inhibitor Cocktail (PIC) and Triton X-100 were from Boehringer Mannheim (8), thiopurines (9, 10) and steroids (5, 11); and (c) ABCG2, also known as Breast Cancer (Ingelheim, Germany). MG132 was from Calbiochem (Germany). Methotrexate (MTX) was Resistance Protein (BCRP), a recently identified ABC transporter that confers resistance to a generous gift from Pharmachemie Haarlem, the Netherlands. Cyclosporin A was a gift amphiphilic drugs, including mitoxantrone and MTX (12, 13). from Novartis (Arnhem, the Netherlands). The ABCG2/BCRP inhibitor Ko143 (23) was kindly

64 65 chapter 4 Characterization of sulfasalazine resistant T-cells

provided by Dr A. Schinkel (Netherlands Cancer Institute, Amsterdam, the Netherlands). microcentrifuge (10 minutes; 14000 rpm; 4oC). Protein content of the supernatant was RPMI-1640 tissue culture medium and fetal calf serum (FCS) were obtained from Gibco determined by Biorad protein assay (32). Fifty microgram of total cell lysates were Chemical Co, Grand Island, NY, USA. fractionated on a 7.5% polyacrylamide gel and transferred on a nitrocellulose membrane. The nitrocellulose membranes were pre-incubated overnight at 4oC in blocking buffer (5% Cell culture and selection of SSZ-resistant CEM (T) cells Biorad Blocker in TBS-T (10 mM Tris-HCl, pH 8.0, 0.15 M NaCl, 0.1% Tween-20) to prevent SSZ resistant human CEM (T) cells were isolated as described previously (22). In short, aspecific antibody binding. After blocking, the membranes were incubated for 1 hour human CEM (T) cells were cultured at an initial density of 3x105 cells/ml in RPMI-1640 at room temperature with the primary antibodies for the multidrug resistance (MDR) medium supplemented with 10% FCS, 2 mM L-glutamine, and 100 µg/ml penicillin and pumps ABCG2/BCRP (BXP21, 1:400) and MRP1 (MRPr1, 1:500) as described by Scheffer

o streptomycin in a 5% CO2 incubator at 37 C (24, 25). Cell cultures were refreshed twice et al (32, 33). As control for protein loading, β-actin (Chemicon International, Ca, USA., weekly and SSZ was added to the cell culture medium at an initial concentration of 0.4 1:3000) was used. After three washing steps with TBS-T, membranes were incubated mM. This concentration of SSZ could be stepwise increased to 1.5 mM SSZ (after 4 months) for 1 hour with horseradish peroxidise labelled anti-rat/mouse (Dako: 1:2000 for ABCG2/ and 2.5 mM SSZ after a period of 6 months. BCRP and ABCC1/MRP1 and 1:3000 for β-actin) as secondary antibody. Detection of the Extensive binding of SSZ to plasma proteins (serum albumin) is known to limit its antibody binding was measured by enhanced chemoluminescence (ECL) according to the therapeutic effect (26, 27). Based on the presence of at least three classes of SSZ binding manufacturers instructions (Amersham International, Buckinghamshire, UK). Protein sites on serum albumin and their association rate constants (28) it can be calculated that levels were determined by densitometric scanning (GelDoc and Molecular Analyst, Biorad a protein concentration of about 100 µmol/l in 10% fetal calf serum can bind 320 µM SSZ. Laboratories) of the X-ray films (Hyperfim ECL, Amersham International, Buckinghamshire, For the two selected SSZ-resistant CEM cells grown the presence of 1500 and 2500 µM SSZ, UK). this would imply that 21% and 13% of SSZ, respectively, is protein bound. It should be recognized that CEM cells are of T cell leukaemic origin, proliferating with a Statistical analysis doubling time of 26-30 hours. Thus CEM cells may not fully representative for normal T Statistical significance of differences was analyzed by analysis of variance, using SPSS 9.0 cells or T cells applicable to RA. However, CEM cells may serve as a valuable in vitro model computer software. A p-value <0.05 was considered to be statistically different. system for RA reasearch as they respond to T cell stimuli, producing pro-inflammatory cytokines like TNFα (22). Furthermore, CEM cells have been exploited to disclose resistance mechanisms to MTX that are clinically encountered (24, 29-31). Results Antiproliferative effects of DMARDs and other drugs were analyzed by plating 1.25x105 cells/ml of parental CEM (T) cells, CEM/SSZ1.5 and CEM/SSZ2.5 cells (in the absence of SSZ) Onset and stability of SSZ-resistance in human CEM (T) cells in individual wells of a 24 well plate containing up to 50 µl of drug solution. Inhibition Acquired resistance to SSZ in human CEM (T) cells could be provoked by culturing cells of cell growth was determined after 72 hours incubation by counting cells with a in stepwise increasing concentrations of SSZ (22). Figure 1A (point A) shows the anti- haemocytometer and cell viability by trypan blue exclusion. The drug concentration proliferative effect of SSZ (IC-50: concentration for 50% growth inhibition) for CEM (T)

required to inhibit cell growth by 50% compared with control growth is defined as IC50 cells at the initial dosing of 0.4 mM SSZ, and for CEM (T) cells that were grown in the concentration. presence of stepwise increasing concentrations of SSZ. After a period of 4 months, CEM (T) could be maintained at an IC-50 concentration of 1.5 mM SSZ (CEM/SSZ1.5) which was Western blotting about fourfold higher than parental CEM (T) cells. When CEM/SSZ1.5 cells were grown in For analysis of expression of ABCG2/BCRPand ABCC1/MRP1, cells were harvested in the the presence of 1.5 mM SSZ for another 2 months, the IC-50 value slightly increased to 1.7 mid-log phase of growth and washed three times with ice cold Hepes buffered saline, pH mM (Figure 1A, point B). When, in parallel, CEM/SSZ1.5 cells obtained after 4 months were 7.4. Total cell lysates of 107 cells were suspended in 500 µl of lysis buffer containing 50 mM exposed to further increasing concentrations of SSZ for 2 months, eventually CEM/SSZ2.5

Tris-HCl (pH 7.6), 5 mM dithiotreitol, 20 µl protease inhibitor cocktail (PIC; 1 tablet/ml H2O), cells were obtained that could grow in the presence of 2.5 mM SSZ (not shown). 20% glycerol and 0.5% NP-40. The suspension was sonicated (MSE sonicator, amplitude 6, To establish whether the SSZ-resistance in CEM/SSZ1.5 cells is a transient or stable for 3x5 seconds with 30 seconds time intervals at 4oC) and centrifuged in an Eppendorf phenotype, CEM/SSZ1.5 cells (at point B) were further grown in the absence of SSZ and IC-

66 67 Figuur_1A.pzf:Data 1 graph - Mon Aug 18 13:28:21 2008

chapter 4 Characterization of sulfasalazine resistant T-cells

Figure 1 A +SSZ -SSZ +SSZ cells resumed their original level of SSZ resistance as seen at point B. These results indicate 2.0 that although SSZ resistance in CEM/SSZ is transiently lost over a period of 6 months, rechallenge of revertant cells to SSZ rapidly reinduces resistance to SSZ. 1.5

1.0 Expression of multidrug resistance (MDR) transporters ABCG2/BCRP and ABCC1/ MRP1 in CEM (T) and CEM/SSZ cells 0.5 IC-50SSZ (mmol/l) Initial studies from our laboratory (22) reported differential expression of multidrug resistance proteins upon development of SSZ resistance in CEM/SSZ cells: induction of 0.0 -1 0 1 3 6 10 12 15 ABCG2/BCRP and down regulation of ABCC1/MRP1 expression. Figure 1B illustrates the Months expression of ABCG2/BCRP and ABCC1/MRP1 at the various times A-D in Figure 1A. Parental A BCD CEM (T) cells (Figure 1A, point A) exhibited undetectable levels of expression of ABCG2/ BCRP protein, but had an appreciable level of ABCC1/MRP1. In contrast, SSZ resistant CEM cells isolated at 1.5 mM SSZ (CEM/SSZ1.5 cells) (Figure 1A, point B) showed a marked Figure 1 B induction of ABCG2/BCRP expression and down regulation of ABCC1/MRP1 expression. After withdrawal of SSZ from the cell culture of CEM/SSZ1.5 for a period of six months (Figure 1A, point C) ABCG2/BCRP and ABCC1/MRP1 expression reverted to the original levels of parental CEM (T) cells. Notably, when revertant CEM/SSZ1.5 cells where re-exposed to 1.5 mM SSZ for 2.5 weeks (Figure 1A, point D), ABCG2/BCRP expression was rapidly up regulated, while ABCC1/MRP1 was again down regulated. As a control, fluctuations in the expression of ABCG2/BCRP paralleled the growth inhibitory effects (IC-50 values) for the cytostatic drug mitoxantrone (MXR), which is a known substrate for efflux by ABCG2/ Figure 1: (A) Time course of sensitivity to sulfasalazine (SSZ) of CEM (T) during development of SSZ BCRP (6, 34) (data not shown). resistance, after withdrawal of SSZ from CEM/SSZ1.5 cells and rechallenge of revertant CEM/SSZ1.5 cells with SSZ. Parental CEM (T) cells (point A) were exposed to a starting concentration of 0.4 mM SSZ that was gradually increased to 1.5 mM over a period of 6 months to yield SSZ resistant CEM/SSZ1.5 cells (point B). Because Figure 1A showed that changes in IC-50 values for CEM/SSZ1.5 cells were From this point on CEM/SSZ1.5 were grown in the absence of SSZ to finally yield revertant CEM/SSZ1.5 cells particularly noted within the first 10 weeks after withdrawal of SSZ (Figure 1A, point B→ that displayed again parental CEM (T) cell sensitivity to SSZ (point C). When revertant cells were re-exposed to 1.5 mM SSZ for 2.5 weeks (point D), CEM/SSZ1.5 resumed their original resistance level as observed at C), we fine-tuned the analysis of IC-50 values for SSZ and changes in expression of ABCG2/ point B. IC-50 values were determined after 72 hours’ exposure of cells to SSZ. BCRP and ABCC1/MRP1 during this period of 10 weeks. IC-50 values for SSZ dropped by (B) ABCG2/BCRP and ABCC1/MRP1 protein expression is shown for CEM (T) cells and (revertant) CEM/SSZ1.5 cells isolated at the various time points A-D. Cell lysates of 2008/MRP1 (32) and MCF7/MR (43) served as approximately 40-50% after growing of CEM/SSZ1.5 cells in the absence of SSZ for 1 week positive controls for ABCC1/MRP1 and ABCG2/BCRP, respectively (E). For CEM (T) and CEM/SSZ1.5 cells, 50 (Figure 2A). From this time point on, IC-50 values for SSZ only slowly declined. At 70 days µg of total cell lysate was applied on the SDS-PAGE gel, for the controls 10 µg protein. ABCC1/MRP1 was after withdrawal of SSZ from CEM/SSZ1.5 cells, resistance to SSZ could still be noted detected by the monoclonal antibody MRPr1 and ABCG2/BCRP was detected by the monoclonal antibody BXP21 (32, 33). β-Actin was used as a control for equal protein loading. compared with parental CEM (T) cells. Figure 2B shows the expression of ABCG2/BCRP and ABCC1/MRP1 in parental CEM (T) cells (lane A) compared with CEM/SSZ1.5 cells before (lane B) and after withdrawal of SSZ for 3 days, 7 days, 4 weeks and 10 weeks (lanes C-F, 50 values for the CEM/SSZ1.5 cells were monitored over time. After 1 week in the absence respectively). ABCG2/BCRP protein expression in CEM/SSZ1.5 cells appeared to be stable of SSZ, IC-50 values for SSZ dropped by 40-50% to stay at this level for up to 4 weeks. for at least 4 weeks upon withdrawal of SSZ, after which a diminished, but still detectable From this time point on, IC-50 values for SSZ gradually declined to reach ultimately the level of expression could be seen after 10 weeks. ABCC1/MRP1 expression, which was original Ievels of SSZ sensitivity of parental CEM (T) cells (point C). To assess whether SSZ down regulated in CEM/SSZ1.5 cells in the presence of SSZ, was rapidly re induced within resistance would be reinduced upon exposure to SSZ, revertant CEM/SSZ1.5 cells were re- 3-7 days after withdrawal of SSZ, even overshooting the level of ABCC1/MRP1 expression in exposed to 1.5 mM SSZ. Within only 2.5 weeks’ exposure to SSZ (D), revertant CEM/SSZ1.5 parental CEM (T) cells after 70 days of SSZ withdrawal from CEM/SSZ1.5 cells. These results

68 69 Figuur_2A.pzf:Data 1 graph - Mon Aug 18 13:28:58 2008 Figuur_2C.pzf:Data 1 graph - Mon Aug 18 13:29:18 2008

chapter 4 Characterization of sulfasalazine resistant T-cells

120 Figure 2 A 120 Figure 2 C inhibitor, Ko143 (23). Blocking of ABCG2/BCRP reversed SSZ resistance in CEM/SSZ1.5

100 100 cells by 50-60%, but not completely to parental CEM (T) cell sensitivity (Figure 2C). After

80 80 growth of CEM/SSZ1.5 cells in the absence of SSZ for 4 weeks, when IC-50 values for SSZ had stabilized at about 50% of the original CEM/SSZ1.5 cells, Ko143 fully reversed the 60 60 residual SSZ resistance of CEM/SSZ1.5 cells to the SSZ sensitivity of CEM (T) cells (Figure 40 40 2C). These results indicate that SSZ resistance in CEM/SSZ1.5 is mediated by at least two 20 20 Cell growth (% of control) Cell growth (% of control) components, each contributing for approximately 50%. One component is rapidly lost 0 0 0.0 0.5 1.0 1.5 2.0 2.5 0.0 0.5 1.0 1.5 2.0 2.5 within one week after withdrawal of SSZ, the other component, which stabilised after [SSZ] (mmol/l) [SSZ] (mmol/l) 1-4 weeks’ growth of CEM/SSZ1.5 cells in the absence of SSZ, can be fully accounted for by ABCG2/BCRP. The latter component is then gradually lost over a period of 5 months’ Figure 2 B culture in the absence of SSZ.

Sensitivity profile of CEM/SSZ cells for DMARDs and other anti-inflammatory drugs In clinical rheumatology, SSZ is applied sequentially in mono- or combination therapies with other DMARDs (19, 20, 35). For this reason, we investigated whether the onset of SSZ resistance affects the sensitivity for other clinically active DMARDs, as well as other drugs with potential anti-inflammatory properties. Drug sensitivities were evaluated by their antiproliferative effects in CEM/SSZ1.5 and CEM/SSZ2.5 cells as compared with parental CEM (T) cells (Table 1). Cross-resistance to mitoxantrone, a topoisomerase II inhibitor that is a prototypical substrate for ABCG2/BCRP (34, 36), for both CEM/SSZ1.5 (4.3-fold, p=0.002 vs. CEM) and Figure 2: (A) Antiproliferative effect of SSZ against CEM/SSZ1.5 cells isolated at various time points (0-10 weeks) after withdrawal of SSZ from CEM/SSZ1.5 cells from point B in Figure 1A: 0 days (closed circles), CEM/SSZ2.5 cells (6.4-fold, p<0.001 vs. CEM) was observed. As expected, mitoxantrone 3 days (open triangles), 1 week (closed triangles), 4 weeks (open diamonds), 10 weeks (closed squares). cross resistance was fully reversed by the ABCG2/BCRP blocker Ko143. Furthermore, Antiproliferative effects/growth inhibition for SSZ were evaluated after 72 hours’ exposure to SSZ and compared with those for parental CEM (T) cells (open circles). (B) Expression of ABCG2/BCRP and ABCC1/MRP1 protein of CEM/SSZ1.5 cells during withdrawal of SSZ for up to 10 weeks as in Figure 2A. Lane A: parental CEM (T) cells, lane B: CEM/SSZ1.5 cells before withdrawal of Table 1: Antiproliferative effects of DMARDs and other drugs against CEM (T) and CEM/SSZ cells. SSZ; lane C-F: CEM/SSZ1.5 cells 3 days, 7 days, 4 weeks and 10 weeks, respectively, after withdrawal of SSZ. IC-50 values were determined after 72 hours DMARD/drug exposure. Values are mean ± SD of 3-6 separate Note: sample E for ABCC1/MRP1 was not tested. experiments. RF: Resistance Factor = IC-50 CEM/SSZ / IC-50 CEM (T). Statistical significance (ANOVA test): IC-50 (C) Reversal of SSZ resistance in CEM/SSZ1.5 cells by the ABCG2/BCRP blocker Ko143 before and after 4 CEM/SSZ vs IC-50 CEM (T): p<0.001***, p<0.01**, p<0.05* weeks’ withdrawal of SSZ. Symbols: (open triangle): parental CEM (T) cells, (open circles): CEM/SSZ1.5 cells, (closed circles): CEM/SSZ cells + 0.5 µM Ko143, (open squares): CEM/SSZ1.5 cells after 4 weeks’ withdrawal of SSZ, (closed squares): CEM/SSZ1.5 cells after 4 weeks’ withdrawal of SSZ + 0.5 µM SSZ. Anti-proliferative DMARD/DRUG CEM (T) CEM/SSZ1.5 CEM/SSZ2.5 effects of SSZ were evaluated after 72 hours’ exposure to SSZ in the absence or presence of Ko143. IC-50 (SD) IC-50 (SD) [RF] IC-50 (SD) [RF]

SSZ (µM) 400 (30) 1720 (170) [4.3]*** 2550 (212) [6.4] *** Mitoxantrone (nM) 0.47 (0.08) 1.94 (0.67) [4.1] ** 3.3 (0.6) [7.0] *** Mitoxantrone (+ 100 nM Ko143) 0.33 (0.07) 0.36 (0.06) [1.1] N.D. indicate that ABCG2/BCRP and ABCC1/MRP1 follow an inverse pattern of expression after Leflunomide (µM) 17.2 (1.9) 37.4 (14.4) [2.2]NS 88.0 (37.4) [5.1] ** SSZ exposure and withdrawal. Methotrexate (nM) 8.4 (0.7) 9.0 (0.7) [1.1]NS 15.1 (5.1) [1.8] * Given the apparent discrepancy of the rapid partial loss of SSZ resistance within 1 week of MG132 (nM) 39.5 (5.0) 68.1 (8.8) [1.7] *** 63.1 (7.9) [1.6] ** NS * withdrawal of SSZ from CEM/SSZ1.5 cells (Figure 2A) and the stable expression of ABCG2/ Chloroquine (µM) 44.5 (7.5) 37.8 (8.7) [0.8] 23.3 (13.9) [0.5] Cyclosporin A (µM) 5.7 (0.8) 3.6 (0.8) [0.6] ** 4.0 (0.2) [0.7] * BCRP during this period (Figure 2B, lanes B-D), we further investigated the functional Dexamethason (nM) 41.5 (13.1) 3.4 (0.6) [0.08] ** 6.4 (0.8) [0.15] ** contribution of ABCG2/BCRP in SSZ resistance by blocking ABCG2/BCRP with a specific

70 71 chapter 4 Characterization of sulfasalazine resistant T-cells

cross resistance of CEM/SSZ cells was noted for the DMARDs leflunomide (up to 5.1-fold been noted upon development of a selected group of topoisomerase inhibitors such as for CEM/SSZ2.5, p=0.005 vs. CEM) and MTX (up to 1.8-fold for CEM/SSZ2.5, p=0.027 vs. mitoxantrone, doxorubicin and topotecan (12, 34, 42-44). CEM). Because SSZ mediates part of its anti-inflammatory effects by targeting of the NFκB ABCG2/BCRP expression in CEM/SSZ cells did not fully account for the resistant phenotype signalling pathway (37, 38) we also determined the drug sensitivity for MG132, an inhibitor as about 50% of the SSZ resistance was lost within one week after withdrawal of SSZ, while of NFκB activation via inhibition of 26S-proteasome (39). A low level of cross-resistance ABCG2/BCRP expression was unchanged. The nature of this latter component has not been for CEM/SSZ cells was noted for MG132, (1.6-fold, p=0.004 for CEM/SSZ2.5 and p<0.001 for identified, but could be related to SSZ-induced transient alterations in the NFκB signalling CEM/SSZ1.5 vs. CEM). Interestingly, enhanced sensitivity of CEM/SSZ cells was noted for pathway (38) that controls the transcription of anti-apoptotic and pro-inflammatory the DMARDs chloroquine (up to 1.9-fold CEM/SSZ2.5, p=0.043 vs. CEM) and cyclosporin cytokine/chemokine genes (45-47). Consistent with this hypothesis is the observation A (up to 1.6-fold, p<0.001 for CEM/SSZ1.5 and p=0.028 for CEM/SSZ2.5 vs. CEM). CEM/SSZ of a two- to threefold enhancement of TNFα production by CEM/SSZ cells compared to cells displayed even more hypersensitivity (up to 13-fold, p=0.002 for CEM/SSZ1.5 and CEM (T) cells (22). It remains to established whether these type of SSZ resistance-induced p=0.003 for CEM/SSZ2.5 vs. CEM) to the glucocorticoid dexamethasone. These results effects may also be responsible for the altered anti-proliferative effects (Table 1) of NFκB indicate that induction of SSZ resistance can coincide with a differentially altered DMARD signalling pathway drugs, including leflunomide (48), MG132 (39) and dexamethasone sensitivity pattern, including on the one hand cross resistance to leflunomide and MTX, (45, 49, 50). but on the other enhanced sensitivity to cyclosporin A and chloroquine, and, most The gradual loss of ABCG2/BCRP expression from cell cultures of CEM/SSZ1.5 cells after notably, to dexamethasone. 6 months (approximately 50 passages) withdrawal of SSZ is consistent with a study by Maliepaard et al (51) who noted loss of ABCG2/BCRP expression from a topotecan-resistant ovarian carcinoma cell line over 30 passages in drug-free medium. These results indicate Discussion that ABCG2/BCRP expression gradually declines when the selective pressure is absent, but can be rapidly resumed upon renewed exposure to the selective drug. Characterization of human CEM (T) cells with acquired resistance to the DMARD Besides the effects on ABCG2/BCRP expression, it is of interest to note that SSZ exposure/ sulfasalazine showed differential expression of two multidrug resistance (MDR) efflux resistance had down regulatory effects on the expression of ABCC1/MRP1. Although this pumps: up regulation of ABCG2/BCRP and down regulation of ABCC1/MRP1. This MDR transporter has an established role in extrusion of various toxic drugs (2, 5, 11), it phenotype could be reverted (slowly) and reinduced (rapidly) after SSZ withdrawal also has important immunological functions in for instance dendritic cells, where, by and SSZ re-exposure, respectively. Beyond this, CEM/SSZ cells were characterised by exporting the cysteinyl leucotriene LTC4, it mediates the signalling for chemokine enhanced sensitivity to the DMARDs cyclosporin A and chloroquine, and most notably for CCL19 chemotaxis and migration of dendritic cells to the lymph nodes (52). Because SSZ dexamethasone, which may provide possible strategies to circumvent resistance to SSZ. is an inhibitor of LTC synthetase (53), impaired synthesis of LTC may parallel reduced expression of its transporter ABCC1/MRP1. Another example of ABCC1/MRP1 down/up- SSZ is commonly used in various DMARD regimens for RA treatment, including regulation was recently identified by our laboratory after depletion/repletion of cellular monotherapy or combination therapy with other DMARDs such as MTX, folate status (54) in CEM (T) cells. Consistent with the notion that SSZ can also exert anti- hydroxychloroquine or in the COBRA regimen (19, 20, 35, 40). SSZ can induce folate effects (55), we obtained preliminary evidence that the differential expression of antiproliferative/apoptotic effects (41) as well as anti-inflammatory effects by inhibiting ABCC1/MRP1 is indeed correlated with the cellular folate status in CEM/SSZ cells (Jansen activation of NFκB (37), which leads to decreased production of pro-inflammatory G, Scheper RJ, Dijkmans BAC, unpublished data). cytokines such as TNFα (38). Although SSZ is a potent DMARD, its efficacy upon long term The present study also provides insight into strategies that may or may not be successful treatment seems to be more compromised as compared to other DMARDs, such as MTX to circumvent/reverse SSZ resistance. Firstly, temporarily discontinuation of DMARD (14, 16, 18). Because lack of efficacy may be related to either persistent/renewed disease treatment after an initial response and resumption of DMARD treatment at the time activity and/or side effects of DMARDs, we suggested that diminished SSZ efficacy could of progressive disease, proved to be effective (56). Whether a similar strategy is also also be associated with the development of resistance. Indeed, as previously reported effective for patients with RA for whom DMARD treatment has failed, has not been (22), we found that the onset of SSZ resistance in human CEM (T) cells coincided with investigated. When such a clinical strategy was mimicked in vitro – the induction of the MDR pump ABCG2/BCRP. Thus far, ABCG2/BCRP induction had only discontinuation of SSZ treatment after development of SSZ resistance and rechallenging

72 73 chapter 4 Characterization of sulfasalazine resistant T-cells

with SSZ at the time SSZ resistance had apparently disappeared–it did not seem to be effective. SSZ resistance, along with ABCG2/BCRP overexpression, was rapidly reinduced (<2.5 weeks). Secondly, development of resistance to SSZ may be accompanied by adverse effects of collateral resistance to other DMARDs (e.g. leflunomide). Potentially positive strategies to target SSZ resistance may include the use of (a) blockers of ABCG2/ BCRP that serve as chemosensitizers to reverse SSZ resistance (e.g. Ko143); (b) DMARDs that were not impaired in their anti-proliferative activity during development of SSZ resistance, such as cyclosporin A and chloroquine. Since chloroquine is a substrate for efflux by ABCC1/MRP1, a reduced expression of ABCC1/MRP1 as observed in CEM/SSZ cells may confer an enhanced sensitivity to chloroquine (57) which could increase the efficacy of hydroxychloroquine containing therapies (20). Finally, most interesting was the observation that CEM/SSZ cells displayed hypersensitivity (13-fold) to the glucocorticoid dexamethasone. One previous report has described collateral sensitivity to dexamethasone (10-fold) in human myeloma cells with acquired resistance to doxorubicin (58). The mechanistic basis for the markedly enhanced dexamethasone sensitivity is not clear, but is not associated with an increase in glucocorticoid receptor levels in CEM/SSZ cells compared to CEM (T) cells (not shown). Altogether, this study shows that prolonged exposure of CEM (T) cells to SSZ can have differential effects on expression levels of at least two important MDR pumps: ABCG2/ BCRP and ABCC1/MRP1. As these MDR pumps are normally expressed on various peripheral blood cell types (59-61), further research is warranted to identify the expression levels of these MDR pumps during treatment of patients with RA with DMARDs which include SSZ. Thus far, only a few studies have investigated blood samples of patients with RA for differential expression of the MDR transporter Pgp (62-65). Although these studies included limited numbers of patients, data were supportive for a role of Pgp in DMARD resistance. Our present study, though based on in vitro data using a T cell model system, demonstrates also that MDR pumps other than Pgp may be relevant for DMARD resistance. Once specific MDR proteins are identified for their contributing role in DMARD (in)efficacy, this information may be exploited to use specific blockers for the various MDR pumps (2) as chemosensitizers for DMARD activity.

74 75 References References

References 19 Landewe,R.B., Boers,M., Verhoeven,A.C., Westhovens,R., van de Laar,M.A., Markusse,H.M., van Denderen,J.C., Westedt,M.L., Peeters,A.J., Dijkmans,B.A.C., Jacobs,P., Boonen,A., van der 1 Bradshaw,D.M. and Arceci,R.J. Clinical relevance of transmembrane drug efflux as a mechanism of Heijde,D.M. and Van der Linden,S. COBRA combination therapy in patients with early rheumatoid multidrug resistance, J.Clin.Oncol., 16: 3674-3690, 1998. arthritis: Long-term structural benefits of a brief intervention, Arthritis Rheum., 46: 347-356, 2 Borst,P. and Oude Elferink,R.P. Mammalian ABC transporters in health and disease, Annu.Rev. 2002. Biochem., 71: 537-592, 2002. 20 O’Dell,J.R., Leff,R., Paulsen,G., Haire,C., Mallek,J., Eckhoff,P.J., Fernandez,A., Blakely,K., 3 Borst,P., Evers,R., Kool,M. and Wijnholds,J. A family of drug transporters: the multidrug resistance- Wees,S., Stoner,J., Hadley,S., Felt,J., Palmer,W., Waytz,P., Churchill,M., Klassen,L. and Moore,G. associated proteins, J.Natl.Cancer Inst., 92: 1295-1302, 2000. Treatment of rheumatoid arthritis with methotrexate and hydroxychloroquine, methotrexate 4 Gottesman,M.M. and Ambudkar,S.V. Overview: ABC transporters and human disease, J.Bioenerg. and sulfasalazine, or a combination of the three medications: Results of a two-year, randomized, Biomembr., 33: 453-458, 2001. double-blind, placebo-controlled trial, Arthritis Rheum., 46: 1164-1170, 2002. 5 Leslie,E.M., Deeley,R.G. and Cole,S.P. Toxicological relevance of the multidrug resistance protein 21 Cutolo,M., Sulli,A., Pizzorni,C., Seriolo,B. and Straub,R.H. Anti-inflammatory mechanisms of 1, MRP1 (ABCC1) and related transporters, Toxicology, 167: 3-23, 2001. methotrexate in rheumatoid arthritis, Ann.Rheum.Dis., 60: 729-735, 2001. 6 Litman,T., Druley,T.E., Stein,W.D. and Bates,S.E. From MDR to MXR: new understanding of 22 Van der Heijden,J., de Jong,M.C., Dijkmans,B.A.C., Lems,W.F., Oerlemans,R., Kathmann,G. multidrug resistance systems, their properties and clinical significance, Cell Mol.Life Sci., 58: 931- A., Schalkwijk,C.G., Scheffer,G.L., Scheper,R.J. and Jansen,G. Development of sulphasalazine 959, 2001. resistance in human T cells induces expression of the multidrug resistance transporter ABCG2 7 Gottesman,M.M., Fojo,T. and Bates,S.E. Multidrug resistance in cancer: role of ATP-dependent (BCRP) and augmented production of TNFalpha, Ann.Rheum.Dis., 63: 138-143, 2004. transporters, Nat.Rev.Cancer, 2: 48-58, 2002. 23 Allen,J.D., van Loevezijn,A., Lakhai,J.M., van der Valk,M., van Tellingen,O., Reid,G., Schellens,J. 8 Hooijberg,J.H., Broxterman,H.J., Kool,M., Assaraf,Y.G., Peters,G.J., Noordhuis,P., Scheper,R.J., H.M., Koomen,G.J. and Schinkel,A.H. Potent and specific inhibition of the breast cancer resistance Borst,P., Pinedo,H.M. and Jansen,G. Antifolate resistance mediated by the multidrug resistance protein multidrug transporter in vitro and in mouse intestine by a novel analogue of fumitremorgin proteins MRP1 and MRP2, Cancer Res., 59: 2532-2535, 1999. C, Molecular Cancer Therapeutics, 1: 417-425, 2002. 9 Wijnholds,J., Mol,C.A., van Deemter,L., de Haas,M., Scheffer,G.L., Baas,F., Beijnen,J.H., Scheper,R. 24 Jansen,G., Mauritz,R., Drori,S., Sprecher,H., Kathmann,I., Bunni,M., Priest,D.G., Noordhuis,P., J., Hatse,S., De Clercq,E., Balzarini,J. and Borst,P. Multidrug-resistance protein 5 is a multispecific Schornagel,J.H., Pinedo,H.M., Peters,G.J. and Assaraf,Y.G. A structurally altered human reduced organic anion transporter able to transport nucleotide analogs, Proc.Natl.Acad.Sci.U.S.A, 97: folate carrier with increased folic acid transport mediates a novel mechanism of antifolate 7476-7481, 2000. resistance, J.Biol.Chem., 273: 30189-30198, 1998. 10 Wielinga,P.R., Reid,G., Challa,E.E., van der Heijden,I., van Deemter,L., de Haas,M., Mol,C., Kuil,A. 25 Mauritz,R., Bekkenk,M.W., Rots,M.G., Pieters,R., Mini,E., Van Zantwijk,C.H., Veerman,A.J., J., Groeneveld,E., Schuetz,J.D., Brouwer,C., De Abreu,R.A., Wijnholds,J., Beijnen,J.H. and Borst,P. Peters,G.J. and Jansen,G. Ex vivo activity of methotrexate versus novel antifolate inhibitors of Thiopurine metabolism and identification of the thiopurine metabolites transported by MRP4 dihydrofolate reductase and thymidylate synthase against childhood leukemia cells, Clin.Cancer and MRP5 overexpressed in human embryonic kidney cells, Mol.Pharmacol., 62: 1321-1331, 2002. Res., 4: 2399-2410, 1998. 11 Renes,J., de Vries,E.G., Jansen,P.L. and Muller,M. The (patho)physiological functions of the MRP 26 Das,K.M. and Dubin,R. Clinical pharmacokinetics of sulphasalazine, Clin.Pharmacokinet., 1: 406- family, Drug Resist.Updat., 3: 289-302, 2000. 425, 1976. 12 Doyle,L.A., Yang,W., Abruzzo,L.V., Krogmann,T., Gao,Y., Rishi,A.K. and Ross,D.D. A multidrug 27 Azadkhan,A.K., Truelove,S.C. and Aronson,J.K. The disposition and metabolism of sulphasalazine resistance transporter from human MCF-7 breast cancer cells, Proc.Natl.Acad.Sci.U.S.A, 95: (salicylazosulphapyridine) in man, Br.J.Clin.Pharmacol., 13: 523-528, 1982. 15665-15670, 1998. 28 Jansen,J.A. Kinetics of the binding of salicylazosulfapyridine to human serum albumin, Acta 13 Volk,E.L., Farley,K.M., Wu,Y., Li,F., Robey,R.W. and Schneider,E. Overexpression of wild-type Pharmacol.Toxicol.(Copenh), 41: 401-416, 1977. breast cancer resistance protein mediates methotrexate resistance, Cancer Res., 62: 5035-5040, 29 Mini,E., Moroson,B.A., Franco,C.T. and Bertino,J.R. Cytotoxic effects of folate antagonists against 2002. methotrexate-resistant human leukemic lymphoblast CCRF-CEM cell lines, Cancer Res., 45: 325- 14 Wolfe,F. The epidemiology of drug treatment failure in rheumatoid arthritis, Baillieres Clin. 330, 1985. Rheumatol., 9: 619-632, 1995. 30 Mauritz,R., Peters,G.J., Priest,D.G., Assaraf,Y.G., Drori,S., Kathmann,I., Noordhuis,P., Bunni,M.A., 15 Wolfe,F. Adverse drug reactions of DMARDs and DC-ARTs in rheumatoid arthritis, Clin.Exp. Rosowsky,A., Schornagel,J.H., Pinedo,H.M. and Jansen,G. Multiple mechanisms of resistance to Rheumatol., 15 Suppl 17: S75-S81, 1997. methotrexate and novel antifolates in human CCRF-CEM leukemia cells and their implications for 16 Maetzel,A., Wong,A., Strand,V., Tugwell,P., Wells,G. and Bombardier,C. Meta-analysis of folate homeostasis, Biochem.Pharmacol., 63: 105-115, 2002. treatment termination rates among rheumatoid arthritis patients receiving disease-modifying 31 Rots,M.G., Pieters,R., Kaspers,G.J., Veerman,A.J., Peters,G.J. and Jansen,G. Classification of ex anti-rheumatic drugs, Rheumatology.(Oxford), 39: 975-981, 2000. vivo methotrexate resistance In acute lymphoblastic and myeloid leukaemia, Br.J.Haematol., 110: 17 Aletaha,D. and Smolen,J.S. Effectiveness profiles and dose dependent retention of traditional 791-800, 2000. disease modifying antirheumatic drugs for rheumatoid arthritis. An observational study, 32 Scheffer,G.L., Kool,M., Heijn,M., de Haas,M., Pijnenborg,A.C., Wijnholds,J., van Helvoort,A., de J.Rheumatol., 29: 1631-1638, 2002. Jong,M.C., Hooijberg,J.H., Mol,C.A., van der Linden,M., de Vree,J.M., van der Valk,P., Elferink,R.P., 18 Morgan,C., Lunt,M., Brightwell,H., Bradburn,P., Fallow,W., Lay,M., Silman,A. and Bruce,I.N. Borst,P. and Scheper,R.J. Specific detection of multidrug resistance proteins MRP1, MRP2, MRP3, Contribution of patient related differences to multidrug resistance in rheumatoid arthritis, Ann. MRP5, and MDR3 P-glycoprotein with a panel of monoclonal antibodies, Cancer Res., 60: 5269- Rheum.Dis., 62: 15-19, 2003. 5277, 2000.

76 77 References References

33 Maliepaard,M., Scheffer,G.L., Faneyte,I.F., van Gastelen,M.A., Pijnenborg,A.C., Schinkel,A.H., Helmers,M.C., Floot,B.G. and Schellens,J.H. Overexpression of the BCRP/MXR/ABCP gene in a van De Vijver,M.J., Scheper,R.J. and Schellens,J.H. Subcellular localization and distribution of the topotecan-selected ovarian tumor cell line, Cancer Res., 59: 4559-4563, 1999. breast cancer resistance protein transporter in normal human tissues, Cancer Res., 61: 3458-3464, 52 Robbiani,D.F., Finch,R.A., Jager,D., Muller,W.A., Sartorelli,A.C. and Randolph,G.J. The leukotriene 2001. C(4) transporter MRP1 regulates CCL19 (MIP-3beta, ELC)- dependent mobilization of dendritic 34 Ross,D.D., Yang,W., Abruzzo,L.V., Dalton,W.S., Schneider,E., Lage,H., Dietel,M., Greenberger,L., cells to lymph nodes, Cell, 103: 757-768, 2000. Cole,S.P. and Doyle,L.A. Atypical multidrug resistance: breast cancer resistance protein messenger 53 Bach,M.K., Brashler,J.R. and Johnson,M.A. Inhibition by sulfasalazine of LTC synthetase and of rat RNA expression in mitoxantrone-selected cell lines, J.Natl.Cancer Inst., 91: 429-433, 1999. liver glutathione S-transferases, Biochem.Pharmacol., 34: 2695-2704, 1985. 35 Goekoop,Y.P., Allaart,C.F., Breedveld,F.C. and Dijkmans,B.A.C. Combination therapy in rheumatoid 54 Assaraf,Y.G., Rothem,L., Hooijberg,J.H., Stark,M., Ifergan,I., Kathmann,G.A., Dijkmans,B.A.C., arthritis, Curr.Opin.Rheumatol., 13: 177-183, 2001. Peters,G.J. and Jansen,G. Loss of multidrug resistance protein 1 (MRP1) expression and folate efflux 36 Litman,T., Brangi,M., Hudson,E., Fetsch,P., Abati,A., Ross,D.D., Miyake,K., Resau,J.H. and Bates,S. activity results in a highly concentrative folate transport in human leukemia cells, J.Biol.Chem., E. The multidrug-resistant phenotype associated with overexpression of the new ABC half- 278: 6680-6686, 2003. transporter, MXR (ABCG2), J.Cell Sci., 113 ( Pt 11): 2011-2021, 2000. 55 Baum,C.L., Selhub,J. and Rosenberg,I.H. Antifolate actions of sulfasalazine on intact lymphocytes, 37 Wahl,C., Liptay,S., Adler,G. and Schmid,R.M. Sulfasalazine: a potent and specific inhibitor of J. Lab. Clin. Med., 97: 779-784, 1981. nuclear factor kappa B, J.Clin.Invest, 101: 1163-1174, 1998. 56 ten Wolde,S., Hermans,J., Breedveld,F.C. and Dijkmans,B.A.C. Effect of resumption of second line 38 Tak,P.P. and Firestein,G.S. NF-kappaB: a key role in inflammatory diseases, J.Clin.Invest, 107: 7-11, drugs in patients with rheumatoid arthritis that flared up after treatment discontinuation, Ann. 2001. Rheum.Dis., 56: 235-239, 1997. 39 Lee,D.H. and Goldberg,A.L. Proteasome inhibitors: valuable new tools for cell biologists, Trends 57 Jansen,G., Scheper,R.J. and Dijkmans,B.A.C. Chloroquine resitance in human CEM (T) cells is Cell Biol., 8: 397-403, 1998. mediated by multidrug resistance protein 1, Annals of the Rheumatic Diseases, 60: 89, 2001. 40 Barrera,P., Haagsma,C.J., Boerbooms,A.M., van Riel,P.L., Borm,G.F., van de Putte,L.B. and Van der 58 Dalton,W.S., Durie,B.G., Alberts,D.S., Gerlach,J.H. and Cress,A.E. Characterization of a new drug- Meer,J.W. Effect of methotrexate alone or in combination with sulphasalazine on the production resistant human myeloma cell line that expresses P-glycoprotein, Cancer Res., 46: 5125-5130, and circulating concentrations of cytokines and their antagonists. Longitudinal evaluation in 1986. patients with rheumatoid arthritis, Br.J.Rheumatol., 34: 747-755, 1995. 59 Legrand,O., Perrot,J.Y., Tang,R., Simonin,G., Gurbuxani,S., Zittoun,R. and Marie,J.P. Expression of 41 Rodenburg,R.J., Ganga,A., van Lent,P.L., van de Putte,L.B. and van Venrooij,W.J. The the multidrug resistance-associated protein (MRP) mRNA and protein in normal peripheral blood antiinflammatory drug sulfasalazine inhibits tumor necrosis factor alpha expression in and bone marrow haemopoietic cells, Br.J.Haematol., 94: 23-33, 1996. macrophages by inducing apoptosis, Arthritis Rheum., 43: 1941-1950, 2000. 60 Laupeze,B., Amiot,L., Payen,L., Drenou,B., Grosset,J.M., Lehne,G., Fauchet,R. and Fardel,O. 42 Bates,S.E., Robey,R., Miyake,K., Rao,K., Ross,D.D. and Litman,T. The role of half-transporters in Multidrug resistance protein (MRP) activity in normal mature leukocytes and CD34-positive multidrug resistance, J.Bioenerg.Biomembr., 33: 503-511, 2001. hematopoietic cells from peripheral blood, Life Sci., 68: 1323-1331, 2001. 43 Taylor,C.W., Dalton,W.S., Parrish,P.R., Gleason,M.C., Bellamy,W.T., Thompson,F.H., Roe,D. 61 Zhou,S., Schuetz,J.D., Bunting,K.D., Colapietro,A.M., Sampath,J., Morris,J.J., Lagutina,I., J. and Trent,J.M. Different mechanisms of decreased drug accumulation in doxorubicin and Grosveld,G.C., Osawa,M., Nakauchi,H. and Sorrentino,B.P. The ABC transporter Bcrp1/ABCG2 is mitoxantrone resistant variants of the MCF7 human breast cancer cell line, Br.J.Cancer, 63: 923- expressed in a wide variety of stem cells and is a molecular determinant of the side-population 929, 1991. phenotype, Nat.Med., 7: 1028-1034, 2001. 44 Scheffer,G.L., Maliepaard,M., Pijnenborg,A.C., van Gastelen,M.A., de Jong,M.C., Schroeijers,A.B., 62 Salmon,S.E. and Dalton,W.S. Relevance of multidrug resistance to rheumatoid arthritis: van der Kolk,D.M., Allen,J.D., Ross,D.D., van der Valk,P., Dalton,W.S., Schellens,J.H. and Scheper,R. development of a new therapeutic hypothesis, J.Rheumatol.Suppl, 44: 97-101, 1996. J. Breast cancer resistance protein is localized at the plasma membrane in mitoxantrone- and 63 Maillefert,J.F., Maynadie,M., Tebib,J.G., Aho,S., Walker,P., Chatard,C., Dulieu,V., Bouvier,M., topotecan-resistant cell lines, Cancer Res., 60: 2589-2593, 2000. Carli,P.M. and Tavernier,C. Expression of the multidrug resistance glycoprotein 170 in the 45 Yamamoto,Y. and Gaynor,R.B. Therapeutic potential of inhibition of the NF-kappaB pathway in peripheral blood lymphocytes of rheumatoid arthritis patients. The percentage of lymphocytes the treatment of inflammation and cancer, J.Clin.Invest, 107: 135-142, 2001. expressing glycoprotein 170 is increased in patients treated with prednisolone, Br.J.Rheumatol., 46 Baldwin,A.S. Control of oncogenesis and cancer therapy resistance by the transcription factor NF- 35: 430-435, 1996. kappaB, J.Clin.Invest, 107: 241-246, 2001. 64 Yudoh,K., Matsuno,H., Nakazawa,F., Yonezawa,T. and Kimura,T. Increased expression of multidrug 47 Chen,F., Castranova,V. and Shi,X. New insights into the role of nuclear factor-kappaB in cell growth resistance of P-glycoprotein on Th1 cells correlates with drug resistance in rheumatoid arthritis, regulation, Am.J.Pathol., 159: 387-397, 2001. Arthritis Rheum., 42: 2014-2015, 1999. 48 Manna,S.K., Mukhopadhyay,A. and Aggarwal,B.B. Leflunomide suppresses TNF-induced cellular 65 Llorente,L., Richaud-Patin,Y., Diaz-Borjon,A., Alvarado,D.L.B., Jakez-Ocampo,J., De La Fuente H., responses: effects on NF- kappa B, activator protein-1, c-Jun N-terminal protein kinase, and Gonzalez-Amaro,R. and Diaz-Jouanen,E. Multidrug resistance-1 (MDR-1) in rheumatic autoimmune apoptosis, J.Immunol., 165: 5962-5969, 2000. disorders. Part I: Increased P-glycoprotein activity in lymphocytes from rheumatoid arthritis 49 Almawi,W.Y. and Melemedjian,O.K. Negative regulation of nuclear factor-kappaB activation and patients might influence disease outcome, Joint Bone Spine, 67: 30-39, 2000. function by glucocorticoids, J.Mol.Endocrinol., 28: 69-78, 2002. 50 Almawi,W.Y., Abou Jaoude,M.M. and Li,X.C. Transcriptional and post-transcriptional mechanisms of glucocorticoid antiproliferative effects, Hematol.Oncol., 20: 17-32, 2002. 51 Maliepaard,M., van Gastelen,M.A., de Jong,L.A., Pluim,D., van Waardenburg,R.C., Ruevekamp-

78 79 chapter 5 Sulfasalazine is a potent inhibitor of the Reduced Folate Carrier: implications for combination therapies with methotrexate in rheumatoid arthritis

Gerrit Jansen1, Joost W. van der Heijden1, Ruud Oerlemans1, Willem F. Lems1, Ilan Ifergan2, Rik J. Scheper3, Yehuda G. Assaraf2 and Ben A.C. Dijkmans1

Departments of 1Rheumatology and 3Pathology, VU University Medical Center, Amsterdam, The Netherlands. 2The Fred Wyszkowski Cancer Research Laboratory, department of Biology, The Technion, Israel Institute of Technology, Haifa, Israel

Arthritis & Rheumatism 2004; 50:2130-2139 chapter 5 Sulfasalazine - Reduced Folate Carrier Interactions

Abstract those with inflammatory bowel disease (9). For the treatment of RA, SSZ (2-3 g/day) is often combined with the anchor drug MTX (7.5-15 mg/week), although this combination therapy Objective: To investigate whether interactions of sulfasalazine (SSZ) with the reduced does not display additive/synergistic effects compared with monotherapy with either SSZ folate carrier (RFC), the dominant cell membrane transporter for natural folates and or MTX (10, 11). In fact, addition of a third component such as hydroxychloroquine (12, 13) or methotrexate (MTX), may limit the efficacy of combination therapy with MTX and SSZ in prednisone (14) is usually required to significantly improve clinical efficacy. Furthermore, patients with rheumatoid arthritis. one notable adverse effect was a persistent increase in plasma homocysteine levels in Methods: Human RFC-(over)expressing CEM cells of T-cell origin were used to analyze the patients with RA who were receiving the combination of MTX and SSZ, compared with effect of SSZ on the RFC-mediated cellular uptake of radiolabeled MTX and the natural those receiving single-drug therapy (15, 16). Elevated plasma homocysteine levels are folate leucovorin. Moreover, both cells with and those without acquired resistance to SSZ associated with cellular folate depletion (17-22), which has been ascribed to the inhibitory were used to assess the antiproliferative effects of MTX in combinations with SSZ. effects of SSZ on several intracellular enzymes in folate metabolism (23, 24) and/or Results: Transport kinetic analyses revealed that SSZ was a potent noncompetitive inhibitor inhibition of intestinal folate uptake (25-27).

of RFC-mediated cellular uptake of MTX and leucovorin with mean Ki (50% inhibitory In this study we present various lines of evidence for a novel site of interaction of SSZ with concentration) values of 36 ± 6 µM and 74 ± 7 µM SSZ, respectively. Consistent with the the reduced folate carrier (RFC) (28-32). RFC is the dominant cell membrane transporter inhibitory interaction of SSZ with RFC, a marked loss of MTX efficacy was observed when for natural folates such as the main circulating folate 5-methyltetrahydrofolate, 5- the latter was co administered with SSZ: up to 3.5-fold for CEM cells in the presence of 0.25 formyltetrahydrofolate/leucovorin and folic acid (29, 32). Importantly, MTX also utilizes

mM SSZ, and >400-fold for SSZ-resistant cells in the presence of 2.5 mM of SSZ. Importantly, RFC for cellular uptake with an affinity (Km: 1-5 µM) close to 5-methyltetrahydrofolate along with a diminished efficacy of MTX, evidence for cellular folate depletion was obtained (29, 32). Given their anionic nature, natural folates and MTX can not traverse the plasma by the demonstration of a SSZ dose-dependent decrease in leucovorin accumulation. membrane; therefore RFC-mediated cellular uptake should be regarded as the first Conclusion: At clinically relevant plasma concentrations, interactions of SSZ with the RFC critical step in any anti-inflammatory/antiproliferative mode of action of MTX (33-36). provide a biochemical rationale for two important clinical observations: (1) the onset of a Consequently, diminished transport activity of RFC has been associated with loss of MTX (sub)clinical folate deficiency during SSZ treatment, and (2) the lack of additivity/synergism efficacy and with folate deficiency (31, 37-40). of the combination of SSZ and MTX when these disease-modifying antirheumatic drugs are Herein we report that SSZ is a potent, non-competitive inhibitor of the RFC. Consistently, administered simultaneously. Thus, when considering use of these drugs in combination inhibition of RFC transport activity by SSZ resulted in a diminished efficacy of MTX when therapies, the present results provide both a rationale for folate supplementation and for coadministered with SSZ and, as a side-effect, a cellular folate depletion. The possible spacing administration of these drugs over time. clinical implications of SSZ-RFC interactions for SSZ/MTX-based therapies for RA are discussed.

Introduction Materials and methods Although novel biologic agents (e.g. anti-tumour necrosis factor α or anti-interleukin- 1) have become available for the treatment of patients with rheumatoid arthritis (RA) Materials (1-3), traditional disease modifying anti-rheumatic drugs (DMARDs) or combinations SSZ, 5’-aminosalicylic acid (5’-ASA), sulfapyridine (SP) and folic acid were purchased from of DMARDs, still find widespread application because of their proven efficacy and low Sigma (St. Louis, MO, USA). Leucovorin was obtained from Eprova Co (Schaffhausen, cost (1, 3, 4). Moreover, long term superiority of expensive biologic agents over current Switzerland). Methotrexate was a generous gift from Pharmachemie (Haarlem, The (combinations of) DMARDs has not yet been proven. Therefore, further research on Netherlands). Trimetrexate was a gift from Dr. D. Fry, (Park-Davis, Ann Arbor, MI, USA).

o combination therapies remains warranted, at the levels of both clinical trials and Stock solutions of 25 mM SSZ were prepared by dissolving SSZ in 50 mM NaHCO3 at 37 C. fundamental research, in particular regarding mechanisms of action and possible drug Following filter sterilization (through a 0.22 µm filter), stock solutions were stored at interactions (5). -20oC. Stock solutions (0.5 M) of 5’-ASA and SP were prepared in DMSO. 3’, 5’,7’-[3H]-MTX Sulfasalazine (SSZ) is commonly prescribed for the treatment of patients with RA (6-8) and (specific activity 20-25 Ci/mmol) and 3’,5’, 7,9-[3H]-Leucovorin (specific activity 15-20 Ci/

82 83 chapter 5 Sulfasalazine - Reduced Folate Carrier Interactions

mmol) were obtained from Moravek Biochemicals (Brea, CA) and purified prior to use by ml ice-cold transport buffer, after which the cell suspension was centrifuged (500g for 5 thin layer chromatography as described previously (41). RPMI 1640 tissue culture medium minutes at 4oC) and the cell pellet was washed once more with 10 ml ice-cold transport (with or without 2.2 µmol/l folic acid) and whole and dialyzed fetal calf serum (FCS) were buffer. The final pellet was suspended in water and processed for radioactivity counting. purchased from Gibco BRL (Grand Island, NY, USA). Quantification of RFC and multidrug resistance-associated protein 1 (MRP1) Cell lines expression by Western blot analysis Cell cultures of human CEM cells (T-cell origin) were maintained in RPMI 1640 medium Triton X-100-soluble microsomal proteins (50 µg) were resolved by electrophoresis (with 2.2 µmol/l folic acid, unless otherwise indicated) supplemented with 10% FCS, 2 mM on 10% polyacrylamide gels containing SDS and electroblotted onto nitrocellulose L-glutamine and 100 U/ml penicillin/streptomycin. Cells were cultured at an initial density nylon membranes. The blots were then blocked for 1 hour at room temperature in Tris

5 of 3x10 cells/ml in a 5% CO2 incubator as described previously (42). A subline of CEM cells buffered saline (TBS) buffer (150 mM NaCl and 0.5% Tween 20 and 10 mM Tris/HCl at pH (CEM-7A) with RFC overexpression was grown in folic acid-free RPMI medium, 10% dialyzed 8.0) containing 1% skim milk. The blots were reacted with a polyclonal antiserum (1:700) FCS, 2 mM L-glutamine, 100 U/ml penicillin/streptomycin and 0.15 nM L-leucovorin as the prepared in mice against a C-terminal hRFC peptide. Blots were then rinsed in the same sole folate source (43-45). CEM cells with acquired resistance to SSZ were obtained by buffer for 10 minutes at room temperature and reacted with horseradish peroxidase- growing CEM cells in stepwise increasing concentrations of SSZ over a period of 6 months conjugated goat anti-mouse IgG (1:40,000 dilution, Jackson Immunoresearch, West (46, 47). CEM cells selected to grow in the presence of 1.5 mM SSZ (CEM/SSZ1.5) and 2.5 Grove, PA) for 1 hr at room temperature (38). Following three 10 minutes washes in TBS at mM SSZ (CEM/SSZ2.5) were used in this study. room temperature, enhanced chemoluminescence detection was performed according to the manufacturer’s instructions (Biological Industries, Kibbutz, Beth Haemek, Israel). Cell growth inhibition/stimulation studies Protein expression of MRP1 was analyzed by Western blotting according to previously The antiproliferative effects of DMARDs were assessed by seeding cells into individual wells described procedures, using MRPr1 (1:500) monoclonal antibody to MRP1 (46, 48). of 24 well plates (1.25x105 cells/ml; 1 ml/well). For each drug, 8 concentrations covering 2-3 Protein was determined by the colorimetric method (Biorad, Hercules, CA) described by logs were added to the individual wells. Thereafter, cells were exposed continuously to the Bradford. drugs for 72 hours after which viable cell numbers were determined by a haemocytometer and trypan blue exclusion. The drug concentration required to inhibit cell proliferation Statistical analysis by 50% (IC-50 value) is determined to assess antiproliferative effects. For folic acid and Results were analyzed by Student’s t-test. P values less than 0.05 were considered leucovorin growth stimulation analysis, cells were suspended in folic acid-free modified significant. RPMI medium supplemented with 10% dialyzed FCS and transferred to individual wells of a 24 well plate containing increasing concentrations of either leucovorin (range: 0-250 nM) or folic acid (0-250 µM). Stimulation of cell growth was analyzed after 72 hours of RESULTS incubation by determining the cell number with a hemocytometer. Effects of SSZ on MTX efficacy Transport studies To test the effect of SSZ on the efficacy of MTX, human T cells (CEM cells) were exposed RFC-mediated uptake of [3H]-MTX or [3H]-LV was analyzed for CEM cells, CEM-7A cells to MTX in the presence of non-toxic concentrations of SSZ (up to 0.25 mM) after which and CEM/SSZ cells in the mid-log phase of growth. Cells were harvested and washed with the degree of abrogation of the antiproliferative capacity of MTX was determined (Figure

transport buffer (140 mM NaCl, 20 mM HEPES, 6 mM KCl, 2 mM MgCl2, and 6.0 mM D- 1A). In the presence of 250 µM SSZ, the antiproliferative efficacy of MTX was markedly glucose, pH 7.4). Cells were suspended to a final density of 5x106/ml for RFC-overproducing decreased (3.5-fold) when compared with that in absence of SSZ (p<0.001); the mean CEM-7A cells, and 1.5x107/ml for CEM or CEM/SSZ cells. Incubations with 0.05-5 µM [3H]- ±SD IC-50 values were 34.1 ±2.1 and 9.7 ±1.5 nM MTX, respectively. In contrast, equimolar MTX or [3H]-LV (final specific activity: 1000 dpm/pmol), in the absence or presence of concentrations (0.25 mM) of the SSZ catabolic products 5’-ASA and sulfapyridine (49), did inhibitors, were made for 1.5 minutes (for CEM-7A cells) and 3 minutes (for CEM and CEM/ not alter MTX efficacy against CEM cells (Figure 1A). SSZ cells) at 37oC in a shaking water bath. Transport was terminated by the addition of 10 We next determined the efficacy of MTX against SSZ-resistant CEM cells (CEM/SSZ),

84 85 Figure_1A.pzf:Data 1 graph - Mon Aug 18 13:38:33 2008 Figure_1B.pzf:Data 1 graph - Mon Aug 18 13:39:10 2008

chapter 5 Sulfasalazine - Reduced Folate Carrier Interactions Figure_1C.pzf:Data 1 graph - Mon Aug 18 13:39:45 2008

Figure 1 A Figure 1 B also affects the efficacy of folate antagonists other than MTX, we performed similar

120 experiments with the folate antagonist trimetrexate. Like MTX, trimetrexate has the 120 CEM No addition 100 CEM/SSZ, no addition same target in folate metabolism, i.e. dihydrofolate reductase (DHFR), but unlike MTX, 100 +0.25mM SSZ CEM/SSZ + 0.25mM SSZ 80 trimetrexate does not require cellular entry through the RFC, because it enters cells by +0.25mM 5'-ASA 80 CEM/SSZ + 1.00mM SSZ +0.25mM sulfapyridine 60 CEM/SSZ + 2.50mM SSZ passive diffusion (32, 33, 50, 51). Strikingly, in combination with increasing concentrations 60 40 of SSZ, markedly increased efficacy (i.e., a decrease in the IC-50 values) of trimetrexate (up 40 to 8-fold; p<0.001) was noted against CEM/SSZ cells as compared with CEM cells (Figure 20 Cell growth (% of control) 20 Cell growth (% of control) 1C). These results demonstrate that the differential effects of SSZ on MTX and trimetrexate 0 0 10 20 30 40 50 0 1 10 100 1000 10000 efficacy cannot be explained at the level of the target enzyme DHFR. Rather, these results [MTX] (nM) [MTX] (nM) suggest that SSZ interferes with the cell membrane transport of MTX via RFC, resulting in Figure 1 C a dose-dependent abrogation of MTX efficacy. 5000 20

1000 Effects of SSZ on RFC-mediated transport of MTX and natural folates 15 To test the hypothesis that SSZ exerts an inhibitory action on RFC transport activity, we 100 10 determined whether SSZ is capable of inhibiting RFC-mediated transport of radiolabeled IC-50 MTX (nM) 10 IC-50 TMQ (nM) MTX in human CEM-7A cells that overexpress RFC (43). Indeed, experiments shown in Figure  5  2A demonstrate that RFC-mediated cellular uptake of radiolabeled MTX was inhibited by 1 0 SSZ in a concentration-dependent manner, whereas the catabolic products of SSZ, 5’- 0 0.25 1.0 2.5 [SSZ] (mM) ASA and sulfapyridine had no effect on RFC-mediated transport of MTX. Similar results were obtained for CEM cells and CEM/SSZ cells (data not shown). Because the physiologic Figure 1 (A) Antiproliferative effect of MTX in combination with SSZ, 5’-ASA or sulfapyridine against CEM cells. CEM cells were incubated with a concentration range of MTX in the absence or presence of 0.25 mM function of RFC is to transport natural folates, we determined whether SSZ could also SSZ, 0.25 mM 5’-ASA or 0.25 mM sulfapyridine. After 72 hours incubation, cell counts were determined by inhibit RFC-mediated transport of leucovorin as a representative natural folate. Indeed, SSZ a hemocytometer. Results are expressed as the percentage of the growth of cells incubated in the absence also inhibited RFC mediated transport of leucovorin, although, when compared with MTX, of drugs. Results are the mean of 3 independent experiments (SD <20%). (B) Antiproliferative effect of MTX in combination with SSZ against CEM/SSZ cells. SSZ-resistant CEM cells were incubated for 72 hours with a 2-fold higher concentration of SSZ was required to elicit a 50% inhibition of leucovorin a concentration range of MTX in the absence or presence of 0.25 mM SSZ, 1.0 mM SSZ or 2.5 mM SSZ. transport (Figure 2B). Interestingly, the potential of SSZ to inhibit RFC-mediated transport Data for parental CEM cells are given for comparison. Analyses for antiproliferative effects were performed as described in (A). Results are the mean of 3 independent experiments (SD <17%). (C) Antiproliferative of MTX was not affected by extracellular concentrations of MTX; at an extracellular potency of MTX and trimetrexate (TMQ) in combination with SSZ against CEM/SSZ cells. CEM/SSZ cells were concentration of either 0.05 µM or 2 µM MTX, the same level of inhibition was observed incubated for 72 hours with a concentration range of MTX (closed squares) or trimetrexate (closed circles) (Figure 2B). in the absence or presence of SSZ (0.25-2.5 mM). Analyses for antiproliferative effects were performed as described in (A). Results are the mean of 3 independent experiments (SD <19%). IC-50 value = 50% inhibitory To identify the mode of inhibition of RFC-dependent transport by SSZ, the inhibitory concentration. effects of SSZ on RFC transport were measured at extracellular concentrations of MTX or

leucovorin (1-5 µM) in the relevant range of substrate affinities (Km) for RFC. Dixon plots, which can tolerate higher concentrations of SSZ than parental CEM cells due to the representing reciprocal transport rates of MTX uptake (Figure 2C) or leucovorin uptake overexpression of the multidrug resistance transporter ABCG2 (46, 47). In the absence of (Figure 2D) at various concentrations of SSZ (0-100 µM), revealed a noncompetitive mode

SSZ, CEM/SSZ cells and parental CEM cells were equally sensitive to MTX, however, in the of inhibition, with mean ±SD Ki (50% inhibitory concentration) values of 36 ±6 µM SSZ for presence of SSZ a significant (p<0.001), concentration-dependent loss of MTX efficacy RFC-mediated MTX transport and 74 ±7 µM SSZ for leucovorin transport. (i.e. increase in IC-50 value) was observed: 3.4-fold at 0.25 mM SSZ, 41-fold at 1.0 mM SSZ and 345-fold at 2.5 mM SSZ (Figure 1B). Effect of SSZ on cellular folate accumulation and folate growth dependency The changes in the IC-50 values for MTX in the presence of increasing concentrations Consistent with the inhibitory interaction of SSZ with RFC, SSZ induced a dose-dependent of SSZ are illustrated in Figure 1C. In order to explore whether coadministration of SSZ inhibition of [3H]-LV accumulation in CEM and CEM/SSZ cells incubated for 24 hours

86 87 Figure_3A.pzf:Data 1 graph - Wed Oct 08 17:33:12 2008 Figure_3B.pzf:Data 1 graph - Mon Aug 18 13:43:17 2008 Figure_2A.pzf:Data 1 graph - Wed Oct 08 17:38:28 2008 Figure_2B.pzf:Data 1 graph - Fri Sep 26 10:02:28 2008

Figure_2C.pzf:Data 1 graph - Mon Aug 18 13:41:28 2008 Figure_2D.pzf:Data 1 graph - Mon Aug 18 13:42:01 2008 Figure_3C.pzf:Data 1 graph - Mon Aug 18 13:43:51 2008 chapter 5 Sulfasalazine - Reduced Folate Carrier Interactions

Figure 2 A Figure 2 B Figure 3 A Figure 3 B

3 30 120 120 CEM-7A +SSZ 120 50 nM H-MTX CEM CEM

3 cells) CEM-7A +5'-ASA 100 2 µM H-MTX 7 100 CEM/SSZ1.5 -SSZ 100 CEM/SSZ1.5 CEM-7A +sulfapyridine µ 3 CEM/SSZ1.5 +SSZ 80 80 2 M H-LV 20 CEM/SSZ2.5 80 H]-LV uptake H]-MTX uptake 3 3 60 60 60

%of control 40 %of control 40 10 40 20 20 H]-MTXor [

3 20 [ H]-LV uptake (pmol/10 Cell growth (% of maximum) 0 3 RFCmediated [ 0 [ 0.001 0.01 0.1 1 10 0.001 0.01 0.1 1 0 0 0.0 0.5 1.0 [SSZ or catabolites] (mM) [SSZ] (mM) 0.01 0.1 1 10 100 1000 [SSZ] (mM) Leucovorin (nM) Figure 2 C Figure 2 D Figure 3 C 10 4 120 CEM 8 100 CEM/SSZ1.5 -SSZ 3 -1 -1 80 CEM/SSZ1.5 +SSZ 6 2 60 (x 0.01) 4 (x 0.01) (LV-uptake)

(MTX uptake) 40 1 2 20 Cell growth (% of maximum) 0 -50 0 25 50 75 100 -100 0 25 20 75 100 0.001 0.01 0.1 1 10 100 µ µ [SSZ] ( M) [SSZ] ( M) Folic acid (µM)

Figure 2 (A): Effect of SSZ, 5’-ASA and sulfapyridine on RFC-mediated uptake of MTX. RFC-mediated uptake Figure 3 (A): Inhibition by SSZ of [3H]-leucovorin accumulation in CEM and CEM/SSZ cells. CEM, CEM/SSZ1.5 of [3H]-MTX was determined in RFC-overexpressing CEM-7A cells following exposure at 37oC for 1.5 minutes and CEM/SSZ2.5 cells were suspended in folate-free RPMI 1640 culture medium containing 50 nM [3H]- to an extracellular concentration of 2 µM [3H]-MTX in the absence or presence of a concentration range leucovorin and various concentrations of SSZ (0-1.0 mM). Accumulation of [3H]-leucovorin was determined (0-1 mM) of SSZ, 5’-ASA or sulfapyridine. Results are the mean of 3 separate experiments (SD <20%) and after 24 hours incubation and expressed as pmoles of [3H]-leucovorin accumulated/107 cells/24 hours. are expressed as a percentage of [3H]-MTX uptake compared to controls without additions. The 100% value Results are the mean of 2 independent experiments performed in duplicate. corresponds to a mean ±SD uptake rate of 85 ±13 pmol [3H]-MTX/minute/107 cells. (B) Leucovorin and (C) folic acid growth stimulation of CEM and CEM/SSZ cells. Cells were suspended in (B) Effect of SSZ on RFC-mediated uptake of MTX and leucovorin (LV). RFC-mediated uptake of [3H]-MTX folate-free RPMI medium with 10% dialyzed FCS and incubated in the absence or presence of 1.5 mM SSZ with and [3H]-leucovorin was determined in CEM-7A cells following exposure at 37oC for 1.5 minutes to an increasing concentrations of leucovorin or folic acid. Cell growth was monitored after 72 hours incubation. extracellular concentration of 50 nM [3H]-MTX, 2 µM [3H]-MTX and 2 µM [3H]-LV, in the absence or presence of a concentration range (0-1 µM) of SSZ. Results are the mean of 3 separate experiments (SD <20%) and are expressed as a percentage of [3H]-MTX/[3H]-LV uptake compared with controls without additions. The with cell growth in the absence of SSZ. These results indicate that the inhibitory action of 100% value for leucovorin uptake corresponds to a mean ±SD uptake rate of 97 ±15 pmol [3H]-LV/minute/107 cells. (C),(D) Dixon plots denoting reciprocal uptake rates of (C) [3H]-MTX and (D) [3H]-LV, at extracellular SSZ on RFC transport activity may lead to a decrease in cellular folate pools. concentrations of 1.0 µM (circles), 2.5 µM (triangles) and 5.0 µM (squares) in the presence of 0-100 µM SSZ. Ki values were calculated from the X-axis intercept. RFC and MRP1 expression in CEM and CEM/SSZ cells At the level of the cell membrane, cellular folate homeostasis is regulated by at least two groups of transporters. The RFC mediates folate transport inward (29, 32), while members with a physiologically representative concentration of 50 nM leucovorin (Figure 3A). of the MRP family (52), in particular MRP1, control cellular export of folates (53-55). In Consequently, this implies that in the presence of SSZ, higher concentrations of folates will order to assess effects of long term exposure to SSZ, we analyzed RFC protein expression be required to support cell growth. This is illustrated for CEM/SSZ cells which required 80- and transport activity and MRP1 protein expression in CEM/SSZ cells. RFC protein fold higher concentrations of leucovorin (EC-50= 16 nM vs. 0.21 nM; p<0.001) (Figure 3B) or expression appeared to be down-regulated 4-fold and 8-fold in CEM/SSZ1.5 cells and CEM/ 27-fold higher concentrations of folic acid (EC-50= 1960 nM vs. 27 nM; p<0.001) (Figure 3C) SSZ2.5 cells, respectively (Figure 4). RFC transport activity, represented by relative rates in order to support half-optimal growth in the presence of 1.5 mM of SSZ when compared of [3H]-MTX uptake, closely corresponded to RFC protein levels in RFC-overproducing

88 89 chapter 5 Sulfasalazine - Reduced Folate Carrier Interactions

dominant cell membrane transport protein for natural folates as well as structurally related CEM-7A, CEM cells and CEM/SSZ cells (Figure 4). Interestingly, MRP1 levels were markedly drugs like MTX. RFC is constitutively expressed in many cell types, particularly those relying decreased in CEM/SSZ cells compared with CEM cells (Figure 4). It is conceivable that this on folate uptake for DNA synthesis and proliferation (29, 32). down-regulation of MRP1 is a response to minimize export of folates after inhibition of RFC-mediated uptake of natural folates by SSZ (53, 55). In the current study, several lines of evidence support RFC inhibition by SSZ. First, the efficacy of MTX was progressively reduced when it was combined with increasing Figure 4 concentrations of SSZ. In fact, the levels of loss of MTX efficacy at high concentrations of SSZ (>400-fold in the presence of 2.5 mM SSZ) were similar to those obtained in cells displaying loss of MTX efficacy due to decreased expression of the RFC protein (28, 38, 39, 56). In this respect, the decreased expression of RFC (up to 8-fold) in CEM/SSZ cells (Figure 4) could have contributed to a diminished efficacy of MTX, but data shown in Figures 1A and 1B did not reveal a difference between MTX efficacy in CEM/SSZ cells and that in CEM cells. This may be explained by the fact that MTX efficacy, as shown in Figures 1A and 1B, is determined mainly by the duration of inhibition of the intracellular target enzyme DHFR by MTX. When sufficient MTX is internalized via RFC to bind DHFR stochiometrically, MTX Figure 4: RFC and MRP1 protein expression of CEM/SSZ cells versus CEM and CEM-7A cells. Western blot analysis of RFC and MRP1 expression in CEM-7A, CEM, CEM/SSZ1.5 and CEM/2.5 cells. A CEM line will start to be efficacious. Experiments described in Figures 1A and 1B included a 72-hour with a mutated RFC gene, CEM/R0.6MTX (38), served as negative control for RFC expression. Na+/K+-ATPase exposure to MTX, a time frame that is permissive to fully saturate DHFR over time, even expression served as control for equal loading. when the internalization rate of MTX is 8-fold lower in CEM/SSZ cells compared with CEM cells. It can also be argued that the decrease in RFC expression in CEM/SSZ cells provokes lower intracellular pools of natural folates, which are also transported by the RFC (see Discussion below). Consequently, less MTX is required to deplete intracellular folate pools as part of the secondary effect of DHFR inhibition. Thus, a lower intracellular folate pool may compensate Notwithstanding current successes with biologic agents, DMARDs continue to have an for any decrease in MTX uptake to confer a similar efficacy of MTX between CEM/SSZ and established place in the treatment of patients with RA. MTX, the anchor drug in RA treatment, CEM cells when analyzed in the absence of SSZ. is effective as a single agent but also serves as the basis for combination therapies with other Second, transport kinetic studies revealed a concentration-dependent inhibition of (RFC- DMARDs such as SSZ. Initial clinical studies with the combination of MTX plus SSZ were mediated) uptake of MTX or leucovorin by SSZ in CEM cells and RFC-overproducing CEM-7A empirically driven by the proven efficacy of both DMARDs as single agents (10, 11), although cells. These observations are not specific for cells of T cell origin, because we observed similar a biochemical basis for the putative additive or synergistic effects of the combination effects of SSZ-RFC interaction in monocytic/macrophage cells (Oerlemans R, Dijkmans BAC, was still lacking. In fact, extensive clinical evaluation of the combination of MTX plus SSZ Jansen G: unpublished observation). revealed no superior activity over mono-therapy with either DMARDs (15). Moreover, side Third, SSZ-mediated inhibition of RFC transport resulted in a diminished uptake of natural effects of the combination of MTX plus SSZ included elevated plasma homocysteine levels, folates required for cell proliferation. To counteract this effect, folate supplementation revealing folate deficiency. Manifestations of (sub)clinical folate deficiency requiring folate may be required whereby leucovorin, concentration-wise, may be more effective than supplementation had been reported previously during SSZ single-drug therapy in patients folic acid, because leucovorin is more efficiently transported via RFC than folic acid (for

with RA (17-22). Thus far, the mechanisms proposed to underlie these effects were inhibitory leucovorin, Km= 1-5 µM; for folic acid, Km= 200-400 µM) (29, 32). SSZ-induced cellular folate effects of SSZ on intestinal folate absorption (25-27) and several enzymes in intracellular deficiency is also reflected by hypersensitivity to the folate antagonist trimetrexate. folate metabolism (23, 24). Hypersensitivity to trimetrexate has been described in cells with low intracellular folate levels due to quantitatively and/or qualitatively defective RFC protein (38, 39, 57, 58). Here In this study, data are presented revealing an as yet unknown mechanism explaining not we demonstrate that direct inhibition of RFC function by SSZ can confer the same phenotype only the lack of additivity/synergism of SSZ when combined with MTX, but also the side of increased sensitivity for the folate antagonist trimetrexate. Hence, these results provide effect of folate deficiency. This mechanism is based on interactions of SSZ with the RFC, the a biochemical rationale for a potential synergistic combination of SSZ plus trimetrexate.

90 91 chapter 5 Sulfasalazine - Reduced Folate Carrier Interactions

Recent studies by Chen et al (59) and Volk et al (60) showed that the multidrug resistance Another strategy of the rheumatologist for enhancing the efficacy of the combination of transporter ABCG2, which is upregulated in CEM/SSZ cells (46, 47), could mediate cellular MTX plus SSZ is the addition of a third drug (e.g. hydroxychloroquine (13) or prednisone export of MTX and contribute to diminished efficacy of MTX. However, recent studies from (14)) to the MTX-SSZ combination. Although the objective of this study was not to our laboratory (61) showed that ABCG2 did not play a major role in MTX resistance upon elucidate a mechanistic basis for the efficacy of the triple DMARD therapy, recent studies prolonged (>72 hrs) exposure to MTX, the conditions that were used in the experiments by our group (47) did indicate that CEM/SSZ cells displayed enhanced sensitivity to both illustrated in Figures 1A and 1B. Of note, ABCG2-mediated MTX resistance was observed chloroquine and dexamethasone. In the case of chloroquine this may be related to down- when MTX exposure was limited to a short time period (<4 hours), during which no long- regulation in the expression of MRP1 (see Figure 4), which, apart from a physiological role chain polyglutamate forms of MTX were formed. of MRP1 in folate homeostasis (53-55), is best known as a drug efflux pump that can export various drugs (52, 66), including chloroquine (67, 68). Down-regulation of MRP1 may Although the effects observed in this in vitro study may be more pronounced because therefore lead to increased intracellular retention of chloroquine, resulting in enhanced cells, in particular SSZ-resistant CEM cells, could be exposed to high concentrations of efficacy. SSZ, the present results are most likely also of clinical relevance. The ability of SSZ to block RFC-mediated transport of MTX and leucovorin by 50% was noted at SSZ concentrations In conclusion, this is the first study to demonstrate that SSZ is a potent noncompetitive of 36 and 74 µM, respectively. Of note, daily administration of SSZ (2-3 g/day) to patients inhibitor of RFC-mediated cellular uptake of MTX and leucovorin. Consistently, the with RA can result in mean plasma levels of up to 100 µM SSZ (24, 49), which is thus clearly inhibitory interaction of SSZ with RFC resulted in a marked loss of MTX efficacy when MTX sufficient to exert an inhibitory effect on RFC-mediated uptake of MTX and natural was coadministered with SSZ. Furthermore, along with diminishing efficacy of MTX, the folates. Consequently, this may trigger a (sub)clinical folate deficiency, especially when inhibition of natural folate uptake by SSZ induced cellular folate depletion. Hence, results dietary intake, absorption or cellular retention of folates is poor. With respect to folate of this study points to the RFC as an important site of interaction with SSZ and downstream absorption, it is important to note that the intestinal folate transporter is structurally and effects on folate and MTX metabolism (see Figure 5). RFC-SSZ interactions warrant further functionally identical to the RFC expressed in CEM cells (62-64). Thus, original reports on consideration for optimal scheduling of MTX/SSZ-based DMARD combination therapies impaired intestinal folate absorption following SSZ treatment (25-27) can be attributed and folate supplementation in the treatment of patients with RA. to direct inhibition of intestinal RFC by SSZ. Complementary inhibitory effects of SSZ on enzymes in the folate metabolism (23, 24) may then further aggravate the folate-deficient Figure 5 status.

It has been well established that a variety of anorganic and organic anions can influence functional RFC transport activity (29, 30). For example, recently we have shown that

prostaglandin A1 (PGA1), which is a charged organic anion at physiological pH, is a potent

noncompetitive inhibitor of RFC-mediated transport of MTX with a Ki of 21 µM (65). Our current study revealed that the monovalent anion SSZ also displays an interaction with the RFC. Of particular interest is that the mode of inhibition of RFC activity by both

PGA1 and SSZ is noncompetitive, indicating that both interact at an RFC domain that is distinct from the folate/MTX binding site. Furthermore, SSZ may interact in an inhibitory mode with the RFC, regardless of whether the latter is loaded with MTX or natural folate substrates. Consequently, this implies that the inhibition of RFC-mediated uptake of MTX Figure 5: Summarizing model of SSZ-RFC interactions and downstream effects. SSZ acts as a non-competitive inhibitor of the RFC by interacting at an RFC domain (triangle groove) that by SSZ cannot be overcome by increasing doses of MTX. Rather, one could speculate that is distinct from the binding/transport site for MTX and natural folates. This blockage of RFC leads to two a possible strategy to improve the efficacy of the combination of MTX plus SSZ may be to different effects: 1) diminished uptake of MTX and consequently abrogation of antiproliferative and anti- inflammatory effects of MTX, and 2) diminished uptake of natural folates. In combination with a low dietary alleviate the block on RFC by discontinuing the daily administrations of SSZ for the one intake of folates, this may result in decreased intracellular folate levels/folate deficiency, which results in by day at which the weekly dose of MTX is administered. elevated levels of homocysteine.

92 93 References References

References plasma homocysteine concentrations in patients with rheumatoid arthritis, Ann.Rheum.Dis., 58: 79- 84, 1999. 16 Krogh,J.M., Ekelund,S. and Svendsen,L. Folate and homocysteine status and haemolysis in patients 1 Lipsky,P.E., van der Heijde,D.M., St Clair,E.W., Furst,D.E., Breedveld,F.C., Kalden,J.R., Smolen,J.S., treated with sulphasalazine for arthritis, Scand.J.Clin.Lab Invest, 56: 421-429, 1996. Weisman,M., Emery,P., Feldmann,M., Harriman,G.R. and Maini,R.N. Infliximab and methotrexate in 17 Grindulis,K.A. and McConkey,B. Does sulphasalazine cause folate deficiency in rheumatoid arthritis?, the treatment of rheumatoid arthritis. Anti-Tumor Necrosis Factor Trial in Rheumatoid Arthritis with Scand.J.Rheumatol., 14: 265-270, 1985. Concomitant Therapy Study Group, N.Engl.J.Med., 343: 1594-1602, 2000. 18 Logan,E.C., Williamson,L.M. and Ryrie,D.R. Sulphasalazine associated pancytopenia may be caused 2 Bathon,J.M., Martin,R.W., Fleischmann,R.M., Tesser,J.R., Schiff,M.H., Keystone,E.C., Genovese,M.C., by acute folate deficiency, Gut, 27: 868-872, 1986. Wasko,M.C., Moreland,L.W., Weaver,A.L., Markenson,J. and Finck,B.K. A comparison of etanercept 19 Swinson,C.M., Perry,J., Lumb,M. and Levi,A.J. Role of sulphasalazine in the aetiology of folate and methotrexate in patients with early rheumatoid arthritis, N.Engl.J.Med., 343: 1586-1593, 2000. deficiency in ulcerative colitis, Gut, 22: 456-461, 1981. 3 Smolen,J.S. and Steiner,G. Therapeutic strategies for rheumatoid arthritis, Nat.Rev.Drug Discov., 2: 20 Shiroky,J.B. Unsubstantiated “risk” of antifolate toxicity with combination methotrexate and 473-488, 2003. sulfasalazine therapy, Arthritis Rheum., 12: 1757-1758, 1993. 4 Verhoeven,A.C., Boers,M. and Tugwell,P. Combination therapy in rheumatoid arthritis: updated 21 Morgan,S.L., Baggott,J.E. and Alarcon,G.S. Methotrexate and sulfasalazine combination therapy: is it systematic review, Br.J.Rheumatol., 37: 612-619, 1998. worth the risk?, Arthritis Rheum., 36: 281-282, 1993. 5 Fathy,N. and Furst,D.E. Combination therapy for autoimmune diseases: the rheumatoid arthritis 22 Longstreth,G.F. and Green,R. Folate status in patients receiving maintenance doses of sulfasalazine, model, Springer Semin.Immunopathol., 23: 5-26, 2001. Arch.Intern.Med., 143: 902-904, 1983. 6 Boers,M., Verhoeven,A.C., Markusse,H.M., van de Laar,M.A., Westhovens,R., van Denderen,J.C., 23 Selhub,J., Dhar,G.J. and Rosenberg,I.H. Inhibition of folate enzymes by sulfasalazine, J.Clin.Invest, 61: van Zeben,D., Dijkmans,B.A.C., Peeters,A.J., Jacobs,P., van den Brink,H.R., Schouten,H.J., van der 221-224, 1978. Heijde,D.M., Boonen,A. and van der Linden.S. Randomised comparison of combined step-down 24 Baggott,J.E., Morgan,S.L., Ha,T., Vaughn,W.H. and Hine,R.J. Inhibition of folate-dependent enzymes prednisolone, methotrexate and sulphasalazine with sulphasalazine alone in early rheumatoid by non-steroidal anti- inflammatory drugs, Biochem.J., 282: 197-202, 1992. arthritis, Lancet, 350: 309-318, 1997. 25 Halsted,C.H., Gandhi,G. and Tamura,T. Sulfasalazine inhibits the absorption of folates in ulcerative 7 Ferraccioli,G.F., Gremese,E., Tomietto,P., Favret,G., Damato,R. and Di Poi,E. Analysis of colitis, N.Engl.J.Med., 305: 1513-1517, 1981. improvements, full responses, remission and toxicity in rheumatoid patients treated with step-up 26 Franklin,J.L. and Rosenberg,H.H. Impaired folic acid absorption in inflammatory bowel disease: combination therapy (methotrexate, cyclosporin A, sulphasalazine) or monotherapy for three years, effects of salicylazosulfapyridine (Azulfidine), Gastroenterology, 64: 517-525, 1973. Rheumatology.(Oxford), 41: 892-898, 2002. 27 Zimmerman,J. Drug interactions in intestinal transport of folic acid and methotrexate. Further 8 Maetzel,A., Wong,A., Strand,V., Tugwell,P., Wells,G. and Bombardier,C. Meta-analysis of treatment evidence for the heterogeneity of folate transport in the human small intestine, Biochem.Pharmacol., termination rates among rheumatoid arthritis patients receiving disease-modifying anti-rheumatic 44: 1839-1842, 1992. drugs, Rheumatology.(Oxford), 39: 975-981, 2000. 28 Westerhof,G.R., Schornagel,J.H., Kathmann,I., Jackman,A.L., Rosowsky,A., Forsch,R.A., Hynes,J.B., 9 Ardizzone,S. and Porro,G.B. Inflammatory bowel disease: new insights into pathogenesis and Boyle,F.T., Peters,G.J., Pinedo,H.M. and Jansen,G. Carrier- and receptor-mediated transport of folate treatment, J.Intern.Med., 252: 475-496, 2002. antagonists targeting folate-dependent enzymes: correlates of molecular structure and biological 10 Haagsma,C.J., Russel,F.G., Vree,T.B., van Riel,P.L. and van de Putte,L.B. Combination of methotrexate activity., Mol.Pharmacol., 48: 459-471, 1995. and sulphasalazine in patients with rheumatoid arthritis: pharmacokinetic analysis and relationship 29 Sirotnak,F.M. and Tolner,B. Carrier-mediated membrane transport of folates in mammalian cells, to clinical response, Br.J.Clin.Pharmacol., 42: 195-200, 1996. Annu.Rev.Nutr., 19: 91-122, 1999. 11 Haagsma,C.J., van Riel,P.L., de Jong,A.J. and van de Putte,L.B. Combination of sulphasalazine and 30 Goldman,I.D. The characteristics of the membrane transport of amethopterin and the naturally methotrexate versus the single components in early rheumatoid arthritis: a randomized, controlled, occurring folates, Ann.N.Y.Acad.Sci., 186: 400-422, 1971. double-blind, 52 week clinical trial, Br.J.Rheumatol., 36: 1082-1088, 1997. 31 Gorlick,R., Goker,E., Trippett,T., Waltham,M., Banerjee,D. and Bertino,J.R. Intrinsic and acquired 12 O’Dell,J.R., Haire,C.E., Erikson,N., Drymalski,W., Palmer,W., Eckhoff,P.J., Garwood,V., Maloley,P., resistance to methotrexate in acute leukemia, N.Engl.J.Med., 335: 1041-1048, 1996. Klassen,L.W., Wees,S., Klein,H. and Moore,G.F. Treatment of rheumatoid arthritis with methotrexate 32 Jansen,G. Receptor- and carrier-mediated transport systems for folates and antifolates, In: Anticancer alone, sulfasalazine and hydroxychloroquine, or a combination of all three medications, N.Engl. Drug Development Guide: Antifolate Drugs in Cancer Therapy (A.L.Jackman, ed), Humana Press Inc., J.Med., 334: 1287-1291, 1996. Totowa, NJ, 293-321, 1999. 13 O’Dell,J.R., Leff,R., Paulsen,G., Haire,C., Mallek,J., Eckhoff,P.J., Fernandez,A., Blakely,K., Wees,S., 33 Bertino,J.R. Karnofsky memorial lecture. Ode to methotrexate, J.Clin.Oncol., 11: 5-14, 1993. Stoner,J., Hadley,S., Felt,J., Palmer,W., Waytz,P., Churchill,M., Klassen,L. and Moore,G. Treatment of 34 Cronstein,B.N. Molecular therapeutics. Methotrexate and its mechanism of action, Arthritis Rheum., rheumatoid arthritis with methotrexate and hydroxychloroquine, methotrexate and sulfasalazine, or 39: 1951-1960, 1996. a combination of the three medications: Results of a two-year, randomized, double-blind, placebo- 35 Cutolo,M., Sulli,A., Pizzorni,C., Seriolo,B. and Straub,R.H. Anti-inflammatory mechanisms of controlled trial, Arthritis Rheum., 46: 1164-1170, 2002. methotrexate in rheumatoid arthritis, Ann.Rheum.Dis., 60: 729-735, 2001. 14 Landewe,R.B., Boers,M., Verhoeven,A.C., Westhovens,R., van de Laar,M.A., Markusse,H.M., 36 Ranganathan,P., Eisen,S., Yokoyama,W.M. and McLeod,H.L. Will pharmacogenetics allow better van Denderen,J.C., Westedt,M.L., Peeters,A.J., Dijkmans,B.A.C., Jacobs,P., Boonen,A., van der prediction of methotrexate toxicity and efficacy in patients with rheumatoid arthritis? Ann.Rheum. Heijde,D.M. and Van der Linden,S. COBRA combination therapy in patients with early rheumatoid Dis., 62: 4-9, 2003. arthritis: Long-term structural benefits of a brief intervention, Arthritis Rheum., 46: 347-356, 37 Rots,M.G., Pieters,R., Kaspers,G.J., Veerman,A.J., Peters,G.J. and Jansen,G. Classification of ex vivo 2002. methotrexate resistance In acute lymphoblastic and myeloid leukaemia, Br.J.Haematol., 110: 791- 15 Haagsma,C.J., Blom,H.J., van Riel,P.L., van’t Hof,M.A., Giesendorf,B.A., Oppenraaij-Emmerzaal,D. 800, 2000. and van de Putte,L.B. Influence of sulphasalazine, methotrexate, and the combination of both on 38 Rothem,L., Ifergan,I., Kaufman,Y., Priest,D.G., Jansen,G. and Assaraf,Y.G. Resistance to multiple

94 95 References References

novel antifolates is mediated via defective drug transport resulting from clustered mutations in the 6680-6686, 2003. reduced folate carrier gene in human leukemia cell lines, Biochem.J., 367: 741-750, 2002. 54 Hooijberg,J.H., Peters,G.J., Assaraf,Y.G., Kathmann,I., Priest,D.G., Bunni,M.A., Veerman,A.J., 39 Jansen,G., Mauritz,R., Drori,S., Sprecher,H., Kathmann,I., Bunni,M., Priest,D.G., Noordhuis,P., Scheffer,G.L., Kaspers,G.J. and Jansen,G. The role of multidrug resistance proteins MRP1, MRP2 and Schornagel,J.H., Pinedo,H.M., Peters,G.J. and Assaraf,Y.G. A structurally altered human reduced MRP3 in cellular folate homeostasis, Biochem.Pharmacol., 65: 765-771, 2003. folate carrier with increased folic acid transport mediates a novel mechanism of antifolate resistance, 55 Stark,M., Rothem,L., Jansen,G., Scheffer,G.L., Goldman,I.D. and Assaraf,Y.G. Antifolate Resistance J.Biol.Chem., 273: 30189-30198, 1998. Associated with Loss of MRP1 Expression and Function in Chinese Hamster Ovary Cells with Markedly 40 Jansen,G. and Pieters,R. The role of impaired transport in (pre)clinical resistance to methotrexate: Impaired Export of Folate and Cholate, Mol.Pharmacol., 64: 220-227, 2003. insights on new antifolates, Drug Resist.Updat., 1: 211-218, 1998. 56 Mauritz,R., Peters,G.J., Priest,D.G., Assaraf,Y.G., Drori,S., Kathmann,I., Noordhuis,P., Bunni,M.A., 41 Jansen,G., Kathmann,I., Rademaker,B.C., Braakhuis,B.J., Westerhof,G.R., Rijksen,G. and Schornagel,J. Rosowsky,A., Schornagel,J.H., Pinedo,H.M. and Jansen,G. Multiple mechanisms of resistance to H. Expression of a folate binding protein in L1210 cells grown in low folate medium, Cancer Res., 49: methotrexate and novel antifolates in human CCRF-CEM leukemia cells and their implications for 1959-1963, 1989. folate homeostasis, Biochem.Pharmacol., 63: 105-115, 2002. 42 Mauritz,R., Bekkenk,M.W., Rots,M.G., Pieters,R., Mini,E., Van Zantwijk,C.H., Veerman,A.J., Peters,G. 57 Sirotnak,F.M., Moccio,D.M., Goutas,L.J., Kelleher,L.E. and Montgomery,J.A. Biochemical correlates J. and Jansen,G. Ex vivo activity of methotrexate versus novel antifolate inhibitors of dihydrofolate of responsiveness and collateral sensitivity of some methotrexate-resistant murine tumors to the reductase and thymidylate synthase against childhood leukemia cells, Clin.Cancer Res., 4: 2399- lipophilic antifolate, metoprine, Cancer Res., 42: 924-928, 1982. 2410, 1998. 58 Jansen,G., Barr,H., Kathmann,I., Bunni,M.A., Priest,D.G., Noordhuis,P., Peters,G.J. and Assaraf,Y.G. 43 Jansen,G., Westerhof,G.R., Jarmuszewski,M.J., Kathmann,I., Rijksen,G. and Schornagel,J.H. Multiple mechanisms of resistance to polyglutamatable and lipophilic antifolates in mammalian Methotrexate transport in variant human CCRF-CEM leukemia cells with elevated levels of the cells: role of increased folylpolyglutamylation, expanded folate pools, and intralysosomal drug reduced folate carrier. Selective effect on carrier-mediated transport of physiological concentrations sequestration, Mol.Pharmacol., 55: 761-769, 1999. of reduced folates, J.Biol.Chem., 265: 18272-18277, 1990. 59 Chen,Z.S., Robey,R.W., Belinsky,M.G., Shchaveleva,I., Ren,X.Q., Sugimoto,Y., Ross,D.D., Bates,S.E. 44 Jansen,G., Mauritz,R.M., Assaraf,Y.G., Sprecher,H., Drori,S., Kathmann,I., Westerhof,G.R., Priest,D. and Kruh,G.D. Transport of methotrexate, methotrexate polyglutamates, and 17beta-estradiol 17- G., Bunni,M., Pinedo,H.M., Schornagel,J.H. and Peters,G.J. Regulation of carrier-mediated transport (beta-D-glucuronide) by ABCG2: effects of acquired mutations at R482 on methotrexate transport, of folates and antifolates in methotrexate-sensitive and-resistant leukemia cells, Adv.Enzyme Regul., Cancer Res., 63: 4048-4054, 2003. 37: 59-76, 1997. 60 Volk,E.L. and Schneider,E. Wild-type breast cancer resistance protein (BCRP/ABCG2) is a methotrexate 45 Drori,S., Sprecher,H., Shemer,G., Jansen,G., Goldman,I.D. and Assaraf,Y.G. Characterization of a polyglutamate transporter, Cancer Res., 63: 5538-5543, 2003. human alternatively spliced truncated reduced folate carrier increasing folate accumulation in 61 Jansen,G., Scheper,R.J. and Dijkmans,B.A.C. Multidrug resistance proteins in rheumatoid arthritis, parental leukemia cells, Eur.J.Biochem., 267: 690-702, 2000. role in disease-modifying antirheumatic drug efficacy and inflammatory processes: an overview, 46 Van der Heijden,J., de Jong,M.C., Dijkmans,B.A.C., Lems,W.F., Oerlemans,R., Kathmann,G.A., Scand.J.Rheumatol., 32, in press:2003. Schalkwijk,C.G., Scheffer,G.L., Scheper,R.J. and Jansen,G. Development of sulphasalazine resistance 62 Said,H.M., Nguyen,T.T., Dyer,D.L., Cowan,K.H. and Rubin,S.A. Intestinal folate transport: in human T cells induces expression of the multidrug resistance transporter ABCG2 (BCRP) and identification of a cDNA involved in folate transport and the functional expression and distribution augmented production of TNFalpha, Ann.Rheum.Dis., 63: 138-143, 2004. of its mRNA, Biochim.Biophys.Acta, 1281: 164-172, 1996. 47 Van der Heijden,J., de Jong,M.C., Dijkmans,B.A.C., Lems,W.F., Oerlemans,R., Kathmann,G.A., 63 Nguyen,T.T., Dyer,D.L., Dunning,D.D., Rubin,S.A., Grant,K.E. and Said,H.M. Human intestinal folate Scheffer,G.L., Scheper,R.J., Assaraf,Y.G. and Jansen,G. Acquired resistance of human T cells to transport: cloning, expression, and distribution of complementary RNA, Gastroenterology, 112: 783- sulphasalazine: stability of the resistant phenotype and sensitivity to non-related DMARDs, Ann. 791, 1997. Rheum.Dis., 63: 131-137, 2004. 64 Rajgopal,A., Sierra,E.E., Zhao,R. and Goldman,I.D. Expression of the reduced folate carrier SLC19A1 48 Scheffer,G.L., Kool,M., Heijn,M., de Haas,M., Pijnenborg,A.C., Wijnholds,J., van Helvoort,A., de in IEC-6 cells results in two distinct transport activities, Am.J.Physiol Cell Physiol, 281: C1579-C1586, Jong,M.C., Hooijberg,J.H., Mol,C.A., van der Linden,M., de Vree,J.M., van der Valk,P., Elferink,R.P., 2001. Borst,P. and Scheper,R.J. Specific detection of multidrug resistance proteins MRP1, MRP2, MRP3, 65 Assaraf,Y.G., Sierra,E.E., Babani,S. and Goldman,I.D. Inhibitory effects of prostaglandin A1 on MRP5, and MDR3 P-glycoprotein with a panel of monoclonal antibodies, Cancer Res., 60: 5269-5277, membrane transport of folates mediated by both the reduced folate carrier and ATP-driven exporters, 2000. Biochem.Pharmacol., 58: 1321-1327, 1999. 49 Smedegard,G. and Bjork,J. Sulphasalazine: mechanism of action in rheumatoid arthritis, 66 Leslie,E.M., Deeley,R.G. and Cole,S.P. Toxicological relevance of the multidrug resistance protein 1, Br.J.Rheumatol., 34 Suppl 2: 7-15, 1995. MRP1 (ABCC1) and related transporters, Toxicology, 167: 3-23, 2001. 50 Lin,J.T. and Bertino,J.R. Update on trimetrexate, a folate antagonist with antineoplastic and 67 Jansen,G., Scheper,R.J. and Dijkmans,B.A.C. Chloroquine resitance in human CEM (T) cells is mediated antiprotozoal properties, Cancer Invest, 9: 159-172, 1991. by multidrug resistance protein 1, Annals of the Rheumatic Diseases, 60: 89, 2001. 51 Hum,M., Holcenberg,J.S., Tkaczewski,I., Weaver,J.W., Wilson,J. and Kamen,B.A. High-dose 68 Vezmar,M. and Georges,E. Direct binding of chloroquine to the multidrug resistance protein trimetrexate and minimal-dose leucovorin: a case for selective protection?, Clin.Cancer Res., 4: (MRP): possible role for MRP in chloroquine drug transport and resistance in tumor cells, Biochem. 2981-2984, 1998. Pharmacol., 56: 733-742, 1998. 52 Borst,P. and Oude Elferink,R.P. Mammalian ABC transporters in health and disease, Annu.Rev. Biochem., 71: 537-592, 2002. 53 Assaraf,Y.G., Rothem,L., Hooijberg,J.H., Stark,M., Ifergan,I., Kathmann,G.A., Dijkmans,B.A.C., Peters,G.J. and Jansen,G. Loss of multidrug resistance protein 1 (MRP1) expression and folate efflux activity results in a highly concentrative folate transport in human leukemia cells, J.Biol.Chem., 278:

96 97 chapter 6 Involvement of Breast Cancer Resistance Protein expression on RA synovial tissue macrophages in resistance to methotrexate and leflunomide

Joost W. van der Heijden1, Ruud Oerlemans1, Paul P. Tak2, Yehuda G. Assaraf3, Maarten C. Kraan2, George L. Scheffer4, Conny J. van der Laken1, Willem F. Lems1, Rik J. Scheper4, Ben A.C. Dijkmans1 and Gerrit Jansen1

Departments of 1Rheumatology and 4Pathology, VU University Medical Center Amsterdam, The Netherlands, 2Division of Clinical Immunology and Rheumatology, Academic Medical Center, University of Amsterdam, The Netherlands and 3The Fred Wyszkowski Cancer Research Laboratory, Department of Biology, The Technion, Israel Institute of Technology, Haifa, Israel

Submitted for publication chapter 6 Expression of multi-drug resistance proteins in synovial tissue of Rheumatoid Arthritis patients

Abstract sulfasalazine and chloroquine could be mediated by the increased expression of the ABC transporters BCRP and MRP1, respectively (10-12). Furthermore, very recently, it was Objective: To determine whether multidrug-resistance efflux transporters are expressed demonstrated that leflunomide is a high affinity substrate for BCRP (13). on immune effector cells in synovial tissue from rheumatoid arthritis (RA) patients and may compromise the efficacy of methotrexate (MTX) and leflunomide. A number of studies have reported on the expression of Pgp and MRP1 (mRNA levels, protein Methods: Synovial tissue biopsies obtained from RA patients before treatment and 4 levels, functional activity) on peripheral blood cells of RA patients. Maillefert et al (14) months after starting treatment with MTX (n=17) and leflunomide (n=13) were examined observed that Pgp levels were increased on peripheral blood cells of prednisolone-treated by immunohistochemical staining and digital image analysis for the expression of the drug RA patients compared to untreated healthy individuals, which may be compatible with efflux transporters: P-glycoprotein (Pgp), Multidrug Resistance-associated Proteins MRP1- the notion that steroids are known substrates for Pgp. Likewise, Llorente et al (15) showed 5, MRP8, MRP9 and Breast Cancer Resistance Protein (BCRP). that RA patients had functionally active Pgp on peripheral blood lymphocytes which was Results: BCRP expression was observed in all RA synovial biopsies, both pre- and post markedly higher in treatment-refractory RA patients than in RA patients with a good treatment, but not in control non-inflammatory synovial tissue of orthopedic patients. clinical response to DMARD therapy. More recently, two studies evaluated the expression BCRP expression was found both in the intimal lining layer as well as on macrophages and of MRP1 (by functional activity and immunohistochemical staining) as a potential MTX endothelial cells in the synovial sublining. Correlation analysis showed a trend towards drug efflux pump in peripheral blood cells of RA patients. In a cross-sectional study, Wolf more abundant BCRP expression at higher disease activity. Furthermore, median BCRP et al (16) counter-intuitively observed that patients with functional MRP1 expression expression was 4-fold higher for MTX-non responders than for MTX-responders (p=0.08), who were treated with MTX had better EULAR response rates than patients with a low and 2.5-fold higher in leflunomide failures compared to leflunomide-responders (p=0.12). MRP1 activity. Hider et al (17) showed that MRP1 expression on peripheral blood cells Low expression of MRP1 was found on synovial macrophages, along with moderate declined from baseline to 6 months therapy with MTX. These results suggest that beyond expression in T-cell areas of synovial biopsies in one-third of RA patients. drug-induced changes in MRP1 expression during dose escalations, basal levels of MRP1 Conclusion: The drug resistance related proteins BCRP and MRP1 are expressed on expression in immune effector cells may also be induced by disease activity/inflammation inflammatory cells in RA synovial tissue. Since MTX is a substrate for both BCRP and MRP1 status (9). In contrast to studies on peripheral blood cells from RA patients, little is known and leflunomide is a high affinity substrate for BCRP, these transporters may contribute to about expression levels of ABC transporters on inflammatory cells in RA synovial tissue. a reduced therapeutic efficacy of these DMARDs. In the present study we assessed the expression of a panel of ABC transporters with an established role in drug resistance in RA synovial tissue biopsies that were taken prior to Introduction and after 4 months of therapy with MTX and leflunomide. Beyond Pgp, we focused on more recently identified ABC transporters: MRP1-5, MRP8 and MRP9, which have MTX among The disease modifying anti-rheumatic drugs (DMARDs) methotrexate (MTX), leflunomide, their substrates (8) and BCRP for which both MTX and leflunomide are known substrates sulfasalazine and (hydroxy)chloroquine have an established place in the treatment of (13), thereby allowing correlation analysis with response to these drugs. patients with Rheumatoid Arthritis (RA), either as single agents or in combination with other DMARDs or biological agents (1, 2). Despite the potent disease modifying effects of these DMARDs, it is well recognized that chronic treatment is often accompanied by a Materials and Methods gradual loss of efficacy, requiring dose escalation, up to a stage where treatment has to be discontinued for reasons of inefficacy and/or toxicity (3-5). From oncology research, Patients it has been established that specific cell membrane-associated drug efflux transporters In this study we analyzed synovial tissue biopsies derived from the same knee joints of 17 RA belonging to the family of ATP Binding Cassette (ABC) proteins, notably P-glycoprotein patients with active disease, both before treatment and after 4 months of treatment with (Pgp; ABCB1), Multidrug Resistance Proteins 1-5 (MRP1-5; ABCC1-5) and Breast Cancer MTX (starting dose of MTX: 7,5 mg/week; increasing stepwise to 15 mg/week over 12 weeks) Resistance Protein (BCRP; ABCG2), can confer resistance to various types of drugs including and 13 RA-patients before and after 4 months of treatment with leflunomide (20 mg/day; MTX (6-9). Studies from our laboratory showed that acquired resistance to the DMARDs loading dose 100 mg/day for the first 3 days). Active disease was defined as ≥6 swollen or

100 101 chapter 6 Expression of multi-drug resistance proteins in synovial tissue of Rheumatoid Arthritis patients

tender joints and levels of moderate or worse on the physician’s and patient’s assessments Double-labeling immunofluorescence of disease activity. All patients had at least 1 clinically involved knee joint. Concomitant ABC transporter protein expression was detected by species specific HRP-labeled treatment with low dose prednisone (<10 mg/day) and stable doses of nonsteroidal anti- secondary reagents (Dako, Glostrup, Danmark), and development was with rhodamine/ inflammatory drugs (NSAIDs) was allowed. None of the patients ever used methotrexate or thyramine (red fluorescence) according to the instructions of the manufacturer. T-cells and leflunomide before enrolling the study. In patients taking other DMARDs, these drugs were macrophages (primary antibodies anti-CD3 and anti-CD68, respectively) were detected by terminated following a washout phase of 28 days. Before treatment and after 4 months anti-mouse biotin-labeled secondary antibodies (Dako, Glostrup, Danmark), Streptavidin of treatment, the DAS28 score was determined (18). Arthroscopies were performed as Alexa-488 serving as substrate (green fluorescence; Molecular Probes, Eugene, OR). Slides described previously (19) as part of a joined study that was approved by the Medical Ethics were mounted with Vectashield, containing 1 µg/ml DAPI (for staining of nuclei) (Vector committees of the Leiden University Medical Center and the Leeds University Medical laboratories Inc., Burlingame, CA). Cells were examined using a fluorescence microscope Center (20). As non-inflammatory control synovial tissue we included 7 samples from (Leica DMRB, Rijswijk, the Netherlands). patients with mechanical joint injury, kindly provided by dr. B.J. van Royen, department of Orthopedic Surgery, VU University Medical Center, Amsterdam, The Netherlands. Statistical analysis Statistical analysis was performed using non-parametric tests. Correlation of BCRP-protein For statistical analysis, RA patients were devided in a group of DMARD-responders and a expression with DAS28 score and BCRP-protein expression for individual patients before group of DMARD-non-responders, according to the DAS28 response criteria (18). Responders treatment and after 4 months of treatment with MTX was calculated by Spearman Rho were patients with a significant decrease in DAS28 score (>1.2) upon treatment and patients analysis. For determining significant differences in macrophage counts before and after with a moderate change in DAS-28 score (≤1.2 and >0.6) and low/moderate disease activity treatment with MTX and BCRP-protein expression between DMARD-responding and (≤5.1). The remaining patients were classified as non-responders. An additional analysis was DMARD-non-responding RA patients, the Mann-Whitney U test was used. P-values <0.05 performed, categorizing patients with a normalized CRP level (<10 mg/l) after 4 months of were considered to represent significant differences. treatment as responders.

Immunohistochemical analysis Results Immunohistochemical staining of cryostat sections (5 µm) of synovial tissue biopsies from RA patients and controls was performed using a 3-step immunoperoxidase method as Expression of drug resistance related transporters on macrophages, T-cells and described previously (21). Sections were stained with the following monoclonal antibodies endothelial cells in RA synovial tissue (5-10 µg/ml): C219 (anti-Pgp) (Alexis, Lausen, Switzerland), MRPr1 (anti-MRP1), M2III6 (anti- Immunohistochemical staining for the drug-resistance related transporters Pgp, MRP1- MRP2), M3II9 (anti-MRP3), M4I10 (anti-MRP4), M5I-1 (anti-MRP5), M8II16 (anti-MRP8), 5, MRP8, MRP9 and BCRP was performed on synovial biopsies obtained from RA patients M9I-27 (anti-MRP9), BXP-21 (anti-BCRP), as described by Scheffer et al (21, 22). T-cells and prior to and after 4 months of treatment with MTX (n=17) and leflunomide (n=13). Negative macrophages were stained with anti-CD3-PE (Dako, Glostrup, Danmark) and 3A5 (23), staining was observed for all isotype controls (Figure 1A). 3A5 staining showed that synovial respectively. Synovial tissue fibroblasts were visualized using anti-CD90-PE (Beckton tissue samples were highly enriched for infiltrated macrophages in the intimal lining layer Dickinson PharMingen, San Diego, USA). and synovial sublining (Figure 1B). The most abundantly expressed drug efflux transporter in RA synovial tissue, both in the intimal lining layer and synovial sublining, appeared to be Analysis of MDR-protein expression BCRP (Figure 1C). Only a few BCRP-positive cells could be identified in ‘non-inflammatory’ Sections stained for BCRP and 3A5 (macrophages) (23) were analyzed by digital image synovial tissues of orthopedic patients (Figure 1F), which also displayed low levels of analysis, as described previously (24). Briefly, for each marker representative regions were infiltrated macrophages (3A5 staining, Figure 1E) as compared to RA synovium (Figure used for image acquisition, using 400x magnification. These regions were divided into 6 1B). More detailed analysis by fluorescence microscopy, demonstrated the co-localization high-power fields (hpfs) with a 3-pixel overlap. Positive cells were evaluated by analyzing 18 of BCRP and CD68 in the intimal lining layer and synovial sublining, proving that BCRP is consecutive hpfs, scoring numbers of positive cells in the intimal lining layer and synovial expressed on RA macrophages. BCRP was also found to be expressed on endothelial cells sublining per mm2. (Figure 2A-H), and double staining techniques showed that BCRP is weakly expressed on

102 103 chapter 6 Expression of multi-drug resistance proteins in synovial tissue of Rheumatoid Arthritis patients

Figure 1 Figure 3

Figure 3: Immunofluorescence double staining of RA synovial tissue for CD3/MRP1 and CD68/MRP1. (A) Light microscopy view of T-cell aggregate (CD3) (B) Detailed immunofluorescence picture for CD3 Figure 1: Light microscopy images of BCRP expression in RA synovial tissue: (A) Mouse isotype staining, staining in a lymphocytic aggregate, (C) MRP1 staining (MRPr1) for the same field and (D) Merge of B and (B) Macrophage staining (3A5), (C) BCRP staining, (D) Non-RA synovial tissue isotype staining, (E) Non-RA C. (E) Light microscopy view of macrophage staining (CD68), (F) Immunofluorescence staining for CD68,(G) synovial tissue macrophage staining (3A5), (F) Non-RA synovial tissue BCRP staining. MRP1 staining (MRPr1) for the same field and (H) Merge of F and G.

peri-vascular CD3 positive T-cells (not shown). Figure 2 With light microscopy, weak MRP1 staining was observed in synovial tissue biopsies of approximately one-third of RA patients. With more sensitive detection methods (immunofluorescence; rhodamine/thyramine development), clear MRP1 expression was observed on CD3-positive lymphocytes, being most abundant in lymphocytic aggregates and less pronounced on neighboring T-cells (Figure 3A-D). With this detection method we also observed moderate MRP1 expression on CD68 positive macrophages in biopsies from the same patients (Figure 3E-F). No co-localization of MRP1 was found with CD90, a marker for synovial tissue fibroblasts (not shown). Expression levels of Pgp, MRP2-5, MRP8 and MRP9 in synovial tissue of RA patients were below the immunohistochemical detection limit.

BCRP expression and response to MTX and leflunomide therapy Since MTX and lefunomide are BCRP substrates (8, 13) and thus can be extruded from synovial tissue macrophages via this transporter, expression levels of BCRP in the synovial Figure 2: Immunofluorescence double staining of RA synovial tissue for CD68 and BCRP. (A) Macrophage staining (CD-68), (B) BCRP staining (BXP-21), (C) nuclei staining (Dapi) and (D) merge of A, sublining were correlated with the response to MTX and leflunomide therapy. B and C. (E-H) more detailed images of synovial lining cells: (E) macrophage staining (CD-68), (F) BCRP Correlation analysis showed a trend towards a more abundant BCRP expression at higher staining (BXP-21), (G) nuclei staining (Dapi) and (H) merge of E, F and G. disease activity (r=0.30, p=0.12) (Figure 4A). BCRP expression for individual patients appeared to be rather constant over time, as a significant correlation was found between BCRP expression before treatment and after 4 months of treatment with MTX (r=0.54, p=0.025) (Figure 4B). The median number of 3A5 macrophages decreased upon MTX treatment: 219/mm2 (range 11-622) to 119/mm2 (range 12-844) (Figure 4C, p=0.14). The

104 105 Figuur_4A.pzf:Data 1 graph - Mon Aug 25 09:46:59 2008 Figuur_4B.pzf:Data 1 graph - Mon Aug 25 09:47:20 2008 Figuur_5B.pzf:Data 1 graph - Mon Aug 25 09:48:34 2008

Figuur_4C.pzf:Data 1 graph - Mon Aug 25 09:47:40 2008 Figuur_5A.pzf:Data 1 graph - Mon Aug 25 09:48:16 2008 chapter 6 Expression of multi-drug resistance proteins in synovial tissue of Rheumatoid Arthritis patients

Figure 4 A Figure 4 B MTX Figure 5 A MTX Figure 5 B 2000 10000 1200 1200 R=0.30; p=0.12 R=0.54; p=0.025* p=0.002* p=0.002* 1500 1000 1000 500 1000 800 p=0.002* p=0.084 800 400 p=0.001* p=0.016* 100 600 600 300

200 10 400 400

BCRPexpression t=0 100 200 200 BCRP expression t=4 months BCRP expression t=4 months 1 BCRP expression t=4 months 0 1 0 0 10

3 4 5 6 7 8 9 100 ls Figuur_5C.pzf:Datas 1 graph - Mon Aug 25 09:49:04 2008 1000 r rs 0 0 o e e ls 1 1 10000 tr o n nd tr ≤ DAS28 t=0 BCRP expression t=0 o o n P P> C pond o R e C C -r CR Resp Non Figure 4 C 1000 Leflunomide Figure 5 C 2 1200 800 1000 p=0.001* 600 293 800

400 Median number of 600 2 p=0.117 BCRP-positivecells/mm p=0.004* 200 400 Number of 3A5 positive 75 synovial macrophages/mm 200 0 BCRP expression t=4 months t=0 t=4 months 0 ls s o r rs tr e e n nd o o pond C e -r Resp Non

Figure 4: (A) Correlation between DAS28 score and BCRP expression (positive cells/mm2; sublining layer) Figure 5: Expression of BCRP (median number of positive cells/mm2; sublining layer) for controls and RA before treatment. Spearman rho: r=0.30, p=0.12. (B) Correlation between BCRP expression (positive cells/ patients after 4 months of treatment with MTX. (A) MTX-responders: 75 (range 0-484) and MTX-non- mm2; sublining layer) before treatment and after 4 months of treatment with MTX for individual patients. responders: 300 (range 2-1159). (B) Number of BCRP positive cells for RA-patients with normalized CRP levels Spearman rho: r=0.54, p=0.025.(C) Median number of 3A5 positive macrophages (positive cells/mm2; (mean CRP: 5 mg/l): 29 (range 0-484) and patients with persistent high CRP levels (mean CRP: 54 mg/l): 224 sublining layer) before treatment: 219, range 11-622 and after 4 months of treatment with MTX: 119, range (range 2-1159). (C) Expression of BCRP (median number of positive cells/mm2; sublining layer) for controls 12-844) (p=0.14). The median expression of BCRP (positive cells/mm2; sublining layer) after 4 months of and RA patients after 4 months of treatment with leflunomide. Median BCRP expression in leflunomide- therapy is depicted for patients with high macrophage counts (upper right square) and low macrophage responders was 127 (range 1-223) and 297 (range 11-1007) for leflunomide-non-responders. Statistical counts (lower right square). analysis: Mann-Whitney U test.

group of patients with a persistent high macrophage count in their synovial biopsies after 4 cells/mm2, range 2-1159) after 4 months of treatment with MTX compared to patients with months of treatment with MTX (Figure 4C, upper right square) had a 3.9-fold higher count normalized CRP levels (median 29 BCRP positive cells/mm2, range 0-484). Also leflunomide of BCRP positive cells (293/mm2) compared to patients with a low macrophage count (75/ non-reponders (n=6) had higher BCRP expression compared to responders (n=7) (median mm2) (p=0.12) (Figure 4C, lower right square). 297 positive cells/mm2, range 11-1007 versus 127 positive cells/mm2, range 1-223; p=0.12) For MTX non-responders (n=8), the median number of BCRP positive cells appeared to (Figure 5C). In synovial tissue of 7 healthy controls, consistent with very low macrophage be 4-fold higher after 4 months of treatment with MTX than for MTX-responders (n=9): counts, only a few BCRP positive cells were calculated. Accordingly, BCRP expression in 300 (range 2-1159) and 75 (range 0-484) cells/mm2, respectively (p=0.08) (Figure 5A). synovial tissue of RA patients was significantly higher (p<0.005) than for controls. The low Additional analysis (Figure 5B) revealed a statistically significant (p=0.016) 8-fold higher incidence of MRP1 expression in the synovial samples of RA patients did not allow correlation BCRP expression for RA-patients with persistent high CRP levels (median 224 BCRP positive analysis with disease activity or response to MTX therapy.

106 107 chapter 6 Expression of multi-drug resistance proteins in synovial tissue of Rheumatoid Arthritis patients

Discussion The present study revealed BCRP expression on synovial tissue macrophages of all tested RA patients. High BCRP expression was associated with the persistence of infiltrated 3A5 This is the first detailed study in RA synovial tissue investigating the expression of a positive synovial tissue macrophages after treatment with MTX (Figure 4C), which might be panel of multidrug transporter proteins from the family of ATP-binding cassette (ABC) related with BCRP-mediated resistance to MTX in these macrophages. Accordingly, a trend transporters, known to confer resistance to multiple anticancer drugs as well as DMARDs but no significant correlation was found between BCRP expression in RA synovial tissue and (9, 25). Immunohistochemical detection of Pgp and MRP2-5, MRP8 and MRP9 in RA a diminished clinical response to MTX and leflunomide therapy, assessed by an attenuated synovial tissue was not observed, in contrast to the drug transporters MRP1 and BCRP, the improvement of DAS28 scores after 4 months of treatment. In secondary analyses, a 8-fold latter being markedly expressed on RA synovial tissue macrophages in the intimal lining significant increase in BCRP expression was observed in patients with a persistent high CRP layer and the synovial sublining and on endothelial cells. Importantly, BCRP expression could level after treatment compared to patients with normalized CRP levels, providing additional already be observed in the synovial tissue of RA patients prior to MTX therapy. This finding support for a role of BCRP in conferring MTX resistance. suggests that BCRP expression may be intrinsically associated with the inflammatory status In addition to BCRP expression on synovial tissue macrophages, moderate expression of RA synovial tissue, rather than being a therapy-induced phenomenon. It should be noted of MRP1 was found on macrophages in synovial tissue of one-third of RA patients, while that some patients had received sulfasalazine as initial treatment prior to a wash-out period more prominent expression of MRP1 could be noted on CD3 positive T-cells in lymphocytic and MTX or leflunomide treatment in the current study. Previous studies from our laboratory aggregates of these patients by more sensitive staining methods. MTX and the anti-malarial have shown that prolonged in vitro exposure of T cells to sulfasalazine may provoke chloroquine are among the DMARD substrates of MRP1, and expression of this transporter induction of BCRP (10), but this induction was transient and reversed upon discontinuation of may thus confer resistance to these drugs (6, 12, 39). However, since MRP1 levels in synovial T- sulfasalazine exposure (11).Thus, it seems unlikely that the marked expression levels of BCRP cells are rather moderate, a major contribution to a reduced response to MTX or anti-malarial on synovial macrophages at the onset of MTX/leflunomide treatment may be reminiscent to drugs remains elusive. Apart from its putative pharmacological role in MTX extrusion, MRP1 any previous sulfasalazine treatment. Consistent with our hypothesis that BCRP expression is also has an important physiological function as it exports inflammatory mediators such as inflammation-driven, is the correlation between BCRP expression and DAS28 scores (Figure leucotriene C4 (LTC4), which triggers antigen-presenting dendritic cell migration to lymph 4A), although statistical significance should be further corroborated in a larger group of RA nodes via chemo-attraction by CCL19 (40, 41). Since high levels of CCL19 have been identified patients. in RA-synovial tissues (42, 43), MRP1 may be instrumental in the homing of inflammatory cells in RA synovial tissues. One pathophysiological condition in RA synovial tissue that could be implicated in BCRP Current experimental strategies to reverse drug resistance associated with efflux transporters expression is its hypoxic environment. It is well recognized that synovial infiltrating cells include the utilization of specific inhibitors of ABC transporters. Small molecule inhibitors of suffer from hypoxic stress (26). Given the notion that BCRP transcription is induced by BCRP have been tested in a laboratory setting (34, 36). Whether such an approach may also hypoxia via the hypoxia-inducible transcription factor complex HIF-1α (27), and that HIF- be of therapeutic benefit for RA treatment remains to be established. Recently, Zaher et al. 1α is particularly expressed in synovial tissue macrophages (28, 29), this may underlie the (44) reported that Iressa, an EGFR receptor antagonist and BCRP transport inhibitor, could expression of BCRP on these cells. markedly enhance the bioavailabity of the DMARD sulfasalazine (a BCRP substrate (10, 11)) by Biodistribution and functional studies indicated that BCRP has important physiological blocking intestinal extrusion of sulfasalazine via BCRP. Based on this proof of principle, BCRP and pharmacological roles, particularly in the clearance of toxic agents from the gastro- expression in RA synovial tissue may also be an interesting target for intervention aimed at intestinal tract, blood-brain barrier, liver, kidney, placenta and mammary glands (21, 30, increasing MTX, leflunomide or sulfasalazine levels in RA macrophages. 31). With respect to hematological cells, BCRP was identified in hematopoietic stem cells In conclusion, along with an established role for the efficacy of cancer therapy, this study (32) and dendritic cells (33). From a therapeutic perspective, BCRP has been recognized to revealed that multidrug resistance related transporters, notably BCRP and to a lesser extent mediate the cellular export of several anticancer drugs (8, 34-36), and has been associated MRP1, may play a role in the efficacy of RA treatment with specific DMARDs. Delineation of with a poor clinical outcome in the treatment of AML (37). Beyond this, DMARDs like MTX, their precise pharmacological and physiological role warrants further evaluation and should sulfasalazine and leflunomide can also be extruded from cells via BCRP (8, 10, 13, 38). Of note, be helpful in improving the efficacy of DMARDs such as MTX, leflunomide, sulfasalazine and BCRP can export MTX as well as its di- and triglutamate metabolites, which could thus lead (hydroxy)chloroquine, being substrates for these drug efflux transporters. to a diminished efficacy of MTX (38).

108 109 References References

References Ann.Rheum.Dis., 64: 564-568, 2004. 17 Hider,S.L., Owen,A., Hartkoorn,R., Khoo,S., Back,D.J., Silman,A. and Bruce,I.N. Down-Regulation 1 Kremer,J.M. Toward a better under-standing of methotrexate, Arthritis Rheum., 50: 1370-1382, Of MRP1 Expression In Early RA Patients Exposed To Methotrexate As A First DMARD, Ann Rheum. 2004. Dis., 65: 1390-1393, 2006. 2 O’Dell,J.R. Therapeutic strategies for rheumatoid arthritis, N.Engl.J.Med., 350: 2591-2602, 18 van Gestel,A.M., Prevoo,M.L., van ‘t Hof,M.A., van Rijswijk,M.H., van de Putte,L.B. and van Riel,P. 2004. L. Development and validation of the European League Against Rheumatism response criteria 3 Wolfe,F. The epidemiology of drug treatment failure in rheumatoid arthritis, Baillieres Clin. for rheumatoid arthritis. Comparison with the preliminary American College of Rheumatology Rheumatol., 9: 619-632, 1995. and the World Health Organization/International League Against Rheumatism Criteria, Arthritis 4 Fleischmann,R.M. Is there a need for new therapies for rheumatoid arthritis?, J.Rheumatol. Rheum., 39: 34-40, 1996. Suppl, 73: 3-7, 2005. 19 Kraan,M.C., Reece,R.J., Smeets,T.J., Veale,D.J., Emery,P. and Tak,P.P. Comparison of synovial 5 Aletaha,D. and Smolen,J.S. Effectivenessprofiles and dose dependent retention of traditional tissues from the knee joints and the small joints of rheumatoid arthritis patients: Implications for disease modifying antirheumatic drugs for rheumatoid arthritis. An observational study, pathogenesis and evaluation of treatment, Arthritis Rheum., 46: 2034-2038, 2002. J.Rheumatol., 29: 1631-1638, 2002. 20 Kraan,M.C., Reece,R.J., Barg,E.C., Smeets,T.J., Farnell,J., Rosenburg,R., Veale,D.J., Breedveld,F.C., 6 Hooijberg,J.H., Broxterman,H.J., Kool,M., Assaraf,Y.G., Peters,G.J., Noordhuis,P., Scheper,R.J., Emery,P. and Tak,P.P. Modulation of inflammation and metalloproteinase expression in synovial Borst,P., Pinedo,H.M. and Jansen,G. Antifolate resistance mediated by the multidrug resistance tissue by leflunomide and methotrexate in patients with active rheumatoid arthritis. Findings in proteins MRP1 and MRP2, Cancer Res., 59: 2532-2535, 1999. a prospective, randomized, double-blind, parallel-design clinical trial in thirty-nine patients at 7 Borst,P. and Oude Elferink,R.P. Mammalian ABC transporters in health and disease, Annu.Rev. two centers, Arthritis Rheum., 43: 1820-1830, 2000. Biochem., 71: 537-592, 2002. 21 Maliepaard,M., Scheffer,G.L., Faneyte,I. F., van Gastelen,M.A., Pijnenborg,A.C., Schinkel,A.H., 8 Assaraf,Y.G. The role of multidrug resistance efflux transporters in antifolate resistance and van De Vijver,M.J., Scheper,R.J. and Schellens,J.H. Subcellular localization and distribution of folate homeostasis, Drug Resist.Updat., 9: 227-246, 2006. the breast cancer resistance protein transporter in normal human tissues, Cancer Res., 61: 3458- 9 Jansen,G., Scheper,R.J. and Dijkmans,B.A.C. Multidrug resistance proteins in rheumatoid 3464, 2001. arthritis, role in disease-modifying antirheumatic drug efficacy and inflammatory processes: an 22 Scheffer,G.L., Kool,M., Heijn,M., de Haas,M., Pijnenborg,A.C., Wijnholds,J., van Helvoort,A., de overview, Scand.J.Rheumatol., 32: 325-336, 2003. Jong,M.C., Hooijberg,J.H., Mol,C.A., van der Linden,M., de Vree,J.M., van der Valk,V., Elferink,R. 10 Van der Heijden,J., de Jong,M.C., Dijkmans,B.A.C., Lems,W.F., Oerlemans,R., Kathmann,G. P., Borst,P. and Scheper,R.J. Specific detection of multidrug resistance proteins MRP1, MRP2, A., Schalkwijk,C.G., Scheffer,G.L., Scheper,R.J. and Jansen,G. Development of sulphasalazine MRP3, MRP5, and MDR3 P-glycoprotein with a panel of monoclonal antibodies, Cancer Res., 60: resistance in human T cells induces expression of the multidrug resistance transporter ABCG2 5269-5277, 2000. (BCRP) and augmented production of TNFalpha, Ann.Rheum.Dis., 63: 138-143, 2004. 23 Jaspars,E.H., Bloemena,E., Bonnet,P., Scheper,R.J., Kaiserling,E. and Meijer,C.J. A new monoclonal 11 Van der Heijden,J., de Jong,M.C., Dijkmans,B.A.C., Lems,W.F.,Oerlemans,R., Kathmann,G.A., antibody (3A5) that recognises a fixative resistant epitope on tissue macrophages and monocytes, Scheffer,G.L.,Scheper,R.J., Assaraf,Y.G. and Jansen,G. Acquired resistance of human T cells to J.Clin.Pathol., 47: 248-252, 1994. sulphasalazine: stability of the resistant phenotype and sensitivity to non-related DMARDs, Ann. 24 Haringman,J.J., Vinkenoog,M., Gerlag,D.M., Smeets,T.J., Zwinderman,A.H. and Tak,P.P. Reliability Rheum.Dis., 63: 131-137, 2004. of computerized image analysis for the evaluation of serial synovial biopsies in randomized 12 Oerlemans,R., van der Heijden J., Vink,J., Dijkmans, B.A.C., Kaspers,G.J., Lems,W.F., Scheffer,G.L., controlled trials in rheumatoid arthritis, Arthritis Res.Ther., 7: R862-R867, 2005. Ifergan,I., Scheper,R.J., Cloos,J., Assaraf,Y.G. and Jansen,G. Acquired resistance to chloroquine in 25 Van der Heijden,J.W., Dijkmans,B.A.C., Scheper,R.J. and Jansen,G. Drug Insight: resistance to human CEM T cells is mediated by multidrug resistance-associated protein 1 and provokes high methotrexate and other disease-modifying antirheumatic drugs-from bench to bedside, Nat. levels of cross-resistance to glucocorticoids, Arthritis Rheum., 54: 557-568, 2006. Clin.Pract.Rheumatol., 3: 26-34, 2007. 13 Kis,E., Nagy,T., Jani,M., Molnar,E., Janossy,J., Ujhelly,O., Nemet,K., Heredi-Szabo,K. and Krajcsi,P. 26 Taylor,P.C. and Sivakumar,B. Hypoxia and angiogenesis in rheumatoid arthritis, Curr.Opin. Leflunomide and A771726, its metabolite are high affinity substrates of B, Ann.Rheum.Dis., Rheumatol., 17: 293-298, 2005. 2008. 27 Krishnamurthy,P., Ross,D.D., Nakanishi,T., Bailey-Dell,K., Zhou,S., Mercer,K.E., Sarkadi,B., 14 Maillefert,J.F., Maynadie,M., Tebib,J.G., Aho,S., Walker,P., Chatard,C., Dulieu,V., Bouvier,M., Sorrentino,B.P. and Schuetz,J.D. The stem cell marker Bcrp/ABCG2 enhances hypoxic cell survival Carli,P.M. and Tavernier,C. Expression of the multidrug resistance glycoprotein 170 in the through interactions with heme, J.Biol.Chem., 279: 24218-24225, 2004. peripheral blood lymphocytes of rheumatoid arthritis patients. The percentage of lymphocytes 28 Hollander,A.P., Corke,K.P., Freemont,A.J. and Lewis,C.E. Expression of hypoxia-inducible factor expressing glycoprotein 170 is increased in patients treated with prednisolone, Br.J.Rheumatol., 1alpha by macrophages in the rheumatoid synovium: implications for targeting of therapeutic 35: 430-435, 1996. genes to the inflamed joint, Arthritis Rheum., 44: 1540-1544, 2001. 15 Llorente,L., Richaud-Patin,Y., Diaz-Borjon,A., Alvarado,D.L.B., Jakez-Ocampo,J., De La Fuente 29 Tak,P.P., Zvaifler,N.J., Green,D.R. and Firestein,G.S. Rheumatoid arthritis and p53: how oxidative H., Gonzalez-Amaro,R. and Diaz-Jouanen,E. Multidrug resistance-1 (MDR-1) in rheumatic stress might alter the course of inflammatory diseases, Immunol.Today, 21: 78-82, 2000. autoimmune disorders. Part I: Increased P-glycoprotein activity in lymphocytes from rheumatoid 30 Ross,D.D., Yang,W., Abruzzo,L.V., Dalton,W.S., Schneider,E., Lage,H., Dietel,M., Greenberger,L., arthritis patients might influence disease outcome, Joint Bone Spine, 67: 30-39, 2000. Cole,S.P. and Doyle,L.A. Atypical multidrug resistance: breast cancer resistance protein 16 Wolf,J., Stranzl,T., Filipits,M., Pohl,G., Pirker,R., Leeb,B.F. and Smolen,J.S. Expression of resistance messenger RNA expression in mitoxantrone-selected cell lines, J.Natl.Cancer Inst., 91: 429-433, markers to methotrexate predicts clinical improvement in patients with rheumatoid arthritis, 1999.

110 111 References References

31 Jonker,J.W., Merino,G., Musters,S., van Herwaarden,A.E., Bolscher,E., Wagenaar,E., Mesman,E., Dale,T.C. and Schinkel,A.H. The breast cancer resistance protein BCRP (ABCG2) concentrates drugs and carcinogenic xenotoxins into milk, Nat.Med., 11: 127-129, 2005. 32 Zhou,S., Schuetz,J.D., Bunting,K.D., Colapietro,A.M., Sampath,J., Morris,J.J., Lagutina,I., Grosveld,G.C., Osawa,M., Nakauchi,H. and Sorrentino,B.P. The ABC transporter Bcrp1/ABCG2 is expressed in a wide variety of stem cells and is a molecular determinant of the side-population phenotype, Nat.Med., 7: 1028-1034, 2001. 33 Szatmari,I., Vamosi,G., Brazda,P., Balint,B.L., Benko,S., Szeles,L., Jeney,V., Ozvegy-Laczka,C., Szanto,A., Barta,E., Balla,J., Sarkadi,B. and Nagy,L. Peroxisome proliferator-activated receptor gamma-regulated ABCG2 expression confers cytoprotection to human dendritic cells, J.Biol. Chem., 281: 23812-23823, 2006. 34 Sarkadi,B., Homolya,L., Szakacs,G. and Varadi,A. Human multidrug resistance ABCB and ABCG transporters: participation in a chemoimmunity defense system, Physiol Rev., 86: 1179-1236, 2006. 35 Doyle,L.A. and Ross,D.D. Multidrug resistance mediated by the breast cancer resistance protein BCRP (ABCG2), Oncogene, 22: 7340-7358, 2003. 36 Robey,R.W., Polgar,O., Deeken,J., To,K. W. and Bates,S.E. ABCG2: determining its relevance in clinical drug resistance, Cancer Metastasis Rev., 26: 39-57, 2007. 37 Wilson,C.S., Davidson,G.S., Martin,S.B., Andries,E., Potter,J., Harvey,R., Ar,K., Xu,Y., Kopecky,K. J., Ankerst,D.P., Gundacker,H., Slovak,M.L., Mosquera-Caro,M., Chen,I.M., Stirewalt,D.L., Murphy,M., Schultz,F.A., Kang,H., Wang,X., Radich,J.P., Appelbaum,F.R., Atlas,S.R., Godwin,J. and Willman,C.L. Gene expression profiling of adult acute myeloid leukemia identifies novel biologic clusters for risk classification and outcome prediction, Blood, 108: 685-696, 2006. 38 Chen,Z.S., Robey,R.W., Belinsky,M.G., Shchaveleva,I., Ren,X.Q., Sugimoto,Y., Ross,D.D., Bates,S. E. and Kruh,G.D. Transport of methotrexate, methotrexate polyglutamates, and 17beta-estradiol 17-(beta-D-glucuronide) by ABCG2: effects of acquired mutations at R482 on methotrexate transport, Cancer Res., 63: 4048-4054, 2003. 39 Assaraf,Y.G. Molecular basis of antifolate resistance, Cancer Metastasis Rev., 26: 153-181, 2007. 40 obbiani,D.F., Finch,R.A., Jager,D., Muller,W.A., Sartorelli,A.C. and Randolph,G.J. The leukotriene C(4) transporter MRP1 regulates CCL19 (MIP-3beta, ELC)- dependent mobilization of dendritic cells to lymph nodes, Cell, 103: 757-768, 2000. 41 van der Ven,R., de Jong,M.C., Reurs,A.W., Schoonderwoerd,A.J., Jansen,G., Hooijberg,J.H., Scheffer,G.L., de Gruijl,T.D. and Scheper,R.J. Dendritic cells require multidrug resistance protein 1 (ABCC1) transporter activity for differentiation, J.Immunol., 176: 5191-5198, 2006. 42 Radstake,T.R., van der Voort R., ten Brummelhuis M., de Waal,M.M., Looman,M., Figdor,C.G., van den Berg,W.B., Barrera,P. and Adema,G.J. Increased expression of CCL18, CCL19, and CCL17 by dendritic cells from patients with rheumatoid arthritis, and regulation by Fc gamma receptors, Ann Rheum.Dis., 64: 359-367, 2005. 43 Timmer,T.C., Baltus,B., Vondenhoff,M., Huizinga,T.W., Tak,P.P., Verweij,C.L., Mebius,R.E. and van der Pouw Kraan, T.C. Inflammation and ectopic lymphoid structures in rheumatoid arthritis synovial tissues dissected by genomics technology: identification of the interleukin-7 signaling pathway in tissues with lymphoid neogenesis, Arthritis Rheum., 56: 2492-2502, 2007. 44 Zaher,H., Khan,A.A., Palandra,J., Brayman,T.G., Yu,L. and Ware,J.A. Breast Cancer Resistance Protein (Bcrp/abcg2) is a major determinant of sulfasalazine absorption and elimination in mouse, Molecular Pharmaceutics, 3: 55-61, 2006.

112 113 chapter 7 Anti-folate Drug Combinations for Inflammatory Diseases

Joost W. van der Heijden, Gerrit Jansen and Ben A.C. Dijkmans

Department of Rheumatology, VU University Medical Center, Amsterdam, The Netherlands

In: Chemistry and Biology of Pteridines and Folates 2007; 365-377 chapter 7 Anti-folate Drug Combinations for Inflammatory Diseases

Abstract Figure 1

Drugs used in daily medical practice to treat inflammatory diseases include the anti- folate methotrexate (MTX) and the anti-pyrimidine leflunomide. Treatment with MTX is widespread for various inflammatory diseases and this drug has become the anchor drug in the treatment of rheumatoid arthritis (RA) patients. In RA-patients MTX is administered as monotherapy, but nowadays it is often combined with other disease modifying antirheumatic drugs (DMARDs) like sulfasalazine, hydroxychloroquine and prednisone, and biologic agents like infliximab, etanercept and adalimumab. The beneficial effects of biologic agents, like anti-tumour necrosis factor α (TNFα) antibodies, have been assessed after partial failure on MTX, as monotherapy or in combination with MTX. Thus, MTX has gained a very important place in the treatment of RA patients; the definite place for various combination strategies is still under investigation. Leflunomide has also proven to be an Time frame of DMARD utilization in the treatment of patients with rheumatoid arthritis. effective drug in the treatment of RA-patients, but has a less established place than MTX.

place in the treatment of patients with RA, in principle in combination with MTX. Introduction This manuscript provides an overview of the current status of MTX and leflunomide in the treatment of RA-patients, as monotherapy or in combination with other DMARDs Rheumatoid arthritis (RA) is a disease with synovitis as hallmark, leading to cartilage and or biologic agents. It is a review of the leading manuscripts involving RA treatment. The bone destruction (1). Moreover, RA is accompanied by general effects of inflammation, efficacy of drugs in the various trials is defined by the ACR response criteria (developed by such as malaise, fatigue and an increased sedimentation rate. It affects on average 1% the American College of Rheumatology), that is the 20%, 50% and 70% improvement in RA of the Dutch population, 70% of patients being women. For decades, therapy of RA- disease activity. In Europe, rheumatologists also commonly use the Disease Activity Score patients consisted of non-steroidal anti-inflammatory drugs (NSAIDs), reducing pain and 28 (DAS28), evaluating swelling and tenderness of 28 joints and counting BSE scores and inflammation but not modifying the course of the disease. In the 1930’s, the first Disease patient-reported disease activity, to scale disease severity of RA patients (5). Modifying Anti-rheumatic Drugs (DMARDs) -gold and sulfasalazine- were introduced, interfering with the course and progression of the disease (2, 3). The modifying effect Methotrexate in rheumatoid arthritis

of DMARDs is reflected in improvement of the Disease Activity Score (DAS), a composite MTX is a folate analogue originally developed in the 1940s as a highly selective inhibitor of index of the patients opinion of disease activity, a count of tender and swollen joints by dihydrofolate reductase (DHFR) (6). Its beneficial effect in reduction of RA synovitis was the physician and the erythrocyte sedimentation rate. The most important DMARDs are first reported in 1951. Subsequently, its efficacy in RA was proven in a series of papers in listed in an historical perspective in figure 1. This figure illustrates that intramuscular gold the mid 1980’s and nowadays it is the anchor drug in the treatment of RA-patients, either is the eldest DMARD, followed by sulfasalazine. Corticoids are used since the 1940’s for as monotherapy or in combination therapy as recommended by the American College of RA, with good clinical response, but it induces many side effects in patients, for example Rheumatology (ACR). osteoporosis, hypertension and diabetes mellitus upon prolonged use. Two developments can be considered as real breakthroughs in the treatment of RA- The following features of MTX are supposed to explain its efficacy in RA: patients: (a) the rediscovery of methotrexate (MTX) in the 1980’s and (b) the introduction of 1. Induction of apoptosis in T cells or monocytic/macrophage cells, biologic agents in the 1990’s. Biologic agents are monoclonal antibodies directed against 2. Inhibition of the production of proinflammatory cytokines: IL1 and IL6, pro-inflammatory cytokines (e.g. TNFα) that play a major role in the pathophysiology of 3. Stimulation of the production of anti-inflammatory cytokines: IL4 and IL10 and RA (4). These agents include infliximab and adalimumab (both antibodies against TNFα) 4. Inhibition of metalloproteinase production, thereby reducing cartilage and bone and etanercept (soluble TNFα receptors). At present, biologic agents have an established destruction (7)

116 117 chapter 7 Anti-folate Drug Combinations for Inflammatory Diseases

MTX is metabolised in the liver to active polyglutamylated forms and inactive 7-hydroxy- Table 1: Combinations of MTX with other DMARDs and biological agents in current clinical methotrexate forms. Approximately 60% is bound to albumin. The elimination half- treatment of Rheumatoid Arthritis. life increases with dose and ranges form 3 to 15 hours. MTX and its metabolites are DMARDs Remark Reference predominantly renally excreted. Hence, drugs that might influence renal function should

be co-administered with caution (8). MTX + SSZ (17) MTX can be administered orally, parenterally (subcutaneously or intramuscularly). The MTX + SSZ + prednisolone COBRA schedule (16, 22) MTX + HCHQ (20) bioavailability of low oral doses MTX (up to 10 mg) is relatively high but decreases with MTX + HCHQ + SSZ O’Dell schedule (21) higher doses. Hence, for a patient not responding to higher doses of MTX (25 mg/week) MTX + leflunomide (34) parenteral administration should be considered. MTX is administered once weekly, with Biological agents Remark Reference a starting dose of 7.5 mg, up to 25-30 mg/week. In the Netherlands, it is recommended to

prescribe folic acid or folinic acid along with MTX-therapy to reduce systemic toxicity (9). MTX + Infliximab (anti-TNFα) Chimeric Moab to TNFα (25) MTX + Etanercept (anti-TNFα) Soluble TNFα receptor II (27) MTX monotherapy MTX + Adalimumab (anti-TNFα) Human Moab to TNFα (30) MTX + Anakinra (anti-IL1β) IL-1R antagonist (35) Open studies in the early 1980’s indicated the efficacy of MTX, up to 15 mg per week, in

patients with RA refractory to other DMARDs (10, 11). The efficacy of MTX was confirmed Abbreviations: SSZ; sulfasalazine, HCHQ; hydroxychloroquine, MTX; methotrexate, TNFα: in placebo-controlled studies assessing dosages up to 25 mg per week (12, 13). A meta- Tumor Necrosis Factor α, IL1-β; Interleukin-1β, analysis of these studies showed that patients treated with MTX had a 39% reduction in painful joints and 29% reduction in swollen joints compared with placebo (14). Long-term observational studies with follow-up periods of more than 10 years showed long-lasting effectiveness with low discontinuation rates, and drug survival rates of more than 5 years in approximately 50% of patients, which compares favourably with 25% or sulfasalazine alone. Prednisolone and methotrexate were tapered and stopped after reported for other DMARDs. Moreover, from these studies it has been proven that MTX 28 and 40 weeks, respectively. The primary efficacy outcome measure was a composite prevents joint damage in RA patients (15). index comprising improvement in erythrocyte sedimentation rate, grip strength, tender joint count, global assessment by the observer, and improvement in functional ability. The MTX in combination with other DMARDs improvement of this pooled index at week 28 was strongly in favour of the combination- So far, five comparative trials have been published investigating combination therapy therapy group, but at week 56 the improvement in both groups was equal. The efficacy of of sulfasalazine and MTX (16-20) (see also Table 1). One trial indicated that triple therapy the COBRA schedule was further demonstrated in a larger cohort of RA patients (22). consisting of sulfasalazine, MTX and hydroxychloroquine was more effective than MTX alone or the sulfasalazine/hydroxychloroquine combination (21). Another comparative At present, there is no evidence that the combination of MTX and sulfasalazine is superior trial showed no efficacy differences between sulfasalazine alone, MTX alone, and to monotherapy with these agents. The initial addition of prednisolone improved the their combination (17). Two trials had a step-down approach (16, 19). In one study the clinical efficacy and might have long-term benefits in the treatment of RA-patients. combination sulfasalazine, MTX, hydroxychloroquine and low-dose prednisolone was Recently in-vitro studies showed that sulfasalazine is a potent inhibitor of the Reduced compared with sulfasalazine alone, which could be replaced by MTX alone (19). In the Folate Carrier (RFC; carrier of MTX), which may explain the lack of potentiation of the combination therapy group, prednisolone and MTX could be discontinued provided combination therapy of MTX and sulfasalazine compared to monotherapy with these remission was achieved during the first year. The remission rate in this group after two drugs (23) years was doubled compared with the sulfasalazine group. MTX in combination with biological agents In the COBRA-trial (16) 56 patients with early active RA were randomised to either If, because of loss of DMARD efficacy, RA-patients switch to ‘third-line’ treatment with treatment with a combination of sulfasalazine, MTX and prednisolone (COBRA-schedule) biological agents, MTX usually remains included in therapeutic protocols. There is evidence

118 119 chapter 7 Anti-folate Drug Combinations for Inflammatory Diseases

that MTX administration prolongs the duration of response to biological agents, probably patients achieving the response criteria was significantly higher in the etanercept 25 mg due to the inhibition of the formation of antibodies against these agents. In several trials group than in the MTX monotherapy group. the efficacy of the combination therapy of MTX plus a biological agent was compared with In 2004 the results of the TEMPO study have been published (29). This study is a comparative monotherapy with MTX or the biological agent (Table 1). double-blind trial evaluating etanercept with or without MTX versus MTX alone. After 1 Infliximab (anti-TNFα antibody) was administered in doses of 1,3 and 10 mg/kg, with or year, the 20% response criteria were met by 85% of patients treated with etanercept plus without weekly MTX (24). Significant clinical benefit was seen in the combination therapy MTX, 76% of patients treated with etanercept alone and 75% of patients treated with MTX. group, with responses up to 55% at week 26 compared with 35% in patients who were 43%, 24% and 19% of the patients receiving the combination, etanercept alone or MTX treated with MTX alone. This trial indeed indicated that MTX administration prolonged the alone, respectively, achieved the 70% response criteria. Of the patients with combination duration of response to infliximab. therapy, 80% experienced no radiographic progression throughout one year, compared In a later trial, 428 patients with established RA who were treated with MTX were randomised with 68% of patients treated with etanercept alone and 57% of patients treated with MTX to either infliximab 3 or 10 mg/kg given monthly or bimonthly, or placebo (25). At week 30, alone. the response criteria were met by 53% and 50% of patients receiving the 3 mg/kg every 4 or 8 weeks, respectively, and 58% and 52% for those receiving 10 mg/kg every 4 or 8 weeks. In FDA-approval of adalimumab (a complete human antibody against TNFα) was based on the placebo group, 20% of the patients met the response criteria at week 30. several randomised double-blind trials. In the ARMADA-trial 271 RA-patients, who had an inadequate response to MTX and had no response to other DMARDs, were treated with The BeSt trial is a multicenter, randomized, single blind trial conducted in the Netherlands, adalimumab (either 20, 40 or 80 mg) or placebo every 2 weeks (30). At week 24, patients including 508 patients with newly diagnosed RA who were not previously treated with receiving treatment with adalimumab 40 mg every other week achieved the 20% response DMARDs (26). In this trial 4 different treatment strategies in early rheumatoid arthritis were criteria in 65% of cases and the 70% response criteria in 24% of cases, compared with compared. The one-year follow-up results indicated that patients on a step-down therapy response rates of 13% and 3% for placebo, respectively. from either MTX + sulfasalazine + prednisone 60 mg tapered to 7.5 mg or a treatment with In summary, there is a strong indication that the combination of MTX plus one of the MTX + Inflimixab showed a faster clinical response (reflected by a reduction in DAS) and mentioned biologic agents is superior above monotherapy with MTX or the biologic agent, less radiological damage than patients on either a sequential monotherapy starting with not only in reducing disease activity but also in reducing joint damage as seen on X-rays of MTX → sulfasalazine → leflunomide or a step-up therapy including MTX + sulfasalazine hands and feet. + hydroxychloroquine. Interestingly, follow-up analysis beyond one year showed that all four therapy arms were equally efficacious in DAS reduction. Leflunomide in rheumatoid arthritis The results of the ASPIRE trial in 1049 patients with early RA indicated that infliximab plus Following oral administration, leflunomide is rapidly converted in the gut wall and liver MTX was more effective than MTX alone in preventing joint destruction and disability to the active metabolite ff A 77 1726. Binding of A77 1726 to the enzyme dihydro-orotate (2). The response criteria were met by 66%, 62% and 54% of patients treated with 3 mg/ dehydrogenase results in inhibition of pyrimidine biosynthesis (31, 32). A consequence of kg plus MTX, 6 mg/kg plus MTX, and placebo plus MTX, respectively. Progression of joint this inhibition is a reversible cell cycle arrest in rapidly dividing cell populations such as destruction in the infliximab plus MTX arms were significantly decreased compared to the activated lymphocytes; the immune cells that mediate joint inflammation. Elimination of placebo plus MTX arm. leflunomide from the body is by both renal and gastro-intestinal routes. The mean half- Studies investigating the combination of Etanercept, (soluble TNFα receptor) and MTX life of leflunomide is 14-16 days. Administration of cholestyramine in a dosage of 4 gram showed that in patients with advanced RA, who were not responding to MTX, the addition 3 times daily reduces the half-life of leflunomide to approximately one day, indicating of etanercept 25 mg twice weekly resulted in a marked clinical improvement (27). At that significant levels of the drug undergo enterohepatic circulation. Due to the long half- 6 months, 71% of the patients treated with etanercept plus MTX achieved the response life of the active metabolite, a starting dose of 100 mg, taken daily for 3 days, has been criteria versus 27% of the patients treated with placebo plus MTX. suggested to reach effective steady-state blood levels within 4-6 weeks. Etanercept was also compared with MTX in another trial involving 632 patients with early RA (28). Patients were either treated with etanercept twice-weekly 10 mg or once weekly Leflunomide monotherapy 25 mg or MTX monotherapy for one year. During the first 6 months, the proportion of Recently, a systemic review and meta-analysis of six double-blind comparative trials

120 121 chapter 7 Anti-folate Drug Combinations for Inflammatory Diseases

with leflunomide was published (33). In these trials, 1144 patients were treated with leflunomide, 680 were on MTX, 132 on sulfasalazine and 312 patients got placebo. Overall, the trials indicated that leflunomide is significantly superior to placebo at 6 and 12 months treatment and has a similar efficacy compared to sulphasalazine and MTX, however leflunomide efficacy has a more rapid onset than MTX and sulfasalazine. After 24 months leflunomide was more effective than sulfasalazine and had equal efficacy compared with MTX.

Combination of leflunomide and MTX The rationale for combination therapy of leflunomide with MTX is the simultaneous inhibition of the purine metabolism by MTX and inhibition of the pyrimidine metabolism by leflunomide, resulting in a subsequently more efficient inhibition of proliferation of inflammatory cells. An open label study, in which leflunomide was added to MTX, showed that the combination was well tolerated and efficacious in 53% of patients. However, liver enzyme elevations were observed in 63% of patients. Subsequently, a 24-week double blind, placebo- controlled trial in RA patients with a partial response to MTX was conducted. A total of 130 patients were treated with leflunomide 100 mg/day for two days followed by 10 mg/day. This dosage could be increased to 29 mg/day in case of persistent active disease. Elevated liver enzymes were seen in 28% of patients and the authors concluded that this combination could be administered safely, provided appropriate laboratory monitoring. The 20% response criteria at week 24 were met by 46% of the leflunomide-treated patients compared to 20% of the placebo treated patients. The 70% response rates were 10% and 2% in patients receiving leflunomide and placebo, respectively (34)

Conclusion

The anti-folate drug MTX and, to a lesser extend, the anti-pyrimidine drug leflunomide have an established place in the treatment of patients with rheumatoid arthritis. At present these DMARDs are often prescribed in combination schedules with other DMARDs or in combination with biological agents. There is accumulating clinical evidence that MTX in the COBRA schedule or MTX in combination with a biological agents is superior above monotherapy, not only in reducing disease activity but also in reducing joint damage. Finally, co-administration of MTX is supposed to prolong drug-survival of the biological agents, probably by preventing antibody formation against these agents.

122 123 References References

References controlled, double-blind, 52 week clinical trial, Br.J.Rheumatol., 36: 1082-1088, 1997. 18 Karger,T. and Rau,R. [Treatment of chronic polyarthritis with low-dose methotrexate], Dtsch. 1 Feldmann,M., Brennan,F.M. and Maini,R.N. Rheumatoid arthritis, Cell, 85: 307-310, 1996. Med.Wochenschr., 113: 839-846, 1988. 2 Smolen,J.S. and Steiner,G. Therapeutic strategies for rheumatoid arthritis, Nat.Rev.Drug 19 Mottonen,T., Hannonen,P., Leirisalo-Repo,M., Nissila,M., Kautiainen,H., Korpela,M., Laasonen,L., Discov., 2: 473-488, 2003. Julkunen,H., Luukkainen,R., Vuori,K., Paimela,L., Blafield,H., Hakala,M., Ilva,K., Yli-Kerttula,U., 3 O’Dell,J.R. Therapeutic strategies for rheumatoid arthritis, N.Engl.J.Med., 350: 2591-2602, Puolakka,K., Jarvinen,P., Hakola,M., Piirainen,H., Ahonen,J., Palvimaki,I., Forsberg,S., Koota,K. 2004. and Friman,C. Comparison of combination therapy with single-drug therapy in early rheumatoid 4 Elliott,M.J., Maini,R.N., Feldmann,M., Kalden,J.R., Antoni,C., Smolen,J.S., Leeb,B., Breedveld,F. arthritis: a randomised trial. FIN-RACo trial group, Lancet, 353: 1568-1573, 1999. C., Macfarlane,J.D., Bijl,H. et al. Randomised double-blind comparison of chimeric monoclonal 20 O’Dell,J.R., Haire,C.E., Erikson,N., Drymalski,W., Palmer,W., Eckhoff,P.J., Garwood,V., antibody to tumour necrosis factor alpha (cA2) versus placebo in rheumatoid arthritis, Lancet, Maloley,P., Klassen,L.W., Wees,S., Klein,H. and Moore,G.F. Treatment of rheumatoid arthritis 344: 1105-1110, 1994. with methotrexate alone, sulfasalazine and hydroxychloroquine, or a combination of all three 5 van der Heijde,D.M., van ‘t Hof,M., van Riel,P.L. and van de Putte,L.B. Validity of single variables medications, N.Engl.J.Med., 334: 1287-1291, 1996. and indices to measure disease activity in rheumatoid arthritis, J.Rheumatol., 20: 538-541, 21 O’Dell,J.R., Leff,R., Paulsen,G., Haire,C., Mallek,J., Eckhoff,P.J., Fernandez,A., Blakely,K., 1993. Wees,S., Stoner,J., Hadley,S., Felt,J., Palmer,W., Waytz,P., Churchill,M., Klassen,L. and Moore,G. 6 Kremer,J.M. Toward a better understanding of methotrexate, Arthritis Rheum., 50: 1370-1382, Treatment of rheumatoid arthritis with methotrexate and hydroxychloroquine, methotrexate 2004. and sulfasalazine, or a combination of the three medications: Results of a two-year, randomized, 7 Cronstein,B.N. Molecular therapeutics. Methotrexate and its mechanism of action, Arthritis double-blind, placebo-controlled trial, Arthritis Rheum., 46: 1164-1170, 2002. Rheum., 39: 1951-1960, 1996. 22 Landewe,R.B., Boers,M., Verhoeven,A.C., Westhovens,R., van de Laar,M.A., Markusse,H.M., 8 Nurmohamed,M.T. and Dijkmans,B.A.C. Efficacy, tolerability and cost effectiveness of disease- van Denderen,J.C., Westedt,M.L., Peeters,A.J., Dijkmans,B.A.C., Jacobs,P., Boonen,A., van der modifying antirheumatic drugs and biologic agents in rheumatoid arthritis, Drugs, 65: 661-694, Heijde,D.M. and Van der Linden,S. COBRA combination therapy in patients with early rheumatoid 2005. arthritis: Long-term structural benefits of a brief intervention, Arthritis Rheum., 46: 347-356, 9 van Ede,A.E., Laan,R.F., Rood,M.J., Huizinga,T.W., van de Laar,M.A., van Denderen,C.J., 2002. Westgeest,T.A., Romme,T.C., de Rooij,D.J., Jacobs,M.J., de Boo,T.M., van der Wilt,G.J., Severens,J. 23 Jansen,G., Van der Heijden,J.W., Oerlemans,R., Lems,W.F., Ifergan,I., Scheper,R.J., Assaraf,Y.G. L., Hartman,M., Krabbe,P.F., Dijkmans,B.A.C., Breedveld,F.C. and van de Putte,L.B. Effect of folic and Dijkmans,B.A.C. Sulfasalazine is a potent inhibitor of the reduced folate carrier: implications or folinic acid supplementation on the toxicity and efficacy of methotrexate in rheumatoid for combination therapies with methotrexate in rheumatoid arthritis, Arthritis Rheum., 50: arthritis: a forty-eight week, multicenter, randomized, double-blind, placebo-controlled study, 2130-2139, 2004. Arthritis Rheum., 44: 1515-1524, 2001. 24 Maini,R.N., Breedveld,F.C., Kalden,J.R., Smolen,J.S., Davis,D., Macfarlane,J.D., Antoni,C., 10 Hoffmeister,R.T. Methotrexate therapy in rheumatoid arthritis: 15 years experience, Leeb,B., Elliott,M.J., Woody,J.N., Schaible,T.F. and Feldmann,M. Therapeutic efficacy of multiple Am.J.Med., 75: 69-73, 1983. intravenous infusions of anti-tumor necrosis factor alpha monoclonal antibody combined with 11 Willkens,R.F., Watson,M.A. and Paxson,C.S. Low dose pulse methotrexate therapy in rheumatoid low-dose weekly methotrexate in rheumatoid arthritis, Arthritis Rheum., 41: 1552-1563, 1998. arthritis, J.Rheumatol., 7: 501-505, 1980. 25 Maini,R., St Clair,E.W., Breedveld,F., Furst,D., Kalden,J., Weisman,M., Smolen,J., Emery,P., 12 Weinblatt,M.E., Coblyn,J.S., Fox,D.A., Fraser,P.A., Holdsworth,D.E., Glass,D.N. and Trentham,D. Harriman,G., Feldmann,M. and Lipsky,P. Infliximab (chimeric anti-tumour necrosis factor alpha E. Efficacy of low-dose methotrexate in rheumatoid arthritis, N.Engl.J.Med., 312: 818-822, 1985. monoclonal antibody) versus placebo in rheumatoid arthritis patients receiving concomitant 13 Williams,H.J., Willkens,R.F., Samuelson,C.O., Jr., Alarcon,G.S., Guttadauria,M., Yarboro,C., methotrexate: a randomised phase III trial. ATTRACT Study Group, Lancet, 354: 1932-1939, Polisson,R.P., Weiner,S.R., Luggen,M.E., Billingsley,L.M. and . Comparison of low-dose oral pulse 1999. methotrexate and placebo in the treatment of rheumatoid arthritis. A controlled clinical trial, 26 De Vries-Bouwstra,J.K., Goekoop-Ruiterman,Y.P.M., Breedveld,F.C., Han,K.H., Kerstens,P. Arthritis Rheum., 28: 721-730, 1985. J.S., Ronday,H.K., Zwinderman,A.H., Dijkmans,B.A.C. and Allaart,C.F. Clinical and radiological 14 Tugwell,P., Bennett,K. and Gent,M. Methotrexate in rheumatoid arthritis. Indications, outcomes after one year follow-up of the BeSt study, a randomized trial comparing four different contraindications, efficacy, and safety, Ann.Intern.Med., 107: 358-366, 1987. treatment strategies in early rheumatoid arthritis (RA). In: Anonymous2003. 15 Jeurissen,M.E., Boerbooms,A.M., van de Putte,L.B., Doesburg,W.H. and Lemmens,A.M. 27 Weinblatt,M.E., Kremer,J.M., Bankhurst,A.D., Bulpitt,K.J., Fleischmann,R.M., Fox,R.I., Jackson,C. Influence of methotrexate and azathioprine on radiologic progression in rheumatoid arthritis. A G., Lange,M. and Burge,D.J. A trial of etanercept, a recombinant tumor necrosis factor receptor: randomized, double-blind study, Ann.Intern.Med., 114: 999-1004, 1991. Fc fusion protein, in patients with rheumatoid arthritis receiving methotrexate, N.Engl.J.Med., 16 Boers,M., Verhoeven,A.C., Markusse,H.M., van de Laar,M.A., Westhovens,R., van Denderen,J. 340: 253-259, 1999. C., van Zeben,D., Dijkmans,B.A.C., Peeters,A.J., Jacobs,P., van den Brink,H.R., Schouten,H.J., 28 Bathon,J.M., Martin,R.W., Fleischmann,R.M., Tesser,J.R., Schiff,M.H., Keystone,E.C., Genovese,M. van der Heijde,D.M., Boonen,A. and Van der Linden,S. Randomised comparison of combined C., Wasko,M.C., Moreland,L.W., Weaver,A.L., Markenson,J. and Finck,B.K. A comparison of step-down prednisolone, methotrexate and sulphasalazine with sulphasalazine alone in early etanercept and methotrexate in patients with early rheumatoid arthritis, N.Engl.J.Med., 343: rheumatoid arthritis, Lancet, 350: 309-318, 1997. 1586-1593, 2000. 17 Haagsma,C.J., van Riel,P.L., de Jong,A.J. and van de Putte,L.B. Combination of sulphasalazine and 29 Klareskog,L., van der Heijde,D., de Jager,J.P., Gough,A., Kalden,J., Malaise,M., Martin,M.E., methotrexate versus the single components in early rheumatoid arthritis: a randomized, Pavelka,K., Sany,J., Settas,L., Wajdula,J., Pedersen,R., Fatenejad,S. and Sanda,M. Therapeutic

124 125 References References

effect of the combination of etanercept and methotrexate compared with each treatment alone in patients with rheumatoid arthritis: double-blind randomised controlled trial, Lancet, 363: 675-681, 2004. 30 Weinblatt,M.E., Keystone,E.C., Furst,D.E., Moreland,L.W., Weisman,M.H., Birbara,C.A., Teoh,L. A., Fischkoff,S.A. and Chartash,E.K. Adalimumab, a fully human anti-tumor necrosis factor alpha monoclonal antibody, for the treatment of rheumatoid arthritis in patients taking concomitant methotrexate: the ARMADA trial, Arthritis Rheum., 48: 35-45, 2003. 31 Breedveld,F.C. and Dayer,J.M. Leflunomide: mode of action in the treatment of rheumatoid arthritis, Ann.Rheum.Dis., 59: 841-849, 2000. 32 Fox,R.I., Herrmann,M.L., Frangou,C.G., Wahl,G.M., Morris,R.E., Strand,V. and Kirschbaum,B.J. Mechanism of action for leflunomide in rheumatoid arthritis, Clin.Immunol., 93: 198-208, 1999. 33 Osiri,M., Shea,B., Robinson,V., Suarez-Almazor,M., Strand,V., Tugwell,P. and Wells,G. Leflunomide for the treatment of rheumatoid arthritis: a systematic review and metaanalysis, J.Rheumatol., 30: 1182-1190, 2003. 34 Kalden,J.R., Smolen,J.S., Emery,P., van Riel,P.L., Dougados,M., Strand,C.V. and Breedveld,F.C. Lefunomide in combination therapy, J.Rheumatol.Suppl, 71: 25-30, 2004. 35 Cohen,S., Hurd,E., Cush,J., Schiff,M., Weinblatt,M.E., Moreland,L.W., Kremer,J., Bear,M.B., Rich,W.J. and McCabe,D. Treatment of rheumatoid arthritis with anakinra, a recombinant human interleukin-1 receptor antagonist, in combination with methotrexate: results of a twenty-four- week, multicenter, randomized, double-blind, placebo-controlled trial, Arthritis Rheum., 46: 614-624, 2002.

126 127 chapter 8 Enhanced capacity of selected methotrexate-analogues to inhibit TNFα production in whole blood from RA patients

Joost W. van der Heijden1, Andreas H. Gerards2, Ruud Oerlemans1, Willem F. Lems1, Rik J. Scheper3, Lucien A. Aarden4, Ben A.C. Dijkmans1 and Gerrit Jansen1.

Departments of 1Rheumatology and 3Pathology, VU University Medical Center, Amsterdam, The Netherlands. 2Department of Rheumatology, Vlietland Hospital, Schiedam, The Netherlands. 4Department of Immunopathology, Sanquin-Research, Amsterdam, The Netherlands.

Submitted for publication chapter 8 Anti-inflammatory properties of novel generation antifolates

Abstract ability to inhibit the release of pro-inflammatory cytokines such as tumor necrosis factor α (TNFα) or interleukin 1β (IL-1β) (4). Multiple mechanisms for these anti-inflammatory Objective: Athough methotrexate (MTX) is the anchor drug in the treatment of patients effects have been postulated (2, 4-6): (a) MTX-induced extracellular release of adenosine with rheumatoid arthritis (RA), many patients experience clinical resistance to MTX upon by inhibition of the enzyme AICARTFase (involved in purine biosynthesis), causing anti- prolonged treatment. Here we explored whether novel generation antifolate drugs elicit inflammatory signalling via the adenosine receptor (7-9), (b) MTX-induced inhibition of improved anti-inflammatory properties compared to MTX, based on their capacity to the first enzyme in purine biosynthesis de novo: amidophosphoribosyltransferase (10), inhibit TNFα production by activated T-cells from RA patients. (c) apoptosis of activated T-cells (11) possibly facilitated by MTX-induced generation of Methods: T-cells in whole-blood from 18 RA patients (including MTX naïve, MTX responsive reactive oxygen species (12, 13), (d) MTX-induced inhibition of methyltransferases leading and MTX non-responsive patients) and 7 healthy volunteers were stimulated with αCD3/ to impairment of Ras signalling (14) and/or (e) direct inhibition of the activation of the αCD28 antibodies and incubated ex-vivo with MTX and eight novel antifolate drugs with transcription factor NFκB that controls the transcription of TNFα and IL1β (15). potential favourable pharmacological properties. Inhibition of TNFα production was measured after 72 hours by ELISA and drug concentrations exerting 50% inhibition of TNFα Notwithstanding the potent anti-rheumatic capacity of MTX, up to 50% of RA patients production (IC-50) were monitored as an estimate for their anti-inflammatory capacity. In ultimately experience clinical resistance to MTX during chronic therapy (16). In this parallel, induction of T-cell apoptosis was studied by FACS analysis. context, still little is known about the mechanism(s) of resistance to MTX in RA patients Results: The novel generation antifolates PT523, PT644, raltitrexed and GW1843 proved (17). For MTX as an anti-cancer agent, several mechanisms of resistance have been to be potent inhibitors of TNFα production in activated T-cells from all three groups of RA identified, including (a) down regulation of the reduced folate carrier (RFC) leading to patients as well as healthy volunteers. Based on IC-50 values, these antifolates were 1.3- impaired cellular uptake of MTX, (b) decreased cellular retention of MTX due to impaired 10.3 fold more potent than MTX. The anti-inflammatory effects were observed at drug- polyglutamylation by folylpolyglutamate synthetase (FPGS), (c) elevated levels of the concentrations that provoked apoptosis in 20-40% of activated T-cells. Beyond this, also target enzymes of MTX: dihydrofolate reductase (DHFR) and thymidylate synthase (TS), suppression of T-cell activation was observed, being partially independent from apoptosis (d) enhanced cellular efflux of MTX by specific members of the ATP-Binding Cassette induction. family of drug efflux transporters, and/or (e) increased folate levels that compete with Conclusion: In an ex-vivo setting, novel generation antifolate drugs elicited effective MTX for cellular uptake and polyglutamylation (18-24). inhibition of TNFα production by induction of apoptosis in activated T-cells from RA patients. Conceivably, further clinical evaluation is warranted to investigate whether low Extended knowledge on the molecular basis of resistance mechanisms to MTX has dosages of one of these novel antifolate drugs can induce immuno-suppressive effects facilitated the rational design of new generation antifolate drugs with the potential equivalent to MTX, or be superior to MTX in patients who do not respond to MTX. ability to circumvent MTX-resistance due to any of the above-mentioned mechanisms. In this context, second and third generation of folate-antagonists exhibit one or more of the following characteristics: (a) are transported more efficiently via the RFC, (b) have higher Introduction affinity for FPGS or being independent of polyglutamylation for their activity, (c) have the ability to target other enzymes in the folate metabolic pathway besides DHFR like TS and For many decades the folate antagonist methotrexate (MTX) is considered the anchor glycinamide ribonucleotide transformylase (GARTFase), (d) exhibiting poor affinity for drug in the treatment of neoplastic diseases (1) as well as chronic inflammatory diseases drug efflux transporters or (e) show retention of activity regardless of extra/intracellular such as rheumatoid arthritis (RA) (2). From an anti-cancer perspective, the molecular folate status (reviewed in (18, 25)). The present study was undertaken to assess the anti- basis for the anti-proliferative activity of MTX relies on inhibition of key enzymes in inflammatory effects of a selected set of these novel generation antifolates. To this end, folate metabolism, e.g. dihydrofolate reductase (DHFR) or thymidylate synthase (TS) we investigated the potency to suppress the release of the pro-inflammatory cytokine and folate dependent enzymes in purine biosynthesis de novo (3). These features lead to TNFα from activated T cells in whole blood cell cultures of RA patients, including those intracellular folate depletion and diminished pools of purines and pyrimidines required who were clinically unresponsive to MTX. for DNA synthesis. Anti-proliferative properties of MTX may also be of relevance in RA treatment, but the anti-inflammatory properties of MTX have most been related to its

130 131 chapter 8 Anti-inflammatory properties of novel generation antifolates

Figure 1 Materials and methods

Patient characteristics All patients included in this study signed an informed consent form and the study on ‘DMARD-resistance’ was approved by the Medical Ethics committee of the VU University Medical Center, Amsterdam, The Netherlands. All patients fulfilled the American College of Rheumatology (formerly, the American College of Rheumatism Association) 1987 revised criteria for RA and were on stable doses of MTX (15-30 mg/week) for at least three months. Newly diagnosed RA patients, who were not yet receiving Disease Modifying Anti-Rheumatic Drugs (DMARDs), were included as MTX-naïve patients. Characteristics of RA patients included in this study were: 18 RA patients (male/female: 5/13; mean age: 52 years; mean DAS28 score: 4.3, range 1.3-7.2). Five patients were MTX-naïve and 13 patients received MTX; RA-patients were defined as MTX-non-responders if they had a DAS28 score of >3.2 at the time of enrolment (n=7), patients with a DAS28 score of ≤3.2 were defined as MTX-responders (n=6). In addition, seven healthy volunteers were included (male/female: 4/3; mean age: 38 years). Figure 1: Folate metabolic pathway and the targets for therapy of MTX and novel folate antagonists. Drugs Block arrows: folate metabolic pathway; black arrows: routes of antifolates (minus character means inhibition of target); grey boxes: (target) enzymes in folate metabolic pathway. Methotrexate was a kind gift of Pharmachemie, Haarlem, The Netherlands. Pemetrexed/ Abbreviations: RFC: reduced folate carrier; DHFR: dihydrofolate reductase; TS: thymidylate synthase; GARTF: ALIMTA® (Eli Lilly) was obtained via the VUmc pharmacy department. The following glycinamide ribonucleotide transformylase; AICARTF: 5-aminoimidazole-4-carboxamide ribonucleotide formyltransferase; FH2: dihydrofolate; FH4: tetrahydrofolate; dUMP: deoxy-uridine monophosphate; 10-CHO- folate antagonist drugs were obtained from the indicated companies/institutions; FH4: 10-formyl-tetrahydrofolate; TMP: thymidine monophosphate; PG: polyglutamate form (polyglutamylation Raltitrexed/Tomudex®/ZD1694 (26) (Astra-Zeneca, UK), PT523 and PT644 (27, 28) (Dr. A. is carried out by the enzyme folylpolyglutamate synthetase/FPGS). Antifolate drugs: DHFR-inhibitors: Rosowsky, Harvard Medical School, Boston, U.S.A), GW1843 (29) (Glaxo-Welcome, USA), Methotrexate (MTX), Trimetrexate (TMQ), PT523 and PT644; TS-inhibitors: Raltitrexed, Pemetrexed, BGC9331 and GW1843; GARTF-inhibitor: AG2037. TMQ (Trimetrexate/Neutrexin®; Warner Lambert Park Davis, now MedImmune Pharma Netherlands) (1, 25), Plevitrexed/BGC9331 (30) (BTG International Ltd) and AG2037 (31) (Agouron/Pfizer, San Diego, U.S.A). Chemical structures of these folate antagonists are The concentration of each drug required to inhibit TNFα production by 50% is defined as provided in the supplementary file. A schematic overview of their targets in the folate the anti-inflammatory IC-50 concentration. metabolism is depicted in Figure 1. Properties regarding affinities of these drugs for RFC and FPGS are shown in Table 1. FACS analysis Peripheral blood mononuclear cells (PBMCs) of RA patients were isolated by Ficoll- T-cell activation and TNFα ELISA procedures Paque Plus density centrifugation and suspended in DMEM-medium supplemented Whole blood of 18 RA patients and 7 healthy volunteers was collected in heparin with 10% autologue donor-serum. Apoptosis in PBMCs was analyzed by flow cytometry containing tubes that were tested to be endotoxin-free (Becton Dickinson; San Jose, CA, (FACS) after 72 hours exposure to MTX and novel anti-folate drugs, using Annexin-V- U.S.A.). Blood was diluted 1:10 with DMEM medium supplemented with heparin (15 U/ml), FITC/7-aminoactinomycin D (7-AAD) staining (APOPTESTTM-FITC A700, VPS Diagnostics, 100 µg/ml penicillin/streptomycin and 0.1% fetal calf serum and incubated ex-vivo with Hoeven, the Netherlands) according to the manufacturer’s protocol. At the highest drug- αCD3/αCD28 antibodies (0.1 µg/ml and 1 µg/ml, respectively) as T-cell activating agents concentrations, 2 µM of the natural reduced folate 5-formyltetrahydrofolate/leucovorin as described previously (32). To these incubates 7 concentrations (range: 0-300 nM) of (Merck Eprova, Switzerland) was added to test folate competition-induced prevention of antifolates were added, followed by an incubation period of 72 hours. Inhibition of TNFα apoptosis. Phycoerythrin labeled anti-CD25 (Becton Dickinson; San Jose, CA, U.S.A.) was production was measured after 72 hours drug exposure by ELISA as described before (32). used for monitoring of T-cell activation.

132 133 chapter 8 Anti-inflammatory properties of novel generation antifolates

Statistical analysis IC-50 values (nM) for inhibition of TNFα production by activated T-cells Median (range) Statistical analysis was performed using non-parametric tests. For determining significant differences in anti-inflammatory IC-50 values between healthy controls and RA patients Drug Target RFC affinity FPGS affinity Controls RA-total DMARD naïve Responders Non-responders n=7 n=18 n=5 n=6 n=7 the two tailed Mann-Whitney test was used. A p-value <0.05 was considered statistically

53 60 55 47 60 significant. MTX DHFR ++ + (18-85) (16-325) (32-102) (28-260) (16-325)

254 232 259 185 157 TMQ DHFR- - (71-300) (24-460) (51-460) (74-278) (24-357) Results

PT523 DHFR +++ - 3.0 7.4 10 7.0 6.2 Novel antifolate drugs are potent inhibitors of TNFα production by activated T-cells (1.9-23) (2.9-121) (4.4-121) (2.9-11) (2.5-37) from healthy volunteers and RA patients 2.0 5.8 29 8.4 2.8 To explore whether novel folate antagonists could elicit potential anti-inflammatory PT644 DHFR +++ - (1.0-15) (2.3-111) (2.7-111) (3.9-31) (2.3-110) effects, the efficacy of these drugs was tested to inhibit TNFα production by activated

4.0 8.8* 46 9.0 8.3 T-cells in whole blood cultures of healthy controls and RA patients, including MTX-naive, Raltitrexed TS +++ +++ (2.6-10) (3.3-132) (3.7-90) (3.3-93) (6.7-132) MTX-responsive and MTX non-responsive patients. TNFα levels were determined after 72 hours of continuous drug exposure, as at this time T-cell activation appeared to be optimal 43 46 75 36 30 Pemetrexed TS +++ +++ (23-88) (7.2-326) (17-247) (25-119) (7.2-326) according to CD25 expression (Figure 2). Basal levels of TNFα production after T-cell stimulation were not significantly different between healthy controls (3208 ± 2554 pg/ml) 3.0 7.6* 23* 6.4 7.0 and (subgroups of) RA patients (3400 ± 2818 pg/ml). For each individual patient, the IC-50 GW1843 TS +++ +++ (2.2-17) (2.5-122) (5.1-122) (2.5-42) (6.0-13) values for TNFα inhibition by all nine folate antagonists are shown in Figure 3. The drugs are grouped according to their target in the folate metabolic pathway (see figure 1): (1) the BGC9331 TS +++ - 27 47 68 58 29 (13-59) (8.7-403) (25-403) (16-119) (8.7-341) group of primary DHFR inhibitors: MTX, TMQ, PT523 and PT644 (Figure 3A-D); (2) the group

104 143 313 132 139 of primary TS inhibitors: Raltitrexed, Pemetrexed, GW1843 and BGC9331 (Figure 3E-H) and AG2037 GARTFase +++ +++ (96-208) (79-1250) (161-455) (114-625) (79-1250) the GARTFase inhibitor AG2037 (Figure 3I). From these figures, an overview of all median IC-50 values is presented in Table 1.

Table 1: Properties of MTX and novel generation folate antagonists. Properties of MTX and novel folate antagonists regarding affinity for the reduced folate carrier (RFC) Four folate antagonists (PT523, PT644, raltitrexed and GW1843) could be identified with and folylpolyglutamate synthetase (FPGS). Kinetic properties (affinity; in µM) of RFC and FPGS for folate a greater potency (up to 10-fold) than MTX in RA T-cells, two had comparable activity antagonist drugs are categorized as follows; RFC affinity Km: <5 µM (+++), Km: 5-25 µM (++), Km: 25-100 µM (+), no substrate (-). FPGS affinity; Km: <5 µM (+++), Km: 5-25 µM (++), Km: 25-100 µM (+), no substrate (-). Anti- (pemetrexed and BGC9331), while two (TMQ and AG2037) were less potent (up to 4-fold) inflammatory properties of MTX and novel folate antagonists in ex-vivo activated T-cells from healthy controls than MTX. Data from Figure 3 further revealed a marked inter-patient variability in sensitivity and RA patients are depicted as median drug concentrations (nM) required to inhibit TNFα production by 50% (IC-50). The RA group is sub-categorized in MTX-naïve patients, MTX-responders and MTX-non-responders. for the drugs. In general, IC-50 values for TNFα release were lower for healthy volunteers *Asterisk denotes significant differences between RA-patients and healthy volunteers (p< 0.05). compared to RA patients, but these differences did only reach statistical significance for the TS inhibitors Raltitrexed and GW1843 (p<0.05). Of note, compared to healthy controls, 5 patients within the total group of 18 RA patients (equally distributed among RA subgroups) were less sensitive to MTX and most of the antifolate drugs. Interestingly, a slightly greater potency in suppressing TNFα production was observed for all folate antagonists in the group of MTX treated RA patients compared to MTX naive patients (Table 1; not statistically significant). Clinical unresponsiveness to MTX was not reflected by higher IC-50 values in the whole blood assay; T-cells from clinically MTX non-responsive RA patients were equally

134 135 H_8_Figure_2.pzf:Data 1 graph - Fri Sep 05 14:29:16 2008

H_8_Figure_5.pzf:Data 1 graph - Fri Sep 05 14:30:46 2008 chapter 8 Anti-inflammatory properties of novel generation antifolates

PT523, TMQ, Raltitrexed and Pemetrexed, suggestive for competitive effects with these antifolates. No rescue effect of leucovorin was observed for PT644, GW1843, BGC9331 70 Figure 2 and AG2037, suggesting that the activity of these anti-folates are not influenced at this 60 concentration of leucovorin. 50

40

30

%CD25+ cells 20

10

0 24 48 72 280 100  Figure 5 Hours after stimulation 90 cells CD25+ of Apoptosis % 120 80 80 Figure 2: Time dependency of T-cell activation by anti-CD3/anti-CD28 antibodies. 70 60 Activation of T-cells from PBMCs of RA patients by CD3/ CD28 was monitored by assessment of CD25 60 α α 50 expression by FACS. Results depicted are the mean ± S.D. of 3 independent experiments. 40 40 30 production (RApatients)

α 20 20 10 TNF IC-50 value (nM) for inhibition of

 0 0 3 4 d d 3 1 7 e e MQ x x 84 33 03 MTX T 1 9 2 PT52 PT64 itre tre C lt W AG sensitive to MTX compared to T-cells from MTX responsive RA patients. Similarly, T-cells a me G R BG Pe from MTX non-responsive RA patients were equal sensitive for each of the eight novel antifolate drugs as T-cells from MTX-responsive RA patients. Figure 5: Relation between inhibition of TNFα production and apoptosis induction in activated T-cells from RA patients. T-cells in PBMCs from RA patients were stimulated with αCD3/αCD28 for 72 hours in the presence of a concentration range of MTX and novel antifolates. T-cell activation (CD25 expression) and induction of MTX and novel antifolates induce apoptosis in activated (CD25+) but not resting apoptosis in CD25+ cells (right Y-axis; Annexin-V/7AAD staining) was determined at the drug concentration (CD25-) T-cells and attenuate T-cell activation that conferred 50% inhibition of TNFα production (left Y-axis; ELISA). Results depicted represent the mean of Since one of the proposed mechanism of action of low dose MTX is induction of apoptosis 3 separate experiments. in auto-reactive T-cells (11), we determined the onset of apoptosis in PBMC cultures of three drug-naïve RA patients after exposure to MTX and novel generation antifolate drugs. Upon stimulation with αCD3/CD28, polyclonal expansion of T-cells was observed microscopically between 48 and 72 hours along with a 1.5-2 fold increase in cell counts. For all antifolates tested, we observed a dose dependent decrease in CD25+ cells, which was partially independent of apoptosis (Figure 4; white bars). Furthermore, we observed apoptosis in up to 70% of activated (CD25+) T-cells (Figure 4; dark grey bars), but not in resting (CD25-) T-cells (Figure 4; light grey bars). To verify the role of apoptosis in inhibition of TNFα production by activated T-cells, we calculated the level of apoptosis at the IC- 50 concentration for TNFα inhibition in RA whole-blood for all nine antifolate drugs. As shown in Figure 5, at the IC-50 value for inhibition of TNFα production, 20-40% apoptosis was observed in activated (CD25+) T-cells, except for PT523, exhibiting a more apoptosis- unrelated anti-inflammatory effect. Addition of the natural reduced folate leucovorin (2 µM) (partially) prevented apoptosis induction and inhibition of T-cell activation by MTX,

136 137

chapter 8 Anti-inflammatory properties of novel generation antifolates

rs

de

-respon

rs non

X- de

MT

-respon

X

T CD25+ cells Apoptosis CD25- cells Apoptosis CD25+ cells Apoptosis CD25+ cells CD25+ cells Apoptosis CD25- cells Apoptosis CD25+ cells

CD25+ cells Apoptosis CD25- cells

M

aive

X-n

PT644 MT

Pemetrexed

AG2037

l

a

ot

-t

A R

33 + LV

300 + LV 300 + LV

ls

ro

nt

o 33

300 300 I C F C 0 0 0

75 50 25 50

125 100 350 300 250 200 150 100 750 500 250

inhibition IC-50 (nM) IC-50 inhibition (nM) IC-50 inhibition TNF TNF 1250 1000 α α

inhibition IC-50 (nM) IC-50 inhibition TNF α 11 100 100 [PT644] (nM) [AG2037] (nM) [Pemetrexed] (nM)[Pemetrexed] 33 33 3.7 0 0 0 I C F

0 0 0 8

80 60 40 20 80 60 40 20 80 60 40 20

% of cells of % cells of % cells of %

rs 8

de

-respon

rs non

X- de

MT

-respon

X

T

33 + LV 33 + LV

M 300 + LV

aive X-n

PT523

MT 33 33 Raltitrexed

300

BGC9331

l

a

ot

-t A

release are depicted as IC-50 value for each patient/control. Horizontal lines R

11 11

100 α

ls

ro [PT523] (nM)

nt [BGC9331] (nM) [Raltitrexed] (nM) o C 33 3.7 3.7 E H B 0 0 0

75 50 25 75 50 25 50

H_8_Figure_3.pzf:Layout 1 - Fri Sep05Fri 14:29:55 H_8_Figure_3.pzf:Layout- 1200

125 100 150 125 100 450 400 350 300 250 200 150 100

inhibition IC-50 (nM) IC-50 inhibition (nM) IC-50 inhibition TNF (nM) IC-50 inhibition TNF TNF CD28 for 72 hours in the absence or presence of MTX and novel generation antifolate α α α α 0 0 0 H_8_Figure_4.pzf:Layout 1 - Fri Sep05Fri 14:30:25 - H_8_Figure_4.pzf:Layout1200 H B E

0 0 0

CD3/ 80 60 40 20 80 60 40 20 80 60 40 20

% of cells of % cells of % cells of %

α

rs de

release from activated T-cells in whole blood cell cultures by antifolate drugs. -respon α

33 + LV

100 + LV

rs non + LV 300

X- de MT 33

100

300

-respon

X

T

M aive MTX

TMQ X-n

33 11

100 MT

GW1843

l (nM) [MTX]

[TMQ] (nM) a

ot [GW1843] (nM)

-t

A

11 R 33 3.7

Inhibition of T-cell activation and apoptosis induction in PBMCs of RA patients by antifolate drugs.

ls

ro

nt 0 0

0 o C A D G 0 0 0 A G D 50 75 50 25

0 0 0

500 400 300 200 100 350 300 250 200 150 100 125 100

80 60 40 20 80 60 40 20

80 60 40 20

inhibition IC-50 (nM) IC-50 inhibition TNF inhibition IC-50 (nM) IC-50 inhibition TNF inhibition IC-50 (nM) IC-50 inhibition TNF % of cells of % cells of % % of cells of % α α α Figure 4: PBMCs of RA patients were stimulated with drugs. FACS analysis was used to monitor T-cell activation (CD25 expression) and apoptosisdrugs inductioninclude; (Annexin-V/7AADDHFR inhibitors: staining). Tested (A) MTX, (B) PT523, (C) BGC9331PT644, and the GARTFase (D) inhibitor:TMQ; AG2037. TS(I) inhibitors: (E) Raltitrexed, (F) WhitePemetrexed, bars (G) GW1843,represent (H) the percentage of activated7AAD+) cells (CD25+) inT-cells; the LightCD25- cell grey population;bars Darkrepresent grey barsthepopulation. showpercentage At thethe highest percentage ofdrug-concentrations, apoptoticof apoptotic reversibility(Annexin-V+/ (Annexin-V+/7AAD+) of apoptosis cells was testedResultsin by depictedadditionthe ofrepresentCD25+ 2 µM ofcell the the natural mean ± S.D. folate of 3 separate leucovorin. experiments. Figure 3: Inhibition of TNF Drug concentrations (nM) establishing 50% inhibition of TNF representmedian values. IC-50 Tested drugs include DHFR inhibitors: MTX, (A) PT523,PT644, (C) (B) TMQ; TS(D) inhibitors: Raltitrexed,(E) (F) Pemetrexed, GW1843, (G) BGC9331 (H) and the GARTFase inhibitor: AG2037. (I)

138 139 chapter 8 Anti-inflammatory properties of novel generation antifolates

Discussion observed in Figure 4, revealing that addition of leucovorin rescued activated T-cells from apoptosis induced by MTX, TMQ, Raltitrexed, Pemetrexed and to some extend PT523, The current study demonstrates that selected novel generation folate antagonists (25) whereas the efficacy of PT644, BGC9331 and GW1843 was not influenced by this natural share with MTX the ability to exert potential anti-inflammatory effects by suppressing folate. On top of high folate levels, exogenous purines may bypass inhibitory effects of the release of TNFα by activated T-cells in whole blood from RA patients. Notably, this AICARTFase and GARTFase inhibitors (40). As a result, the presence of exogenous purines/ property is observed at low drug dosages and continuous exposure (72 hours) for: (a) non- pyrimidines in whole blood samples of RA patients could have attenuated the efficacy of polyglutamatable DHFR inhibitors (TMQ, PT523 and PT644), (b) folate-based TS inhibitors; the GARTFase inhibitor AG2037 in this study, as shown in Figure 4. both polyglutamatable (Raltitrexed, Pemetrexed, GW1843) and non-polyglutamatable (BGC9331), and (c) a folate-based GARTFase inhibitor (AG2037). Moreover, the current From the above considerations, it is anticipated that an enhanced therapeutic effect may study indicates that induction of apoptosis in activated T-cells by antifolate drugs be elicited from novel antifolates that have the following properties: (a) high RFC affinity explains at least part of the the anti-inflammatory properties, as described previously for and (b) being less influenced in their activity by fluctuations in the patient’s folate status. MTX (11). Finally, we observed attenuation of T-cell activation at low concentrations of The drugs identified from this study that harbor these features are PT644, GW1843 and anti-folate drugs, an effect that was partially unrelated to apoptosis induction. This latter BGC9331. With respect to the non-polyglutamatable folate antagonists (PT523, PT644 and observation might be supportive for additional mechanisms of action conveyed by MTX BGC9331), it should be taken into account that continuous drug exposure will be required and other anti-folate drugs (7, 9, 15). for optimal activity of these drugs, as these drugs may otherwise be rapidly effluxed from cells when a short term exposure is followed by a drug-free period (17, 25, 41). Where T- We observed a marked inter-patient variability in anti-inflammatory activity of novel cells almost exclusively depend on RFC for cellular uptake of novel antifolate drugs, it antifolates, which could not be related to differences in basal levels of TNFα production should be mentioned that alternative folate transport routes are operative in activated or clinical response to MTX treatment. However, the whole-blood assay (32) proved to macrophages in RA synovial tissue. These activated macrophages utilize a folate-receptor be an useful clinical mimick to assess differential potencies of drugs as described here for (FR) mediated uptake mechanism (42). Given the marked differences in affinities for a series of folate antagonists. Nonetheless, it should be anticipated that besides T-cells, RFC and FR, novel antifolates with a good affinity for RFC are not necessarily effective monocytes/macrophages are contributors to TNFα production in this assay, as they get against FR expressing activated macrophages. From this perspective GW1843, having a stimulated by cytokines (like IL-17) produced by activated T-cells (33). high affinity for both RFC and FR, may be an effective drug for targeting both T-cells and activated synovial tissue macrophages (J.W. van der Heijden, manuscript accepted for Four folate antagonists (PT523, PT644, raltitrexed and GW1843) could be identified with publication in Arthritis & Rheumatism 2008). a greater potency (up to 10-fold) than MTX to inhibit TNFα production. Since the RFC is the dominant folate antagonist transport route in T-cells, at least part of the enhanced In light of one of the proposed mechanisms of action of MTX in RA, i.e. via AICARTFase activity by these novel folate antagonists over MTX can be attributed to their more inhibition, adenosine release and adenosine receptor stimulation (5, 43), it is interesting efficient cellular uptake via this transporter (18, 19, 34, 35). to note that also non-polyglutamatable folate antagonists displayed potential anti- inflammatory effects. By definition, non-polyglutamatable DHFR inhibitors such as TMQ, One important parameter for the efficacy of folate antagonists ex-vivo and in-vivo is the PT523 and PT644 can not be converted to polyglutamate forms and consequently do not extracellular and intracellular folate status, as natural folates may compete for cellular directly target AICARTFase. This suggests that their mechanism of action is probably uptake and polyglutamylation, respectively (36). The wide-spread application of folate indirect via dihydrofolate that is built up due to DHFR inhibition (Figure 2), whereby supplementation to RA patients (37) along with folate food fortification programmes of polyglutamate forms of dihydrofolate are potent inhibitors of AICARTFase. Consistent with the past decade resulted in markedly higher plasma folate levels in RA patient populations this notion may be the potential anti-inflammatory properties of non-polyglutamatable (38), which could confer diminished efficacy of MTX. Beyond MTX, the activity of the DHFR inhibitors such as MX-68 in animal models of arthritis (44) and CH-1054 in a controlled TS inhibitor pemetrexed and the lipophilic DHFR inhibitor TMQ was also found to be clinical trial for RA treatment (45). Interestingly, the non-polyglutamatable and specific TS influenced by changes in folate status, while other drugs (e.g. BGC9331 and GW1843) retain inhibitor ZD9331 (46) also exerted, at low dosages, potent TNFα inhibitory effects. While potent activity regardless of folate status (22, 36, 39). Consistently, such a profile was also this drug does not convey any inhibitory effects on AICARTFase, this finding suggests that

140 141 chapter 8 Anti-inflammatory properties of novel generation antifolates

beyond inhibition of AICARTFase, inhibition of other key-enzymes in the folate metabolic Supplementary figure pathway such as TS also facilitates a potential anti-inflammatory response.

Conclusion

The present study indicates that novel generation antifolates, in particular PT523, PT644, Raltitrexed and GW1843, elicit potential anti-inflammatory effects at low dosages. Even though the clinically accepted good window for MTX efficacy could hold back a timely introduction of these novel generation folate antagonists, some of their superior pharmacological properties over MTX may be exploited in future RA treatment. This option may particularly hold for clinical conditions of MTX resistance (17) or in strategies to improve RA remission. In this context, some recently described prediction models for MTX efficacy and toxicity in RA treatment (47-50) may also be helpful to assess the potential clinical value of second/third generation of folate-based therapeutic drugs. Furthermore many of the drugs presented in this study have been registered for application as anti- cancer drugs, with known efficacy and toxicity profiles (25).

Chemical strucures of folic acid, leucovorin, methotrexate and novel generation anti-folate drugs.

142 143 References References

References resistance to methotrexate in acute leukemia, N.Engl.J.Med., 335: 1041-1048, 1996. 22 Jansen,G., Mauritz,R., Drori,S., Sprecher,H., Kathmann,I., Bunni,M., Priest,D.G., Noordhuis,P., 1 Bertino,J.R. Karnofsky memorial lecture. Ode to methotrexate, J.Clin.Oncol., 11: 5-14, 1993. Schornagel,J.H., Pinedo,H.M., Peters,G.J. and Assaraf,Y.G. A structurally altered human reduced 2 Kremer,J.M. Toward a better understanding of methotrexate, Arthritis Rheum., 50: 1370-1382, folate carrier with increased folic acid transport mediates a novel mechanism of antifolate 2004. resistance, J.Biol.Chem., 273: 30189-30198, 1998. 3 Peters,G.J. and Jansen,G. Antimetabolites. In: R. L. Souhami, I. Tannock and P. H. J. C. Hohenberger 23 Hooijberg,J.H., Broxterman,H.J., Kool,M., Assaraf,Y.G., Peters,G.J., Noordhuis,P., Scheper,R.J., (eds.), Oxford textbook of Oncology, pp. 663-713. Oxford University Press: Oxford, 2001. Borst,P., Pinedo,H.M. and Jansen,G. Antifolate resistance mediated by the multidrug resistance 4 Cutolo,M., Sulli,A., Pizzorni,C., Seriolo,B. and Straub,R.H. Anti-inflammatory mechanisms of proteins MRP1 and MRP2, Cancer Res., 59: 2532-2535, 1999. methotrexate in rheumatoid arthritis, Ann.Rheum.Dis., 60: 729-735, 2001. 24 Peters,G.J. and Jansen,G. Folate homeostasis and antiproliferative activity of folates and 5 Cronstein,B.N. Low-dose methotrexate: a mainstay in the treatment of rheumatoid arthritis, antifolates, Nutrition, 17: 737-738, 2001. Pharmacol.Rev., 57: 163-172, 2005. 25 Walling,J. From methotrexate to pemetrexed and beyond. A review of the pharmacodynamic 6 Genestier,L., Paillot,R., Quemeneur,L., Izeradjene,K. and Revillard,J.P. Mechanisms of action of and clinical properties of antifolates, Invest New Drugs, 24: 37-77, 2006. methotrexate, Immunopharmacology, 47: 247-257, 2000. 26 Jackman,A.L., Taylor,G.A., Gibson,W., Kimbell,R., Brown,M., Calvert,A.H., Judson,I.R. and 7 Cronstein,B.N. Molecular therapeutics. Methotrexate and its mechanism of action, Arthritis Hughes,L.R. ICI D1694, a quinazoline antifolate thymidylate synthase inhibitor that is a potent Rheum., 39: 1951-1960, 1996. inhibitor of L1210 tumor cell growth in vitro and in vivo: a new agent for clinical study, Cancer 8 Cronstein,B.N., Naime,D. and Ostad,E. The antiinflammatory mechanism of methotrexate. Res., 51: 5579-5586, 1991. Increased adenosine release at inflamed sites diminishes leukocyte accumulation in an in vivo 27 Rhee,M.S., Galivan,J., Wright,J.E. and Rosowsky,A. Biochemical studies on PT523, a potent model of inflammation, J.Clin.Invest, 92: 2675-2682, 1993. nonpolyglutamatable antifolate, in cultured cells, Mol.Pharmacol., 45: 783-791, 1994. 9 Riksen,N.P., Barrera,P., van den Broek,P.H., van Riel,P.L., Smits,P. and Rongen,G.A. Methotrexate 28 Rosowsky,A., Wright,J.E., Vaidya,C.M., Bader,H., Forsch,R.A., Mota,C.E., Pardo,J., Chen,C.S. and modulates the kinetics of adenosine in humans in vivo, Ann.Rheum.Dis., 65: 465-470, 2006. Chen,Y.N. Synthesis and potent antifolate activity and cytotoxicity of B-ring deaza analogues of 10 Fairbanks,L.D., Ruckemann,K., Qiu,Y., Hawrylowicz,C.M., Richards,D.F., Swaminathan,R., the nonpolyglutamatable dihydrofolate reductase inhibitor Nalpha-(4-amino-4-deoxypteroyl)- Kirschbaum,B. and Simmonds,H.A. Methotrexate inhibits the first committed step of purine Ndelta-hemiphthaloyl- L-ornithine (PT523), J.Med.Chem., 41: 5310-5319, 1998. biosynthesis in mitogen-stimulated human T-lymphocytes: a metabolic basis for efficacy in 29 Duch,D.S., Banks,S., Dev,I.K., Dickerson,S.H., Ferone,R., Heath,L.S., Humphreys,J., Knick,V., rheumatoid arthritis?, Biochem.J., 342: 143-152, 1999. Pendergast,W., Singer,S. and . Biochemical and cellular pharmacology of 1843U89, a novel 11 Genestier,L., Paillot,R., Fournel,S., Ferraro,C., Miossec,P. and Revillard,J.P. Immunosuppressive benzoquinazoline inhibitor of thymidylate synthase, Cancer Res., 53: 810-818, 1993. properties of methotrexate: apoptosis and clonal deletion of activated peripheral T cells, J.Clin. 30 Jackman,A.L., Kimbell,R., Aherne,G.W., Brunton,L., Jansen,G., Stephens,T.C., Smith,M.N., Invest, 102: 322-328, 1998. Wardleworth,J.M. and Boyle,F.T. Cellular pharmacology and in vivo activity of a new anticancer 12 Herman,S., Zurgil,N. and Deutsch,M. Low dose methotrexate induces apoptosis with reactive agent, ZD9331: a water-soluble, nonpolyglutamatable, quinazoline-based inhibitor of oxygen species involvement in T lymphocytic cell lines to a greater extent than in monocytic thymidylate synthase, Clin.Cancer Res., 3: 911-921, 1997. lines, Inflamm.Res., 54: 273-280, 2005. 31 Boritzki,T.J., Barlett,C.A., Zhang,C. and Howland,E.F. AG2034: a novel inhibitor of glycinamide 13 Phillips,D.C., Woollard,K.J. and Griffiths,H.R. The anti-inflammatory actions of methotrexate ribonucleotide formyltransferase, Invest New Drugs, 14: 295-303, 1996. are critically dependent upon the production of reactive oxygen species, Br.J.Pharmacol., 138: 32 Gerards,A.H., de Lathouder, S., De Groot,E.R., Dijkmans,B.A.C. and Aarden,L.A. Inhibition 501-511, 2003. of cytokine production by methotrexate. Studies in healthy volunteers and patients with 14 Winter-Vann,A.M., Kamen,B.A., Bergo,M.O., Young,S.G., Melnyk,S., James,S.J. and Casey,P.J. rheumatoid arthritis, Rheumatology.(Oxford), 42: 1189-1196, 2003. Targeting Ras signaling through inhibition of carboxyl methylation: an unexpected property of 33 McInnes,I.B. and Schett,G. Cytokines in the pathogenesis of rheumatoid arthritis, Nat.Rev. methotrexate, Proc.Natl.Acad.Sci.U.S.A, 100: 6529-6534, 2003. Immunol., 7: 429-442, 2007. 15 Majumdar,S. and Aggarwal,B.B. Methotrexate suppresses NF-kappaB activation through 34 Westerhof,G.R., Schornagel,J.H., Kathmann,I., Jackman,A.L., Rosowsky,A., Forsch,R.A., Hynes,J. inhibition of IkappaBalpha phosphorylation and degradation, J.Immunol., 167: 2911-2920, 2001. B., Boyle,F.T., Peters,G.J., Pinedo,H.M. and Jansen,G. Carrier- and receptor-mediated transport of 16 Wolfe,F. The epidemiology of drug treatment failure in rheumatoid arthritis, Baillieres Clin. folate antagonists targeting folate-dependent enzymes: correlates of molecular structure and Rheumatol., 9: 619-632, 1995. biological activity., Mol.Pharmacol., 48: 459-471, 1995. 17 Van der Heijden,J.W., Dijkmans,B.A.C., Scheper,R.J. and Jansen,G. Drug Insight: resistance to 35 Jansen,G. Receptor- and carrier-mediated transport systems for folates and antifolates, In: methotrexate and other disease-modifying antirheumatic drugs-from bench to bedside, Nat. Anticancer Drug Development Guide: Antifolate Drugs in Cancer Therapy (A.L.Jackman, ed), Clin.Pract.Rheumatol., 3: 26-34, 2007. Humana Press Inc., Totowa, NJ, 293-321, 1999. 18 Assaraf,Y.G. Molecular basis of antifolate resistance, Cancer Metastasis Rev., 26: 153-181, 2007. 36 Jansen,G., Barr,H., Kathmann,I., Bunni,M.A., Priest,D.G., Noordhuis,P., Peters,G.J. and Assaraf,Y.G. 19 Zhao,R. and Goldman,I.D. Resistance to antifolates, Oncogene, 22: 7431-7457, 2003. Multiple mechanisms of resistance to polyglutamatable and lipophilic antifolates in mammalian 20 Rots,M.G., Pieters,R., Kaspers,G.J., Veerman,A.J., Peters,G.J. and Jansen,G. Classification of ex cells: role of increased folylpolyglutamylation, expanded folate pools, and intralysosomal drug vivo methotrexate resistance In acute lymphoblastic and myeloid leukaemia, Br.J.Haematol., sequestration, Mol.Pharmacol., 55: 761-769, 1999. 110: 791-800, 2000. 37 Whittle,S.L. and Hughes,R.A. Folate supplementation and methotrexate treatment in 21 Gorlick,R., Goker,E., Trippett,T., Waltham,M., Banerjee,D. and Bertino,J.R. Intrinsic and acquired rheumatoid arthritis: a review, Rheumatology.(Oxford), 43: 267-271, 2004.

144 145 References References

38 Arabelovic,S., Sam,G., Dallal,G.E., Jacques,P.F., Selhub,J., Rosenberg,I.H. and Roubenoff,R. Preliminary evidence shows that folic acid fortification of the food supply is associated with higher methotrexate dosing in patients with rheumatoid arthritis, J.Am.Coll.Nutr., 26: 453-455, 2007. 39 Zhao,R., Gao,F. and Goldman,I.D. Marked suppression of the activity of some, but not all, antifolate compounds by augmentation of folate cofactor pools within tumor cells, Biochem. Pharmacol., 61: 857-865, 2001. 40 Rots,M.G., Pieters,R., Kaspers,G.J., Van Zantwijk,C.H., Noordhuis,P., Mauritz,R., Veerman,A.J., Jansen,G. and Peters,G.J. Differential methotrexate resistance in childhood T- versus common/ pre B-acute lymphoblastic leukemia can be measured by an in situ thymidylate synthase inhibition assay, but not by the MTT assay, Blood, 93: 1067-1074, 1999. 41 Assaraf,Y.G. The role of multidrug resistance efflux transporters in antifolate resistance and folate homeostasis, Drug Resist.Updat., 9: 227-246, 2006. 42 Nakashima-Matsushita,N., Homma,T., Yu,S., Matsuda,T., Sunahara,N., Nakamura,T., Tsukano,M., Ratnam,M. and Matsuyama,T. Selective expression of folate receptor beta and its possible role in methotrexate transport in synovial macrophages from patients with rheumatoid arthritis, Arthritis Rheum., 42: 1609-1616, 1999. 43 Tian,H. and Cronstein,B.N. Understanding the mechanisms of action of methotrexate: implications for the treatment of rheumatoid arthritis, Bull.NYU.Hosp.Jt.Dis., 65: 168-173, 2007. 44 Montesinos,M.C., Desai,A., Delano,D., Chen,J.F., Fink,J.S., Jacobson,M.A. and Cronstein,B.N. Adenosine A2A or A3 receptors are required for inhibition of inflammation by methotrexate and its analog MX-68, Arthritis Rheum., 48: 240-247, 2003. 45 Castaneda,O. and Nair,M.G. Controlled trial of methotrexate versus CH-1504 in the treatment of rheumatoid arthritis, J.Rheumatol., 33: 862-864, 2006. 46 Jackman,A.L., Melin,C.J., Kimbell,R., Brunton,L., Aherne,G.W., Theti,D.S. and Walton,M. A rationale for the clinical development of the thymidylate synthase inhibitor ZD9331 in ovarian and other solid tumours, Biochim.Biophys.Acta, 1587: 215-223, 2002. 47 Dervieux,T., Greenstein,N. and Kremer,J. Pharmacogenomic and metabolic biomarkers in the folate pathway and their association with methotrexate effects during dosage escalation in rheumatoid arthritis, Arthritis Rheum., 54: 3095-3103, 2006. 48 Weisman,M.H., Furst,D.E., Park,G.S., Kremer,J.M., Smith,K.M., Wallace,D.J., Caldwell,J. R. and Dervieux,T. Risk genotypes in folate-dependent enzymes and their association with methotrexate-related side effects in rheumatoid arthritis, Arthritis Rheum., 54: 607-612, 2006. 49 Wessels,J.A., De Vries-Bouwstra,J.K., Heijmans,B.T., Slagboom,P.E., Goekoop-Ruiterman,Y. P., Allaart,C.F., Kerstens,P.J., van Zeben,D., Breedveld,F.C., Dijkmans,B.A.C., Huizinga,T.W. and Guchelaar,H.J. Efficacy and toxicity of methotrexate in early rheumatoid arthritis are associated with single-nucleotide polymorphisms in genes coding for folate pathway enzymes, Arthritis Rheum., 54: 1087-1095, 2006. 50 Wessels,J.A., van der Kooij,S.M., le Cessie S., Kievit,W., Barerra,P., Allaart,C.F., Huizinga,T.W. and Guchelaar,H.J. A clinical pharmacogenetic model to predict the efficacy of methotrexate monotherapy in recent-onset rheumatoid arthritis, Arthritis Rheum., 56: 1765-1775, 2007.

146 147 chapter 9 Folate receptor-β as potential delivery route for novel folate antagonists to macrophages in synovial tissue of rheumatoid arthritis patients

Joost W van der Heijden1, Ruud Oerlemans1, Ben AC Dijkmans1, Huiling Qi, Conny van der Laken1, Willem F Lems1, Ann L Jackman2, Maarten C Kraan3, Paul P Tak3, Manohar Ratnam4 and Gerrit Jansen1

1Department of Rheumatology, VU University Medical Center, Amsterdam, The Netherlands, 2Institute of Cancer Research, Sutton, Surrey, United Kingdom, 3Division of Clinical Immunology and Rheumatology, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands and 4Department of Biochemistry and Cancer Biology, The University of Toledo, Toledo (OH), U.S.A.

Accepted for publication in Arthritis & Rheumatism 2008 chapter 9 Targeting RA synovial macrophages via the FR-β

Abstract transport routes harbour physiological and pharmacological relevance for immune- competent cells by facilitating the uptake of natural reduced folate cofactors and folate Purpose: To determine (a) the expression of folate receptor-β (FR-β) in human synovial antagonists such as MTX (6). The RFC and MFR differ considerably in the mechanism of tissue biopsies and peripheral blood lymphocytes from rheumatoid arthritis (RA) patients (anti)folate uptake (transmembrane carrier vs. endocytosis), substrate specificity (low and (b) to identify novel folate antagonists being more selective in FR-β targeting and affinity folic acid/high affinity MTX vs. high affinity folic acid/low affinity MTX) and tissue internalization than methotrexate (MTX). specificity (constitutive vs. restricted expression). For MFR 3 isoforms (α, β and γ) have Methods: Immunohistochemistry and computer-assisted digital imaging was used for been identified. The α-isoform of MFR is overexpressed in specific types of cancer (e.g. the detection of FR-β protein expression on immune-competent cells in synovial biopsies ovarian cancer) while the γ-isoform is a secreted protein from haematopoietic cells (8). of RA patients with active disease and in non-inflammatory control synovial tissue. FR-β Interestingly, selective expression of the FR-β isoform has been described on activated mRNA levels were determined by RT-PCR. Binding affinities of FR-β for folate antagonists macrophages in inflamed synovial fluid of RA patients (11) and animal models of arthritis were assessed by competition experiments for [3H]-folic acid binding on FR-β transfected (12, 13). Subsequently, FR-β was recognized as an attractive target for imaging of arthritis cells. Efficacy of FR-β-mediated internalization of folate antagonists was evaluated by and therapeutically for selective antibody-guided or folate-conjugate guided delivery assessment of anti-proliferative effects against FR-β transfected cells. of toxins and other small/macro-molecules (13-15). Thus far, targeting of FR with folate Results: Immunohistochemical staining of RA synovial tissue showed high expression of antagonists has only been explored on cancer cells/tissues overexpressing FR-α (16, 17). FR-β on macrophages in the intimal lining layer and synovial sublining, while no staining was observed in T-cell areas or control synovial tissue. Consistently, FR-β mRNA levels Over the past two decades, a second generation of folate antagonists has been designed were highest in synovial tissue extracts and RA monocyte-derived macrophages, but and clinically evaluated from a perspective of circumventing common mechanisms of low in peripheral blood T-cells and monocytes. Screening of 10 novel generation folate resistance to MTX, including impaired transport via the RFC, defective polyglutamylation, antagonists revealed 4 compounds for which FR-β had a high binding affinity (20-77 fold increased activity of the target enzyme DHFR and/or enhanced drug efflux (7). Based on this higher than for MTX). One of these, the thymidylate synthase inhibitor BGC945, displayed background, second generation antifolates include compounds that are more efficiently selective targeting against FR-β transfected cells. transported via the RFC, are more efficiently polyglutamylated or being independent of Conclusion: Abundant FR-β expression on activated macrophages in synovial tissue polyglutamylation, or target other key enzymes in folate metabolism than DHFR, e.g. from RA patients deserves further exploration for selective therapeutic interventions thymidylate synthase (TS) or glycinamide ribonucleotide transformylase (6, 18, 19). In the with high affinity binding folate antagonists, of which BGC945 may be a prototypical present study we set out to investigate whether distinct second generation antifolates representative. may serve as selective targeting drugs for FR-β expressing cells in synovial tissue and/ or immune-competent cells of RA patients. We identified the TS inhibitor BGC945 as a prototypical antifolate drug that fulfilled the criteria of a high FR-β binding affinity and a Introduction low RFC affinity, thereby enabling selective uptake by FR-β expressing cells.

The folate antagonist methotrexate (MTX) is the most widely applied disease modifying antirheumatic drug (DMARD) in the treatment of patients with rheumatoid arthritis (RA) Materials and methods (1). It is used either as single agent or in combination with other DMARDs (e.g. sulfasalazine and hydroxychloroquine) and MTX use is obligate in most treatment strategies involving Drugs biological agents (anti-TNFα or anti-CD20 monoclonal antibodies) (2-5). Folic acid was obtained from Sigma Chem Co, St .Louis, Mo. L-Leucovorin was purchased from Merck Eprova, Schaffhausen Switzerland. Methotrexate was a kind gift of Pharmachemie, The first pivotal step in the cellular pharmacology of MTX is its cell entry, which can be Haarlem, The Netherlands. Pemetrexed/ALIMTA® (Eli Lilly) (20) was obtained via the mediated by at least 3 different routes; the reduced folate carrier (RFC) (6, 7), membrane- VUmc pharmacy department. The following folate antagonist drugs were obtained from associated folate receptors (MFR) (8, 9) or a proton-coupled (low pH) folate transporter the indicated companies/ institutions; Raltitrexed/Tomudex®/ZD1694 (21) (AstraZeneca, (PCFT) (10). The latter transporter is mainly involved in intestinal folate uptake, the other UK), PT523 and PT644 (22, 23) (Dr. A. Rosowsky, Harvard Medical School, Boston, MA),

150 151 chapter 9 Targeting RA synovial macrophages via the FR-β

GW1843 (24) (Glaxo-Welcome, USA), CB300635 (25) (Institute of Cancer Research, Sutton, University Medical Center, Amsterdam, The Netherlands. UK), Plevitrexed/BGC 9331 (26) and BGC 945 (6R,S and 6S) (27) (BTG International Ltd), 5,10- dideazatetrahydrofolate (DDATHF) (28) (Eli Lilly, Indianapolis, IN), and AG2034 (29) (Agouron/ FR-β immunohistochemistry Pfizer Pharmaceuticals, San Diego, Ca). The chemical structures of these folate antagonists Immunohistochemical staining of cryostat sections (5 µm) of synovial tissue biopsies are provided in Supplementary Figure. Preclinical and clinical background information on from RA patients and controls was performed using a 3-step immunoperoxidase method these antifolates is published in (6, 18). [3’,5’,7,9-3H]-folic acid (20-40 Ci/mmol, MT783) was as described previously (33, 34). Sections were stained with a specific antibody for FR- purchased from Moravek, Brea, Ca. β (30) (dilution 1: 3000) (isotype control: normal rabbit-serum). Macrophages and T- cells were stained with 3A5 (dilution 1: 100) (35) and anti-CD3-PE (dilution 1: 25; Dako, Cell lines Glostrup, Denmark) monoclonal antibodies (isotype control: mouse immunoglobulin), Wild type Chinese Hamster Ovary (CHO/WT) cells, CHO cells transfected with FR-β (CHO/ respectively. Biotinylated swine anti-rabbit IgG (Dako; dilution 1:200) and rabbit anti- FR-β) (30) and human nasopharyngeal epidermoid KB cells, expressing FR-α (17) (American mouse IgG (Dako; dilution 1:300) were used as secondary antibodies. Color development Type Culture Collection, Manassas, VA) were grown in folic-acid free RPMI 1640 medium was performed using 0.4 mg/ml AEC (aminoethyl carbazole). After counterstaining with (Gibco, Grand Island, NY), supplemented with 10% fetal calf serum, 2 mM L-glutamine, 0.15 hematoxylin, slides were mounted. Stained sections were analysed for FR-β, 3A5 and CD3 mg/ml proline and 100 units/ml penicillin and streptomycin. [3H]-folic acid binding capacities expression by digital image analysis, as described previously (36). In short, for each marker of CHO/FR-β and KB cells were 0.5-1 pmol/106 cells and 20-40 pmol/106 cells, respectively. representative regions were used for image acquisition, using 400x magnification. These Human monocytic-macrophage THP1 cells (American Type Culture Collection, Manassas, regions were divided into 6 high-power fields (hpfs) with a 3-pixel overlap. Positive cells VA) were grown in RPMI 1640 medium (Gibco, Grand Island, NY) containing 2.2 µM folic were evaluated by analysing 18 consecutive hpfs, scoring numbers of positive cells in the acid, supplemented with 10% fetal calf serum, 2 mM L-glutamine, 0.15 mg/ml proline and intimal lining layer and the synovial sublining per mm2. 100 units/ml penicillin and streptomycin. All cell lines were kept at 37°C in a humidified

atmosphere containing 5% CO2 Double-labeling immunofluorescence FR-β was detected by swine-anti-rabbit HRP-labelled antibodies (dilution 1:200; Dako, Synovial tissue samples Glostrup, Denmark) and development was with rhodamine/thyramine (red fluorescence) In this study we analyzed synovial tissue biopsies derived from the knee joints of 15 RA- according to the instructions of the manufacturer (dilution 1:1000) (37). CD68 was detected patients with active disease before treatment and after 4 months of treatment with MTX by goat-anti-mouse biotinylated antibodies (dilution 1:100; Dako, Glostrup, Denmark) (starting dose of MTX: 7,5 mg/week; increasing stepwise to 15 mg/week over 12 weeks). utilizing streptavidin Alexa-488 as a substrate (dilution 1:750; green fluorescence; Molecular Active disease was defined as ≥ 6 swollen or tender joints and levels of moderate or worse Probes, Eugene, OR). Slides were mounted with Vectashield, containing 1 µg/ml DAPI (for on the physician’s and patient’s assessments of disease activity (Disease Activity Score-28; staining of nuclei) (Vector laboratories Inc., Burlingame, CA). Cells were examined using a DAS-28). All patients had at least 1 clinically involved knee joint. Concomitant use of low fluorescence microscope (Leica DMRB, Rijswijk, the Netherlands). dose prednisone (< 10 mg/day) and stable doses of nonsteroidal anti-inflammatory drugs (NSAID) was allowed. None of the patients ever used MTX before enrolling the study. In Isolation of peripheral blood cells of RA-patients and culture conditions patients taking other DMARDs, the treatment was terminated following a washout phase Peripheral blood mononuclear cells were isolated from freshly obtained blood samples of 28 days. The arthroscopy procedure was performed as described previously (31) as part of by gradient centrifugation (35’ at 400xg) on Ficoll-Paque Plus (Amersham Pharmacia a joined study that was approved by the Medical Ethics committees of the Leiden University Biotechnologies, UK). After centrifugation the interphase was carefully collected and Medical Center (The Netherlands) and the Leeds University Medical Center (United washed 3 times using Phosphate Buffered Salt solution (PBS) supplemented with 1% Kingdom) (32). As non-inflammatory control synovial tissue we included 7 samples from BSA. The lymphocyte fraction was counted and resuspended in IMDM culture medium patients with mechanical joint injury, kindly provided by dr. B.J. van Royen, department (Invitrogen, Breda, The Netherlands) which contained 10% FCS, 2 mM L-glutamine and 100 of Orthopedic Surgery, VU University Medical Center, Amsterdam, The Netherlands. For µg/ml penicillin and streptomycin. Monocytes were isolated by adherence after 2 hours peripheral blood sample collection, all patients signed an informed consent form and the incubation at 37o C in culture flasks followed by RNA extraction or macrophage differentiation study on ‘DMARD resistance’ was approved by the Medical Ethics committee of the VU by culturing the monocytes for 7 days in the presence of 10 ng/ml Macrophage Colony

152 153 chapter 9 Targeting RA synovial macrophages via the FR-β

Stimulating Factor (M-CSF) (Strathmann Biotech, Hamburg, Germany). Peripheral blood expressing cells) were detached by incubation in PBS + 1 mM EDTA. Detached cells were lymphocytes (PBLs) remaining in suspension after monocyte adherence were collected for suspended in ice-cold HEPES-buffered saline (140 mM NaCl, 20 mM HEPES, 6 mM KCl, 2

6 6 RNA isolation or used for T-cell activation by incubating them at a density of 1·10 cells/ml mM MgCl2, 6 mM D-glucose, pH 7.4 with NaOH) to a cell concentration of 3 x 10 and 1 x with monoclonal anti-CD28 (5 µg/ml, CLB-CD28/1, Sanquin, Amsterdam, The Netherlands) 106 cells/ml, respectively. One ml of cell suspension was added to a series of Eppendorf and anti-CD3 (1 µg/ml, CLB-T3/4.E, Sanquin, The Netherlands) in goat-anti-mouse (Dako, tubes containing 100 pmol [3H]-folic acid (specific radioactivity: 2,000 dpm/pmol) in the Glostrup, Denmark) coated 24 well plates. After 48 hours stimulation, activated T-cells absence or presence of increasing concentrations of natural folate or folate antagonists. were harvested for RNA isolation and the activation status was determined by measuring Following 10 minutes at 4°C, cells were centrifuged in an Eppendorf centrifuge (30 sec CD25 expression using flow-cytometry (FACScalibur, Beckton & Dickinson). 10,000 rpm), the supernatant was aspirated and cell pellets were resuspended in 200 µl water and analyzed for radioactivity (Optima Gold scintillation fluid, United Technologies, FR-β mRNA expression in synovial tissue and peripheral blood cells of RA-patients Packard, Brussels, Belgium). Non-specific binding of [3H] (usually < 2% of specific binding) RNA from synovial tissue (n=7), PBLs (n=9), monocytes (n=9), macrophages (n=25), and was determined by measuring cell-associated radioactivity in the presence of a 1000-fold activated T-cells (n=22) from RA patients was isolated using the Qiagen RNeasy Plus molar excess of unlabeled folic acid. Concentrations of natural folates and selected folate isolation kit (Qiagen, Venlo, The Netherlands) following the instructions provided by the antagonists required to displace 50% of [3H]-folic acid from FR-β or FR-α were determined manufacturer. Prior to RNA isolation, the frozen synovial tissue samples were powdered and presented as binding affinities relative to folic acid. For comparison, data for affinities by grinding them in a liquid nitrogen pre-chilled mortar where after RPE buffer was added. of the RFC for natural folates and folate antagonists were presented from previous studies Total RNA concentrations were determined using a Nanodrop ND-1000 spectrophotometer (16, 27). (Nanodrop Technologies, Wilmington, USA). Real-time reverse transcription-PCR (RT-PCR) methodology described previously by Qi et al (38) was used to measure simultaneously mRNA Cell proliferation inhibition assay levels for FR-β and glyceraldehyde-3-phosphate dehydrogenase (GAPDH; reference gene). CHO/WT and CHO/FRβ cells were seeded in individual wells of a 24-well tissue culture plate The reverse transcription step was carried out using Taqman Reverse Transcript Reagents at a density of 1 x 104/cm2. After 24 hours, 8 concentrations (with 2.5-fold increments) of from Applied Biosystems (Foster City, CA), following the protocol of the manufacturer. folate antagonist drugs were added in the absence or presence of 1 µM folic acid (to block FR) Briefly, 400 ng of total RNA was mixed with random hexamer primers (50 μmol/L), RNase or 20 nM leucovorin. After 72 hours incubation, cells were harvested by trypsinization and inhibitor (1 unit/μL), MultiScribe reverse transcriptase (5 units/μL), and deoxynucleoside counted for cell viability as described before (39). In other experiments human, monocytic- triphosphate mix (2.5 mmol/L each) in reverse transcriptase buffer. The 10-μL reaction macrophage THP1 cells were tested for antiproliferative effects of folate antagonists. To mixture was first incubated at 25°C for 10 minutes, then at 48°C for 30 minutes and finally this end, 1 ml of cell suspension containing 1.25 x 105 cells were plated in individual wells of a at 95°C for 5 minutes. The subsequent real-time PCR step for FR-β was carried out in the 24-well tissue culture plate and incubated with 8 concentrations (with 2.5-fold increments) presence of 12.5 μL of PCR Mastermix (Applied Biosystems), 0.5 μL each of forward primer of folate antagonist drugs. After 72 hours drug exposure, cell counts were performed with (CTGGCTCCTTGGCTG-AGTTC) and reverse primer (GCCCAGCCTGGTTATCCA), and 0.5 μL of a hemocytometer and viability was checked by trypan blue exclusion. Taqman probe (6FAM-TCCTCCCAGACTACCTGCCCTCAGC-TAMERA). The primers and the Taqman probe for control GAPDH gene were purchased from Applied Biosystems. The PCR conditions were 2 minutes at 50°C, 10 minutes at 95°C, followed by 40 cycles of 15 seconds Results each at 95°C, and finally 1 minute at 60°C. Fluorescence data generated were monitored and recorded by the Gene Amp 5700 sequence detection system (Applied Biosystems). All FR-β synovial tissue immunohistochemistry samples were set up in triplicate and normalized to GAPDH values. Immunohistochemical staining of all RA synovial tissue showed high expression of FR-β in the intimal lining layer as well as in the synovial sublining. The staining pattern for FR-β Analysis of FR-β/ FR-α and RFC binding affinity for novel generation folate antagonists was consistent with 3A5 (macrophage) staining, whereas T-cell areas showed no staining An intact cell binding assay for competitive binding of [3H]-folic acid and novel anti- (figure 1 A-C). In fact, more detailed fluorescence microscopic analysis demonstrated folate drugs to FR-β and FR-α was performed essentially as described previously (16, 39). co-localization of FR-β and CD68 (macrophage marker) on the cellular membranes of Briefly, CHO/FR-β cells (FR-β transfected Chinese Hamster Ovary cells) and KB cells (FR-α synovial tissue infiltrating macrophages and intimal macrophages (figure 1 D-F). In non-

154 155 H9_2a.pzf:Data 1 graph - Thu Jul 31 15:23:10 2008 H9_2b.pzf:Data 1 graph - Thu Jul 31 15:24:38 2008

chapter 9 Targeting RA synovial macrophages via the FR-β

Figure 2 A Figure 2 B Figure 1 700 350 R=0.64 R=0.32

2 600 p=0.004 2 300 p=0.12

500 250

400 200

300 150

200 100

FR-bpositive cells/mm 100 FR-bpositive cells/mm 50

0 0 0 100 200 300 400 500 600 700 -2 -1 0 1 2 3 4 3A5 positive cells/mm2 delta DAS28

Figure 2: (A) Correlation between 3A5 (macrophages) and FR-β positive cells/mm2 in synovial tissue of RA patients (n=15) before treatment with MTX. Median positive cell counts in the synovial sublining layer/ mm2 were 126 (range: 9-630) for FR-β and 219 (range: 11-622) for 3A5. Linear regression: R=0.64; p=0.004. (B) Correlation between DAS28 improvement (∆DAS28) and FR-β expression on macrophages (positive cells/ mm2) after 4 months treatment with MTX (R=0.32; p=0.12).

Figure 1: Immunohistochemistry of RA-synovial tissue: Light microscopy of (A) rabbit isotype staining, (B) FR-β staining and (C) macrophage staining (3A5). CHO-β cells) > peripheral blood lymphocytes (PBLs) (0.7%) >> monocytes (0.02%) and ex- Immunofluorescence staining of (D) FR-β, (E) CD68 (macrophages) and (F) merge of D and E. Of note: nuclei are stained blue (Dapi-staining). vivo activated T-cells (< 0.001%) (Figure 3).

Binding affinities of FR-β vs. FR-α for folate antagonists inflammatory synovial tissue of orthopedic controls, no staining for FR-β was observed, Binding affinities of FR-α for selected folate antagonists were previously reported (16) but consistent with low numbers of macrophages (not shown). to what extent they overlap or differ for the FR-β isoform has not been established. To this end, binding affinities (relative to folic acid) to FR-β versus FR-α for a series of folate- Staining results were analyzed by computer-assisted digital image analysis. A significant based inhibitors of DHFR, TS and GARTFase were determined and shown in Figure 4. correlation was found between 3A5 and FR-β expression in the synovial sublining layer (cell Compared to folic acid, both FR-β and FR-α displayed a rather low affinity for the group of counts/mm2) (p=0.04) (Figure 2A). Median positive cell counts in the synovial sublining DHFR inhibitors. Binding affinity of FR-β for MTX is approximately 50-fold lower than for layer/mm2 were 126 (range 9-630) for FR-β and 219 (range 11-622) for 3A5. Median numbers folic acid. The binding affinity for PT523 is markedly lower than for MTX (0.3% of folic acid) of macrophages decreased upon 4 months of treatment with MTX (from 219 to 119 positive while PT644, the 5-methyl analogue of PT523, showed an affinity comparable to MTX. Of cell counts in the synovial sublining layer/mm2, p=0.14). FR-β expression after 4 months note, FR-α exhibits a good binding affinity for all tested folate-based TS inhibitors, but of treatment with MTX was positively correlated (R=0.32) with DAS28 improvement binding affinities of FR-β for pemetrexed, raltitrexed and BGC9331 were markedly lower (∆DAS28), but did not reach statistical significance (p=0.12) (Figure 2B). (16-30 fold) than for FR-α. High binding affinity of FR-β was observed for the TS inhibitors CB300635 (161% of folic acid) and (6R,S)-BGC945 (89% of folic acid) and (6S)-BGC945 (46% FR-β mRNA expression in RA-synovial tissue and peripheral blood cells of RA patients of folic acid). FR-β also exhibited a proficient binding affinity for the GARTFase inhibitors To further confirm the differential expression of FR-β in synovial tissue, FR-β mRNA levels DDATHF (27% of folic acid) and AG2034 (54% of folic acid) even though FR-α binding were determined by PCR analysis in synovial tissue biopsies and compared to those in affinities for these compounds were 2.5-fold higher. Together, these results demonstrate peripheral blood cells of RA-patients, including lymphocytes, ex-vivo activated T-cells, a broad differential in binding affinities of FR-β for folate antagonists, among which peripheral blood monocytes and ex-vivo monocyte-derived macrophages. FR-β mRNA several folate antagonists revealed a markedly higher binding affinity compared to MTX. levels were presented relative to CHO/FR-β cells (30) (expression in these cells was set to 100%). Ranking of FR-β mRNA expression was highest for RA-synovial tissue (median of 17% Folate antagonist induced growth inhibition of FR-β expressing cells compared to CHO/FR-β cells) > ex-vivo monocyte-derived macrophages (3% compared to To investigate whether the folate antagonists used in the current study would convey a

156 157 H9_3.pzf:Data 1 graph - Thu Jul 31 15:22:39 2008

chapter 9 Targeting RA synovial macrophages via the FR-β

Figure 3 Figure 4 1000 Folic acid (+) L-leucovorin (++++)

100 MTX (+++)

DHFR- inhibitors PT523 (++++) PT644 (++++) 10 17% 3.4% GW1843 (++++)

Pemetrexed (++++) 1 %of expression inCHO/FR-b Raltitrexed (++++) MedianFR-b expression + range 0.7% BGC9331 (++++) TS-inhibitors <0.01 0.03% CB300635 (+/-) 0.1 BGC945 (6R,S) (+/-) e lls tes es u e y g s c c PBLs BGC945 (6S) (+/-) o ha . T- p t on ro Ac M c novial Tis Ma y S DDATHF (+++) GARTFase-inhibitors Figure 3: FR-β mRNA expression in synovial tissue and ex-vivo cultured RA peripheral mononuclear AG2034 (6S) (++++) cells (PBMC’s). 0.1 1 10 100 500 Median FR-β mRNA expression was determined in peripheral blood lymphocytes (PBLs; n=9), monocytes Relative affinity (% of Folic acid) (n=9), ex-vivo monocyte-derived macrophages (n=25), ex-vivo activated T-cells (n=22) and synovial tissue of RA patients (n=7). FR-β expression in these samples is shown as a percentage of the expression in CHO/FR-β Figure 4: Relative binding affinities of FR-α (white bars) and FR-β (grey bars) for novel generation cells (Chinese Hamster Ovarian cells transfected with FR-β; expression in these cells was set to 100%). folate antagonists. FR-α and FR-β binding affinities of the drugs were determined by [3H]-folic acid displacement on FR-α expressing KB-cells and CHO/FR-β cells as described in the Materials and Methods section. Binding affinities are presented as percentage relative to folic acid binding. Results are the mean ± SD of 3-5 separate experiments (SD< 30%). Table 1: Growth inhibitory effects of folate antagonists against human monocytic-macrophage For comparison, affinities of RFC for folate antagonists are presented as: ++++ (high affinity), +++ (moderate THP1 cells. affinity), ++ (low affinity), + (poor affinity), +/- (very poor affinity) based on previously published data (6, 16, 27).

Folate antagonist IC (nM) 50 potential growth inhibitory effects against macrophage-like type of cells, this parameter

MTX 7.1 ± 0.5 was investigated for human monocytic-macrophage THP1 cells (Table 1). Consistent with PT523 2.2 ± 0.3 RFC as the dominant transport route in THP1 cells, potent growth inhibitory effects were PT644 1.0 ± 0.4 observed for all folate antagonists, except CB300635 and BGC945, two compounds that

Raltitrexed 2.4 ± 0.5 have a poor affinity for RFC (25, 27). Pemetrexed 10.7 ± 2.2 Three folate antagonists for which FR-β displayed the highest binding affinity (CB300635, GW1843 1.6 ± 0.1 AG2034 and BGC945) were evaluated for their potency to target FR-β by provoking cell BGC9331 8.7 ± 0.8 CB300635 4850 ± 285 growth inhibition of CHO/FR-β cells. Against CHO/WT cells, BGC945 only induced growth BGC945 3630 ± 350 inhibition at extracellular concentrations of > 1000 nM (Figure 5A). Remarkably, growth inhibition of CHO/FR-β cells was induced at markedly lower concentrations (10-50 nM) of DDATHF 9.8 ± 0.8 BGC945. The addition of folic acid to these cell cultures completely abrogated the activity AG2034 3.2 ± 0.2 of BGC945, consistent with a blockade of FR. Despite displaying the highest binding affinity THP-1 cells were grown in RPMI-1640 medium containing 2.2 µM for FR-β, CB300635 was not markedly potent in inducing growth inhibition in CHO/FR-β folic acid (which would block any FR activity). Drug exposure time: cells (Figure 5B). Finally, AG2034 may utilize both the constitutively expressed RFC and FR 72 hours. Results are the mean ±SD of 3 separate experiments. as route for cell entry (Figure 4). As such, AG2034 displayed a growth inhibitory potential

158 159 Figuur_5A.pzf:Data 1 graph - Mon Sep 22 10:01:45 2008 Figuur_5A.pzf:*Data 1 graph - Mon Sep 22 10:02:22 2008

chapterFiguur_5A.pzf:*Data 9 1 graph - Mon Sep 22 10:02:45 2008 Figuur_5A.pzf:*Data 1 graph - Mon Sep 22 10:03:02 2008 Targeting RA synovial macrophages via the FR-β

Figure 5 A Figure 5 B Here we showed that FR-β expression primarily co-localized with macrophages in the intimal lining layer and the synovial sublining of RA patients and may therefore be an

CHO/WT - FA 100 100 CHO/WT - FA attractive target for folate antagonists. It has been previously reported that both FR-α CHO/WT + FA 80 80 CHO/FRβ - FA CHO/FRβ - FA and FR-β isoforms exhibit a rather low affinity for MTX as compared to folic acid (16, 42). CHO/FRβ + FA 60 CHO/FRβ + FA 60 In this context, it would be worthwhile to investigate whether folate antagonists with a 40 40 higher binding affinity to FR would provide a greater degree of selectivity and thereby 20 20 elicit a potentially improved therapeutic response. Screening for binding affinities of a Cell growth (% of control) Cell growth (% of control) 0 0 0.1 1 10 100 1000 10000 0.1 1 10 100 1000 10000 series of second generation folate antagonists, some of which with proven anticancer [BGC945] (nM) [CB300635] (nM) activity (18), revealed that the group of DHFR inhibitors all had a rather low FR-β affinity. This is consistent with previously reported structure/activity relationship, demonstrating Figure 5 C Figure 5 D that the α isoform of FR had low affinities for folate antagonists with a 2,4-diamino-based

100 CHO/WT - FA 100 CHO/WT - LV structure (see Supplementary File). Interestingly, while FR-α demonstrated a relatively CHO/WT + FA CHO/WT + LV 80 80 high binding affinity for all tested folate-based inhibitors of thymidylate synthase, for FR- CHO/FRβ - FA CHO/FRβ - LV this was only retained for 3 compounds (CB300635, GW1843 and BGC945) that share a 60 CHO/FRβ + FA 60 CHO/FRβ + LV β 40 40 common chemical property of 3-ring structures and/or glutamate side chain modifications.

20 20 The latter modification also markedly suppresses its ability to be transported via the RFC Cell growth (% of control) Cell growth (% of control) 0 0 and thus contributes to a greater FR-selectivity. In fact, selective targeting by BGC945 for 0.1 1 10 100 1000 0.1 1 10 100 1000 [AG2034] (nM) [AG2034] (nM) FR-α and not RFC was demonstrated in FR-α over expressing tumour cell lines. (17, 27). In addition to folate-based TS inhibitors, FR-β also exhibited moderate to high binding Figure 5: Growth inhibition of CHO/WT and CHO/FR-β cells by BGC945 with or without 1 µM folic acid affinities for the folate-based GARTFase inhibitors AG2034 and DDATHF, which classifies (A), CB300635 (B) and AG2034 (C/D) with or without supplementation of folic acid (1 µM) or leucovorin them as folate antagonist drugs that can be transported both via RFC (16) and FR. (20 nM). Results are the mean of 3 experiments (SD< 20%). FR-β-transfected CHO cells were used as a model system to evaluate the efficiency of FR-β- mediated cellular uptake of folate antagonists by conveying anti-proliferative effects (43). against CHO/WT cells and to a greater extent to CHO/FR-β cells. Consistently, abrogation This cell line model may be clinically representative, based on [3H]-folic acid binding levels of AG2034 growth inhibitory effects by FR-β blocking (with folic acid) and RFC blocking and FR-β mRNA levels that are compatible with FR-β mRNA levels in synovial tissue of RA (with leucovorin) are only partial (Figure 5C/D). patients (Figure 3). The largest differential in activity between control (RFC-expressing) CHO cells and FR-β transfected cells was observed for BGC945, consistent with a poor affinity for transport via RFC and a high FR-β binding affinity. FR-β mediated uptake of Discussion BGC945 could be inhibited by blocking of the receptor with excess folic acid, implying that circulating natural folates in synovial tissue/plasma could attenuate the potential Since MTX is the anchor-drug in many therapeutic regimens for RA treatment, delineation activity of BGC945 in vivo either by receptor occupancy/competition or by receptor down- of genetic, biochemical and metabolic parameters that could assist in predicting and/or regulation. In this context, it may be anticipated that e.g. folate food fortification may improving the therapeutic response to MTX have received considerable recent interest reduce the activity of this folate antagonist whereas restrictions in dietary folate intake (1, 19, 40, 41). This study focussed specifically on the role of cell membrane transport of could further enhance the therapeutic efficacy of these type of drugs (44). An intriguing MTX, which, in activated synovial macrophages, is mediated predominantly by the folate observation was that the compound CB300635 for which FR-β had the highest affinity receptor β isoform (11). Given the notion that the molecular and functional properties of (even 1.5-fold higher than folic acid) was not differentially active against FR-β transfected FRs and the constitutively expressed RFC differ considerably, a better understanding of cells. This may suggest that the high affinity binding may have a downside, which is the properties of FR-β may facilitate a better therapeutic window by selective targeting the poor release of the compound from the receptor in an acidic compartment during of FR-β over RFC. endocytic cycling of the receptor (45). Hence, to allow optimal targeting of FR-β by folate

160 161 chapter 9 Targeting RA synovial macrophages via the FR-β

antagonists, both conditions for efficient binding to the receptor and efficient intracellular Supplementary figure release from the receptor for target delivery should be fulfilled. The GARTFase inhibitors OH O COOH OH CHO O COOH

N CH2 NH C NH CH N CH2 NH C NH CH DDATHF and AG2034 harboured dual specificity for RFC and FRα/β as folate transport N N CH2 H CH2 CH CH 2 CH systems. Whether this property renders a different type of activity profile as compared to H2N N N 2 H2N N N 2 COOH H COOH FR-selective compounds awaits further investigations. In this respect, two recent studies Folic acid Leucovorin NH2 NH2 O COOH NH2 CH3 O COOH CH3 O COOH N N CH2 NH C NH CH CH2 NH C NH CH (46, 47) showed that two folate-based GARTFase inhibitors could confer suppression of N CH2 N C NH CH N N CH2 CH2 CH2 H2N N N H2N N N CH2 H2N N N CH2 rat adjuvant arthritis and collagen-induced arthritis in mice. Moreover, in a preliminary CH2 CH2 CH2 COOH account, we showed that novel generation of folate antagonists were potent suppressors NHCO NHCO of TNFα production from activated T cells of RA patients (48) COOH COOH MTX PT523 PT644 COOH O O CH O COOH H3C NH O 3 C CH 2 O COOH C CH2 N C NH CH Although MTX is the drug of first choice in RA treatment, there is room for improving the HN HN S N NH CH2 CH2 CH2 CH2 C NH CH CH2 N N CH H2N N CH2 H3C N CH2 efficacy of MTX, for instance in clinical cases of primary and acquired MTX failures (19). C H COOH CH2 COOH Further evaluation of folate-antagonists with properties of high FR-β binding affinity and O COOH GW1843 Pemetrexed Raltitrexed CH CH CH F O a low affinity for the RFC, prototypically harbored by BGC945 (27), may pave the road for a C C O COOH C O COOH O F O CH2 N C NH CH N C NH CH CH O COOH HN C 2 more selective targeted therapy of activated synovial macrophages. Even in cases where CH2 HN CH2 HN CH2 N C NH CH H2N N CH3 CH2 COOH CH2 COOH CH HO-CH2 N 2 C NH CH C NH CH FR-β expression would be low, there may be strategies to increase FR-β expression by H3C N CH3 CH2 O O CH2 CH2 CH pretreatment with retinoids and/or HDAC inhibitors (38, 49, 50). Thus, beyond exploiting N N CH2 2 CH COOH N N 2 H COOH FR-β for delivery of therapeutic agents or for imaging purposes (12, 14), the present study BGC9331 CB300635 BGC945 O COOH O COOH also supports therapeutic strategies that include FR-β mediated uptake of clinically active O O C CH2 CH2 C NH CH C S CH2 CH2 C NH CH HN HN S CH CH H 2 H 2 CH CH2 second generation folate antagonists. 2 CH CH H2NN N 2 H2NN N 2 H COOH H COOH DDATHF AG2034

Chemical structures of folic acid, leucovorin (5-formyltetrahydrofolate) and folate-based inhibitors of dihydrofolate reductase (DHFR), thymidylate synthase (TS) and glycinamide ribonucleotide transformylase (GARTFase).

162 163 References References

References biological activity., Mol.Pharmacol., 48: 459-471, 1995. 17 Jackman,A.L., Theti,D.S. and Gibbs,D.D. Antifolates targeted specifically to the folate receptor, 1 Kremer,J.M. Toward a better understanding of methotrexate, Arthritis Rheum., 50: 1370-1382, Adv.Drug Deliv.Rev., 56: 1111-1125, 2004. 2004. 18 Walling,J. From methotrexate to pemetrexed and beyond. A review of the pharmacodynamic 2 Landewe,R.B., Boers,M., Verhoeven,A.C., Westhovens,R., van de Laar,M.A., Markusse,H.M., and clinical properties of antifolates, Invest New Drugs, 24: 37-77, 2006. van Denderen,J.C., Westedt,M.L., Peeters,A.J., Dijkmans,B.A.C., Jacobs,P., Boonen,A., van der 19 Van der Heijden,J.W., Dijkmans,B.A.C., Scheper,R.J. and Jansen,G. Drug Insight: resistance to Heijde,D.M. and Van der Linden,S. COBRA combination therapy in patients with early rheumatoid methotrexate and other disease-modifying antirheumatic drugs-from bench to bedside, Nat. arthritis: Long-term structural benefits of a brief intervention, Arthritis Rheum., 46: 347-356, Clin.Pract.Rheumatol., 3: 26-34, 2007. 2002. 20 Shih,C., Chen,V.J., Gossett,L.S., Gates,S.B., MacKellar,W.C., Habeck,L.L., Shackelford,K.A., 3 O’Dell,J.R. Therapeutic strategies for rheumatoid arthritis, N.Engl.J.Med., 350: 2591-2602, Mendelsohn,L.G., Soose,D.J., Patel,V.F., Andis,S.L., Bewley,J.R., Rayl,E.A., Moroson,B.A., 2004. Beardsley,G.P., Kohler,W., Ratnam,M. and Schultz,R.M. LY231514, a pyrrolo[2,3-d]pyrimidine- 4 Smolen,J.S., Aletaha,D., Koeller,M., Weisman,M.H. and Emery,P. New therapies for treatment of based antifolate that inhibits multiple folate-requiring enzymes, Cancer Res., 57: 1116-1123, rheumatoid arthritis, Lancet, 2007. 1997. 5 Goekoop-Ruiterman,Y.P., De Vries-Bouwstra,J.K., Allaart,C.F., van Zeben,D., Kerstens,P. 21 Jackman,A.L., Taylor,G.A., Gibson,W., Kimbell,R., Brown,M., Calvert,A.H., Judson,I.R. and J., Hazes,J.M., Zwinderman,A.H., Ronday,H.K., Han,K.H., Westedt,M.L., Gerards,A.H., van Hughes,L.R. ICI D1694, a quinazoline antifolate thymidylate synthase inhibitor that is a potent Groenendael,J.H., Lems,W.F., van Krugten,M.V., Breedveld,F.C. and Dijkmans,B.A.C. Clinical and inhibitor of L1210 tumor cell growth in vitro and in vivo: a new agent for clinical study, Cancer radiographic outcomes of four different treatment strategies in patients with early rheumatoid Res., 51: 5579-5586, 1991. arthritis (the BeSt study): a randomized, controlled trial, Arthritis Rheum., 52: 3381-3390, 2005. 22 Rhee,M.S., Galivan,J., Wright,J.E. and Rosowsky,A. Biochemical studies on PT523, a potent 6 Jansen,G. Receptor- and carrier-mediated transport systems for folates and antifolates, In: nonpolyglutamatable antifolate, in cultured cells, Mol.Pharmacol., 45: 783-791, 1994. Anticancer Drug Development Guide: Antifolate Drugs in Cancer Therapy (A.L.Jackman, ed), 23 Rosowsky,A., Wright,J.E., Vaidya,C.M., Bader,H., Forsch,R.A., Mota,C.E., Pardo,J., Chen,C.S. and Humana Press Inc., Totowa, NJ, 293-321, 1999. Chen,Y.N. Synthesis and potent antifolate activity and cytotoxicity of B-ring deaza analogues of 7 Assaraf,Y.G. Molecular basis of antifolate resistance, Cancer Metastasis Rev., 26: 153-181, 2007. the nonpolyglutamatable dihydrofolate reductase inhibitor Nalpha-(4-amino-4-deoxypteroyl)- 8 Elnakat,H. and Ratnam,M. Distribution, functionality and gene regulation of folate receptor Ndelta-hemiphthaloyl- L-ornithine (PT523), J.Med.Chem., 41: 5310-5319, 1998. isoforms: implications in targeted therapy, Adv.Drug Deliv.Rev., 56: 1067-1084, 2004. 24 Duch,D.S., Banks,S., Dev,I.K., Dickerson,S.H., Ferone,R., Heath,L.S., Humphreys,J., Knick,V., 9 Salazar,M.D. and Ratnam,M. The folate receptor: what does it promise in tissue-targeted Pendergast,W., Singer,S. and . Biochemical and cellular pharmacology of 1843U89, a novel therapeutics?, Cancer Metastasis Rev., 26: 141-152, 2007. benzoquinazoline inhibitor of thymidylate synthase, Cancer Res., 53: 810-818, 1993. 10 Qiu,A., Jansen,M., Sakaris,A., Min,S.H., Chattopadhyay,S., Tsai,E., Sandoval,C., Zhao,R., 25 Bisset,G.M., Bavetsias,V., Thornton,T.J., Pawelczak,K., Calvert,A.H., Hughes,L.R. and Jackman,A. Akabas,M.H. and Goldman,I.D. Identification of an intestinal folate transporter and the L. The synthesis and thymidylate synthase inhibitory activity of L-gamma-L-linked dipeptide and molecular basis for hereditary folate malabsorption, Cell, 127: 917-928, 2006. L-gamma-amide analogues of 2-desamino-2-methyl-N10-propargyl-5,8-dideazafolic acid (ICI 11 Nakashima-Matsushita,N., Homma,T., Yu,S., Matsuda,T., Sunahara,N., Nakamura,T., Tsukano,M., 198583), J.Med.Chem., 37: 3294-3302, 1994. Ratnam,M. and Matsuyama,T. Selective expression of folate receptor beta and its possible role 26 Jackman,A.L., Kimbell,R., Aherne,G.W., Brunton,L., Jansen,G., Stephens,T.C., Smith,M.N., in methotrexate transport in synovial macrophages from patients with rheumatoid arthritis, Wardleworth,J.M. and Boyle,F.T. Cellular pharmacology and in vivo activity of a new anticancer Arthritis Rheum., 42: 1609-1616, 1999. agent, ZD9331: a water-soluble, nonpolyglutamatable, quinazoline-based inhibitor of 12 Paulos,C.M., Turk,M.J., Breur,G.J. and Low,P.S. Folate receptor-mediated targeting of therapeutic thymidylate synthase, Clin.Cancer Res., 3: 911-921, 1997. and imaging agents to activated macrophages in rheumatoid arthritis, Adv.Drug Deliv.Rev., 56: 27 Gibbs,D.D., Theti,D.S., Wood,N., Green,M., Raynaud,F., Valenti,M., Forster,M.D., Mitchell,F., 1205-1217, 2004. Bavetsias,V., Henderson,E. and Jackman,A.L. BGC 945, a novel tumor-selective thymidylate 13 Turk,M.J., Breur,G.J., Widmer,W.R., Paulos,C.M., Xu,L.C., Grote,L.A. and Low,P.S. Folate-targeted synthase inhibitor targeted to alpha-folate receptor-overexpressing tumors, Cancer Res., 65: imaging of activated macrophages in rats with adjuvant- induced arthritis, Arthritis Rheum., 46: 11721-11728, 2005. 1947-1955, 2002. 28 Beardsley,G.P., Moroson,B.A., Taylor,E.C. and Moran,R.G. A new folate antimetabolite, 5,10- 14 Low,P.S., Henne,W.A. and Doorneweerd,D.D. Discovery and development of folic-acid-based dideaza-5,6,7,8-tetrahydrofolate is a potent inhibitor of de novo purine synthesis, J.Biol.Chem., receptor targeting for imaging and therapy of cancer and inflammatory diseases, Acc.Chem. 264: 328-333, 1989. Res., 41: 120-129, 2008. 29 Boritzki,T.J., Barlett,C.A., Zhang,C. and Howland,E.F. AG2034: a novel inhibitor of glycinamide 15 Nagayoshi,R., Nagai,T., Matsushita,K., Sato,K., Sunahara,N., Matsuda,T., Nakamura,T., Komiya,S., ribonucleotide formyltransferase, Invest New Drugs, 14: 295-303, 1996. Onda,M. and Matsuyama,T. Effectiveness of anti-folate receptor beta antibody conjugated with 30 Ross,J.F., Chaudhuri,P.K. and Ratnam,M. Differential regulation of folate receptor isoforms truncated Pseudomonas exotoxin in the targeting of rheumatoid arthritis synovial macrophages, in normal and malignant tissues in vivo and in established cell lines. Physiologic and clinical Arthritis Rheum., 52: 2666-2675, 2005. implications, Cancer, 73: 2432-2443, 1994. 16 Westerhof,G.R., Schornagel,J.H., Kathmann,I., Jackman,A.L., Rosowsky,A., Forsch,R.A., Hynes,J. 31 Kraan,M.C., Reece,R.J., Smeets,T.J., Veale,D.J., Emery,P. and Tak,P.P. Comparison of synovial B., Boyle,F.T., Peters,G.J., Pinedo,H.M. and Jansen,G. Carrier- and receptor-mediated transport of tissues from the knee joints and the small joints of rheumatoid arthritis patients: Implications folate antagonists targeting folate-dependent enzymes: correlates of molecular structure and for pathogenesis and evaluation of treatment, Arthritis Rheum., 46: 2034-2038, 2002.

164 165 References References

32 Kraan,M.C., Reece,R.J., Barg,E.C., Smeets,T.J., Farnell,J., Rosenburg,R., Veale,D.J., Breedveld,F.C., Rheumatol., 21: 719-725, 2003. Emery,P. and Tak,P.P. Modulation of inflammation and metalloproteinase expression in synovial 47 Chintalacharuvu,S., Evans,G.F., Shih,C., Bryant,H.U., Sandusky,G.E. and Zuckerman,S.H. tissue by leflunomide and methotrexate in patients with active rheumatoid arthritis. Findings in Inhibition of glycinamide ribonucleotide formyltransferase results in selective inhibition of a prospective, randomized, double-blind, parallel-design clinical trial in thirty-nine patients at macrophage cytokine secretion in vitro and in vivo efficacy in rat adjuvant arthritis, Clin.Exp. two centers, Arthritis Rheum., 43: 1820-1830, 2000. Rheumatol., 23: 438-446, 2005. 33 Maliepaard,M., Scheffer,G.L., Faneyte,I.F., van Gastelen,M.A., Pijnenborg,A.C., Schinkel,A.H., 48 Van der Heijden,J.W., Gerards,A.H., Oerlemans,R., Lems,W.F., Scheper,R.J., Aarden,L.A., van De Vijver,M.J., Scheper,R.J. and Schellens,J.H. Subcellular localization and distribution of the Dijkmans,B.A.C. and Jansen,G. Inhibition of tumour necrosis factor alpha production by activated breast cancer resistance protein transporter in normal human tissues, Cancer Res., 61: 3458- T cells of rheumatoid arthritis patients by novel anti-folate drugs: an ex vivo pilot study, Arthritis 3464, 2001. Research & Therapy, 7: S32-S33, 2005. 34 Scheffer,G.L., Kool,M., Heijn,M., de Haas,M., Pijnenborg,A.C., Wijnholds,J., van Helvoort,A., de 49 Ratnam,M., Hao,H., Zheng,X., Wang,H., Qi,H., Lee,R. and Pan,X. Receptor induction and Jong,M.C., Hooijberg,J.H., Mol,C.A., van der Linden,M., de Vree,J.M., van der Valk,P., Elferink,R. targeted drug delivery: a new antileukaemia strategy, Expert.Opin.Biol.Ther., 3: 563-574, 2003. P., Borst,P. and Scheper,R.J. Specific detection of multidrug resistance proteins MRP1, MRP2, 50 Wang,H., Zheng,X., Behm,F.G. and Ratnam,M. Differentiation-independent retinoid induction MRP3, MRP5, and MDR3 P-glycoprotein with a panel of monoclonal antibodies, Cancer Res., 60: of folate receptor type beta, a potential tumor target in myeloid leukemia, Blood, 96: 3529- 5269-5277, 2000. 3536, 2000. 35 Jaspars,E.H., Bloemena,E., Bonnet,P., Scheper,R.J., Kaiserling,E. and Meijer,C.J. A new monoclonal antibody (3A5) that recognises a fixative resistant epitope on tissue macrophages and monocytes, J.Clin.Pathol., 47: 248-252, 1994. 36 Haringman,J.J., Vinkenoog,M., Gerlag,D.M., Smeets,T.J., Zwinderman,A.H. and Tak,P.P. Reliability of computerized image analysis for the evaluation of serial synovial biopsies in randomized controlled trials in rheumatoid arthritis, Arthritis Res.Ther., 7: R862-R867, 2005. 37 Raap,A.K., van de Corp, Vervenne,R.A., van Gijlswijk,R.P., Tanke,H.J. and Wiegant,J. Ultra- sensitive FISH using peroxidase-mediated deposition of biotin- or fluorochrome tyramides, Hum.Mol.Genet., 4: 529-534, 1995. 38 Qi,H. and Ratnam,M. Synergistic induction of folate receptor beta by all-trans retinoic acid and histone deacetylase inhibitors in acute myelogenous leukemia cells: mechanism and utility in enhancing selective growth inhibition by antifolates, Cancer Res., 66: 5875-5882, 2006. 39 Westerhof,G.R., Rijnboutt,S., Schornagel,J.H., Pinedo,H.M., Peters,G.J. and Jansen,G. Functional activity of the reduced folate carrier in KB, MA104, and IGROV-I cells expressing folate-binding protein, Cancer Res., 55: 3795-3802, 1995. 40 Dervieux,T., Greenstein,N. and Kremer,J. Pharmacogenomic and metabolic biomarkers in the folate pathway and their association with methotrexate effects during dosage escalation in rheumatoid arthritis, Arthritis Rheum., 54: 3095-3103, 2006. 41 Wessels,J.A., van der Kooij,S.M., le Cessie S., Kievit,W., Barerra,P., Allaart,C.F., Huizinga,T.W. and Guchelaar,H.J. A clinical pharmacogenetic model to predict the efficacy of methotrexate monotherapy in recent-onset rheumatoid arthritis, Arthritis Rheum., 56: 1765-1775, 2007. 42 Wang,X., Shen,F., Freisheim,J.H., Gentry,L.E. and Ratnam,M. Differential stereospecificities and affinities of folate receptor isoforms for folate compounds and antifolates, Biochem.Pharmacol., 44: 1898-1901, 1992. 43 Ross,J.F., Wang,H., Behm,F.G., Mathew,P., Wu,M., Booth,R. and Ratnam,M. Folate receptor type beta is a neutrophilic lineage marker and is differentially expressed in myeloid leukemia, Cancer, 85: 348-357, 1999. 44 Mauritz,R., Peters,G.J., Kathmann,I., Teshale,H., Noordhuis,P., Comijn,E.M., Pinedo,H.M. and Jansen,G. Dynamics of antifolate transport via the reduced folate carrier and the membrane folate receptor in murine leukaemia cells in vitro and in vivo, Cancer Chemother.Pharmacol., 2008. 45 Rijnboutt,S., Jansen,G., Posthuma,G., Hynes,J.B., Schornagel,J.H. and Strous,G.J. Endocytosis of GPI-linked membrane folate receptor-alpha, J.Cell Biol., 132: 35-47, 1996. 46 Nagayoshi,R., Nakamura,M., Ijiri,K., Yoshida,H., Komiya,S. and Matsuyama,T. LY309887, antifolate via the folate receptor suppresses murine type II collagen-induced arthritis, Clin.Exp.

166 167 chapter 10 The proteasome inhibitor bortezomib inhibits the release of NFκB-inducible cytokines and induces apoptosis of activated T cells from rheumatoid arthritis patients

Joost W. van der Heijden1, Ruud Oerlemans1, Willem F Lems1, Rik J Scheper2, Ben AC Dijkmans1, and Gerrit Jansen1

Departments of 1Rheumatology and 2Pathology, VU University Medical Center, Amsterdam, The Netherlands

Accepted for publication in Clinical and Experimental Rheumatology 2008 chapter 10 Anti-inflammatory effects of bortezomib

Abstract patients (12, 13). Bortezomib is used as single agent and in drug combination regimens where it has displayed additive/ synergistic anti-tumor effects when combined with glucocorticoids Objective : The proteasome is a multicatalytic proteinase complex regulating the intracellular (dexamethasone) or TNF-related apoptosis-inducing ligand (TRAIL) (13, 14). Interestingly, breakdown of many proteins, including those mediating the activation of pro-inflammatory patients with relapsed or refractory lymphoma who responded to bortezomib had a marked signaling pathways (e.g. NFκB), cell proliferation and survival. Conceptually, proteasome reduction in plasma TNFα levels as compared to bortezomib non-responders (15). Of additional inhibitors may therefore elicit potential anti-inflammatory properties by inhibiting these interest from a potential anti-inflammatory perspective were preliminary data from animal processes and thereby impair the cellular release of pro-inflammatory cytokines such as models for arthritis (16, 17) which revealed that bortezomib treatments conveyed clear Tumor Necrosis Factor α (TNFα) in RA patients. therapeutic effects. In fact, a recent report by Neubert et al showed that bortezomib targets Methods: Whole-blood from 19 RA patients (including methotrexate-responsive and non- both short and long-lived plasma cells in mice with lupus-like disease, thereby abrogating responsive patients) and 7 healthy volunteers was incubated ex-vivo with the proteasome the production of antibodies to double-stranded DNA and preventing the onset of nephritis inhibitor bortezomib after T-cell stimulation with αCD3/CD28. Inhibition of cytokine (18). The present study was undertaken to assess the potential anti-inflammatory effects of production by bortezomib was measured after 24 and 72 hours by ELISA. Effects of bortezomib bortezomib on the basis of inhibiting the production of TNFα and other pro-inflammatory on apoptosis and T-cell activation (CD25 expression) were measured by FACS-analysis. cytokines by activated T cells from RA patients. Results: Bortezomib proved to be a rapid (<24 hour) and potent inhibitor of the release of several NFκB-inducible cytokines (including TNFα, IL-1β, IL-6 and IL-10) by activated T-cells from healthy volunteers and RA patients, regardless of their clinical responsiveness to Materials & methods methotrexate. Median concentrations of bortezomib required to inhibit TNFα production by 50% (mIC-50) were 12 nM (range: 8-50 nM) for healthy volunteers and 46 nM (range: 18-60 nM) Patient characteristics for RA patients. A reduction of T cell activation and a marked induction of T-cell apoptosis All patients signed an informed consent form, and the study on ‘DMARD-resistance’ was were revealed as late effects after bortezomib incubations beyond 24 hours. approved by the Medical Ethics committee of the VU University Medical Center, Amsterdam, Conclusion: Proteasome inhibitors, represented by bortezomib, may elicit potential anti- The Netherlands. Characteristics of 19 RA patients included in this study were: male/female: inflammatory properties that deserve further exploration in experimental therapies for RA. 5/14; mean age: 52.5 years; mean DAS28 score (Disease Activity Score for 28 joints): 4.3 (range 1.3-7.2). Five patients had no prior treatment with Disease Modifying Anti-Rheumatic Drugs (DMARD-naive), 13 patients used methotrexate (MTX) 15-30 mg/week, and 1 patient used Introduction the combination of sulfasalazine and hydoxychloroquine. Patients were on DMARDs for at least 3 months (range: 12 weeks to 9 years). Patients who were on DMARD therapy were The proteasome is a multimeric proteinase complex that facilitates the degradation of defined as DMARD responders if their DAS28 was ≤3.2 at the time of enrollment (n=7); RA approximately 80% of intracellular proteins. The catalytic activities of the proteasome not patients with a DAS28 score of >3.2 were defined as DMARD non-responders (n=7). Finally, 7 only ensure protein homeostasis; they also provide a fine-tuned mechanism of controlled healthy volunteers were included (male/female: 4/3; mean age: 38 years). Additional patient (in)activation of proteins involved in cell proliferation, signaling processes and the generation characteristics are described in Table 1. of antigenic peptides to be presented on MHC class I molecules (1-3). In this context, for example, activation and nuclear translocation of the transcription factor NFκB intimately Materials depends on proteasome-mediated breakdown of its natural inhibitor protein IκBα and Bortezomib was kindly provided by Millennium Pharmaceuticals; Cambridge, MA, U.S.A. thereby mediates the transcriptional regulation of several pro-inflammatory cytokines such Stock solutions of 1 mM were prepared in dimethylsulfoxide (DMSO) and stored at -20oC. as TNFα and IL1β (4, 5) as well as anti-apoptotic genes (6). From this perspective, it has been αCD3/αCD28 antibodies were a generous gift from Prof. L. Aarden (Sanquin, Amsterdam, anticipated that inhibitors of the proteasome may counteract the NFκB activation process the Netherlands). Anti-CD25-PE was obtained from Becton Dickinson; San Jose, CA, U.S.A. and elicit a potential anti-inflammatory response (7-11). Cytokine ELISA assays (PeliKine) for TNFα, IL-1β, IL-6 and IL-10 were obtained from Sanquin, Bortezomib (Velcade®, PS341), a boronic acid dipeptide, is the first specific proteasome Amsterdam, The Netherlands, and utilized according to the manufacturers’ instructions and inhibitor that is registered in cancer chemotherapy for therapy-refractory multiple myeloma as described previously (19).

170 171 chapter 10 Anti-inflammatory effects of bortezomib

Table 1: Baseline characteristics of (subgroups of) RA patients and controls and outcome of TNFα Statistical analysis inhibition from activated T-cells by bortezomib and methotrexate. Statistical analysis was performed with the Mann-Whitney test to analyze differences between RA patients and healthy volunteers. Controls RA pts DMARD- DMARD- DMARD- (n=7) (n=19) naïve responders non-responders (n=5) (n=7) (n=7) Results Mean age (yr) (SD) 38 (13) 53 (17)NS Mean ESR (mm/hr) (SD) 27 (27) Bortezomib-induced inhibition of cytokine release from activated T-cells after 24 hours Median DAS28 score (range) 5.5 (4.4-6.9) 2.1 (1.3-2.9) 5.5 (3.9-7.2) Median IC50 bortezomib 12 (8-50) 46 (18-60)* 47 (32-59)* 48 (24-60)* 27 (18-59) In a pilot setting, the ability of bortezomib was assessed to inhibit the release of cytokines (nM) (range) from αCD3/αCD28 activated T-cells from controls (Figure 1A) and RA patients (Figure 1B). Median IC50 MTX 53 (18-85) 59 (16-325) 55 (32-102) 44 (28-260) 60 (16-325) Production of TNFα, IL-1β, IL-6 and IL-10 production in controls was half maximally inhibited (nM) (range) at bortezomib concentrations between 5-25 nM (Fig 1A). In activated T-cells of RA patients IC50: drug concentration required to inhibit TNFα production by 50%. *p<0.05, NS Not Significant. DAS28: (Figure 1B),H10_1A.pzf:Data bortezomib 1 graph also - Thu Jul inhibited 31 16:19:13 2008 the production of IL-6,H10_1B.pzf:Data IL-1β and 1 graph TNF - Thuα ,Jul although 31 16:17:06 2008 for the Disease Activity Score (28 joints), ESR: Erythrocyte Sedimentation Rate. latter cytokine, slightly higher concentrations of bortezomib were required. Production of IL- 10 in activated T-cells of RA patients was less efficiently inhibited as compared to controls.

Inhibition of TNFα release from activated T-cells by bortezomib and methotrexate after 72 hours Procedures & T-cell activation The potency of bortezomib to inhibit the production of TNFα was further investigated in a Whole blood (1:10 diluted with heparin-containing DMEM medium, supplemented with 0.1% larger group of RA patients (n=19) which could be subcategorized in DMARD-naïve patients fetal calf serum) of 19 RA patients and 7 healthy volunteers were incubated ex vivo with αCD3/ (n=5), DMARD-responsive patients (DAS28 <3.2, n=7) and DMARD non-responsive RA αCD28 antibodies (0.1 µg/ml and 1 µg/ml respectively) as T-cell activating agents as described patients (DAS28 >3.2, n =7). For comparison, inhibition of TNFα production was also analyzed previously (19). T-cell activation was monitored by CD25-PE staining by flow cytometry. The for methotrexate (MTX) as a reference drug. Following αCD3/αCD28 stimulation of T-cells, whole blood incubates were then supplemented with a concentration range (0-100 nM) of absolute levels of baseline TNFα production were not significantly different in whole blood bortezomib, and, as a reference, 33-300 nM MTX, followed by an incubation period of 24-72 hours. Inhibition of TNFα production was measured after 72 hours drug exposure by ELISA Figure 1 A Figure 1 B

as described before (19). Beyond TNFα, production of other NFκB-inducible cytokines (i.e. 100 100 IL-1β IL-1β IL-1β, IL-6 and IL-10) was measured in whole blood from 3 separate healthy controls and 3 RA IL-6 IL-6 75 75 patients after T-cell stimulation and 24 hour exposure to bortezomib. IL-10 IL-10 TNF-α TNF-α 50 50 %of control Apoptosis assay %of control 25 25 Peripheral blood lymphocytes (PBLs) of RA patients and healthy volunteers were isolated by Inhibition ofcytokine production Ficoll-Paque Plus density centrifugation and suspended in DMEM-medium supplemented Inhibition ofcytokine production 0 0 0 11 33 100 0 11 33 100 with 10% fetal calf serum. Apoptosis in PBLs was analyzed by flow cytometry (FACS) after 24 [bortezomib] (nM) [bortezomib] (nM) and 48 hours exposure to bortezomib using Annexin-V-FITC/7-aminoactinomycin D (7-AAD) Figure 1: Bortezomib-induced inhibition of TNFα, IL-1β, IL-6 and IL-10 production in activated T-cells TM staining (APOPTEST -FITC A700, VPS Diagnostics, Hoeven, the Netherlands) according to from controls and RA patients. the manufacturer’s protocol. Phycoerythrin labeled T-cell markers (CD4/CD8/CD25) were Bortezomib-induced inhibition of TNFα, IL-1β, IL-6, and IL-10 production from activated T-cells of (A) healthy volunteers (n=3) and (B) DMARD-naïve RA patients (n=3) was analyzed after 24 hour stimulation of T-cells in obtained from Becton Dickinson; San Jose, CA, U.S.A. whole blood by αCD3-α/CD28 in the presence of the indicated concentrations of bortezomib. Results are depicted as the mean percentage of cytokine production relative to controls incubated without bortezomib.

172 173 H10_2.pzf:Data 1 graph - Thu Jul 31 16:18:51 2008

chapter 10 Anti-inflammatory effects of bortezomib

7500 incubates from 7 healthy volunteers (baseline TNFα production: 3208± 2554 pg/ml; range: Figure 2 H10_3.pzf:Data 1 graph - Thu Jul 31 16:18:25 2008 623-7361 pg/ml) and 3 groups of RA patients; DMARD-naive (n=5), DMARD-responders (n=7) 5000 and DMARD non-responders (n=7) (Figure 2). Upon co-incubation with bortezomib, median concentrations of this drug required to inhibit TNFα production by 50% (mIC50) were 3.8-fold 2500 lower (p=0.01) for healthy controls (IC50: 12 nM, range: 8-50 nM) as compared to the total after stimulation (pg/ml)

group of RA patients (IC50: 46 nM, range: 18-60 nM) (Table 1, Figure 3). Sub-analysis for the Mean baselineTNFa production 0 l ls s o ta r rs tr o e e DMARD non-responsive RA patients showed a 1.7-fold greater potency of bortezomib to n nd o A t o C R espond No DMARD Resp -r inhibit TNFα production compared to DMARD-naive and DMARD-responsive RA patients, but n o N this difference was not significant (p=0.32) (Figure 4). Median concentrations of MTX to inhibit Figure 2: Baseline TNFα production by activated T-cells of RA-patients and healthy volunteers. TNFα production by 50% were not significantly different (p=0.64) between healthy controls Baseline TNFα production by αCD3/αCD28 activated T-cells from healthy controls (n=7), total group of RA patients (IC50: 53 nM, range 18-85 nM) and RA patients (IC50: 59 nM, range: 16-325 nM) (Table 1). (n=19), and after sub-classification in DMARD-naïve (n=5), DMARD-responders (n=7) and DMARD-non-responding RA patients (n=7). Data are presented as mean values (pg/ml) ±SD. Differences in baseline TNFα production in the tested groups were not statistically different. Note: no TNFα production was observed in unstimulated whole Bortezomib-induced induction of apoptosis and inhibition of T-cell activation blood cell cultures. Along with a rapid reduction in TNFα release, a marked induction of apoptosis was observed 160 Figure 3 in peripheral blood lymphocytes (PBLs) of RA patients at a later stage of bortezomib 140 120 exposure: 10-21 % Annexin-V positive cells in control conditions versus 62-77% Annexin- 100 V/7AAD-positive cells after exposure to 10-100 nM bortezomib for 48 hours, respectively 80 H10_4.pzf:Data 1 graph - Wed Aug 20 10:07:29 2008 60 (Figure 5A). A representative FACS analysis of bortezomib-induced T cell apoptosis in PBLs %of control 40 from a RA patient is shown in Figure 5B. Without bortezomib (control), only 7% of CD4/CD8 20 Inhibitionof TNFa production 0 positive (activated) T-cells were apoptotic, while after incubation with 33 nM bortezomib 0 11 33 100 for 48 hours (representing IC-50 value for inhibition of TNFα production in RA whole-blood), [Bortezomib] nM Figure 3: Bortezomib-induced inhibition of TNFα production by activated T-cells from RA patients and 60% of CD4/CD8 positive T-cells were apoptotic based on Annexin-V staining. Apoptosis healthy volunteers. induction by bortezomib could be partially prevented, in a concentration dependent Dose-response curve (72 hours incubation) for bortezomib-induced inhibition of TNFα production by αCD3- manner, by pre-incubation with the broad-spectrum-caspase inhibitor ZVAD-fmk (10-50 α/CD28 activated T-cells from healthy volunteers (squares, n=7) and RA patients (triangles, n=19). Results are presented as median value and range (bars) for each bortezomib concentration tested. µM), suggesting that cell death was mediated via apoptosis rather than by necrosis (data not

shown). For comparison, no induction of apoptosis was observed after 48 hours exposure 80 p=0.01 Figure 4

70 to 300 nM concentrations of MTX (Figure 5C). Evaluation of the T-cell activation status by p=0.32 CD25 expression showed that as off 48 hours incubation with bortezomib, CD25 expression 60 decreased in a concentration dependent manner (Figure 5D). 50 40

30

20 Bortezomib IC-50 values(nM) Discussion forinhibition of TNFa production 10

0

ls s o r rs Nowadays, the ubiquitin-proteasome system is recognized as the major pathway for tr e e n nd o o C espond No DMARD Resp -r degradation of intracellular proteins, many of which play a key role in regulation of pro- n o N inflammatory cytokines (7, 11). Data from the present study further support the proof of Figure 4: Bortezomib-induced inhibition of TNFα release by activated T-cells from (subgroups of) RA principle that bortezomib can confer inhibition of TNFα production in whole blood from RA patients and healthy volunteers. Concentrations of bortezomib (72 hours incubation) required for 50% inhibition (IC50) of TNFα production patients after T-cell stimulation. Mechanistically, this effect can be explained by inhibition of in αCD3/αCD28-stimulated whole blood-cell cultures of individual healthy volunteers (n=7; squares) and RA the NFκB signalling pathway as a downstream effect of proteasome inhibition in conjunction patients (n=19; subgroups; triangels). The horizontal lines depict the median IC-50 value for each group.

174 175 H10_5A.pzf:Data 1 graph - Thu Jul 31 16:18:05 2008

chapter 10 Anti-inflammatory effects of bortezomib H10_5C.pzf:Data 1 graph - Thu Jul 31 16:19:32 2008 H10_5D.pzf:Data 1 graph - Thu Jul 31 16:17:38 2008

Figure 5 A Figure 5 B concentrations of bortezomib and/or longer exposure times. In fact, concentrations of 80 24 hours 70 bortezomib that provoke apoptosis induce p38 MAPK activity along with a release of anti- 48 hours 60 inflammatory cytokines, such as IL-10 (22). The incomplete inhibition of IL-10 production 50 at higher concentrations of bortezomib (Figure 1) may be consistent with this notion. 40 30 While bortezomib-induced inhibitory effects on NFκB activity could be observed rather 20 rapidly (within 24 hour, Figure 1), bortezomib-induced apoptosis of activated T-cells was 10 recognized as a later effect, emerging between 24 and 48 hours of bortezomib exposure. %Annexin-V/7AAD positivecells 0 0 11 33 100 In this respect, our data are consistent with Blanco et al showing that bortezomib was [Bortezomib] (nM) particularly active against alloreactive (CD25+) T-cells and not resting T-cells (23). Based on Figure 5 C Figure 5 D the profile of inhibition of TNFα production, bortezomib exhibited potent ex vivo activity 80 50 24 hours 24 hours for both DMARD-naive and clinically DMARD-responsive RA patients, and an even slightly 70 48 hours 40 48 hours 60 greater activity for DMARD non-responsive patients. This result may point to the fact that

50 30 proteasome targeting may bypass or circumvent common mechanisms of loss of efficacy to 40 DMARDs after chronic administration (24). 30 20 20 Apart from the inter-patient variability in ex vivo response to bortezomib, one other 10

10 % of CD-25 positive cells intriguing observation was that bortezomib displayed a greater potency in blocking of

%Annexin-V/7AAD positivecells 0 0 0 33 100 300 0 11 33 100 TNFα production by activated T-cells from healthy controls than from RA patients. An [MTX] (nM) [Bortezomib] (nM) explanation for this is not readily available but could relate to possible differences between healthy controls and RA patients with respect to (i) quantitative and qualitative differences Figure 5: Effects of bortezomib on T-cell activation and induction of apoptosis. (A) Induction of apoptosis by bortezomib in αCD3/αCD28-stimulated peripheral blood lymphocytes of RA in proteasomal catalytic activity in T-cells from healthy controls versus patients with RA or patients after 24 hours (white bars) and 48 hours (black bars) drug exposure. (B) Representative FACS analysis other autoimmune diseases (25-27), or (ii) higher constitutive NFκB activity in activated T- depicting induction of apoptosis (Annexin-V positivity in the CD4/CD8 positive population of (activated) T-cells in control conditions (without bortezomib) and after 48 hours exposure to 33nM bortezomib. (C) Induction of cells from RA patients (4), which would require higher bortezomib dosages for inhibition. apoptosis by MTX in αCD3/αCD28-stimulated peripheral blood lymphocytes of RA patients after 24 hours (white In addition, a recent study (28) indicated that plasma pharmacokinetics and activity of bars) and 48 hours (black bars) drug exposure. (D) Percentage of activated (CD25 positive) T-cells in CD3/ CD28- α α bortezomib can be influenced by uptake of bortezomib in red blood cells. In our study, stimulated peripheral blood lymphocytes after incubation with bortezomib. Results presented are the mean ±SD of 3 individual RA patients. we made use of (1:10) diluted whole blood cell samples (19) within which variability in erythrocyte concentrations could have influenced residual concentrations of bortezomib to some degree. Notably, pharmacokinetics of bortezomib in phase I clinical trials showed peak with induction of apoptosis in activated T-cells (12-14, 17). In the whole blood assay employed plasma levels of 50-1000 nM and steady state plasma levels of 10-20 nM (29, 30), which are in this study, it is anticipated that beside T-cells, monocytes/macrophages will be the main within the concentration range where bortezomib showed inhibition of TNFα production producers of IL-1, IL-6 and TNFα, as they get stimulated by cytokines (like IL-17) produced by by activated T-cells from RA patients (Table 1, Figure 1) and induction of T-cell apoptosis. It activated T-cells (20). Since we observed that bortezomib abrogated T-cell activation at low remains to be established whether this induction of apoptosis is based solely on the apoptotic concentrations, it can be speculated that the production of these cytokines by monocytes/ effects of bortezomib itself or whether it also involves previously reported bortezomib- macrophages is inhibited simultaneously. induced sensitization of apoptotic effects of TRAIL or TNFα (14). Obviously, any design of Consistent with inhibition of NFκB were observations (Figures. 1, 3, 4) that besides TNFα, a trial for bortezomib in RA treatment should address a number of issues: (a) assessment of bortezomib also inhibited other NFκB-inducible cytokines (IL-1β, IL-6, IL-10) (Figure 1) in a optimal dosing/therapeutic window for RA treatment, (b) it is anticipated that commonly concentration range (10-50 nM) that was previously shown to abrogate NFκB activity (21). long term drug administration to RA patients will require special precautions with respect Given the central role of the ubiquitin-proteasome system in regulating signalling pathways to drug safety (31), (c) it should be considered that proteasome inhibition may partially (11), it is anticipated that signalling pathways other than NFκB may be affected at higher abrogate intracellular degradation of (citrinullated) proteins in RA patients (32). Finally, (d)

176 177 chapter 10 Anti-inflammatory effects of bortezomib

it remains to be revealed to which extent other pro-inflammatory cytokine producing cells (monocytes, macrophages) in peripheral blood or in synovial tissue are subject to targeting by bortezomib as demonstrated for activated T cells in this study. Despite the current success of DMARDs and biologic agents, there is still room for improvement of RA therapy (33). Given their unique profile of action, proteasome inhibitors (34, 35) certainly deserve further evaluation for future clinical application in the treatment of chronic inflammatory diseases. As a proof of concept, this pilot study demonstrated that proteasome targeting by specific inhibitors such as bortezomib, can suppress production of pro-inflammatory cytokines from activated T cells of RA patients. Thus, beyond demonstrations of good clinical activity against certain types of cancer, proteasome inhibitors may represent a new generation of targeted small molecule drugs in the therapeutic armour, in particular for patients with DMARD-refractory RA.

178 179 References References

References Immunol., 7: 429-442, 2007. 21 Chauhan,D., Catley,L., Li,G., Podar,K., Hideshima,T., Velankar,M., Mitsiades,C., Mitsiades,N., 1 Glickman,M.H. and Ciechanover,A. The ubiquitin-proteasome proteolytic pathway: destruction Yasui,H., Letai,A., Ovaa,H., Berkers,C., Nicholson,B., Chao,T.H., Neuteboom,S.T., Richardson,P., for the sake of construction, Physiol Rev., 82: 373-428, 2002. Palladino,M.A. and Anderson,K.C. A novel orally active proteasome inhibitor induces apoptosis 2 Rock,K.L., York,I.A. and Goldberg,A.L. Post-proteasomal antigen processing for major in multiple myeloma cells with mechanisms distinct from Bortezomib, Cancer Cell, 8: 407-419, histocompatibility complex class I presentation, Nat.Immunol., 5: 670-677, 2004. 2005. 3 Kloetzel,P.M. and Ossendorp,F. Proteasome and peptidase function in MHC-class-I-mediated 22 Mitra-Kaushik,S., Harding,J.C., Hess,J.L. and Ratner,L. Effects of the proteasome inhibitor PS-341 antigen presentation, Curr.Opin.Immunol., 16: 76-81, 2004. on tumor growth in HTLV-1 Tax transgenic mice and Tax tumor transplants, Blood, 104: 802-809, 4 Firestein,G.S. NF-kappaB: Holy Grail for rheumatoid arthritis?, Arthritis Rheum., 50: 2381-2386, 2004. 2004. 23 Blanco,B., Perez-Simon,J.A., Sanchez-Abarca,L.I., Carvajal-Vergara,X., Mateos,J., Vidriales,B., 5 Tak,P.P. and Firestein,G.S. NF-kappaB: a key role in inflammatory diseases, J.Clin.Invest, 107: 7-11, Lopez-Holgado,N., Maiso,P., Alberca,M., Villaron,E., Schenkein,D., Pandiella,A. and San,M. 2001. J. Bortezomib induces selective depletion of alloreactive T lymphocytes and decreases the 6 Karin,M. and Lin,A. NF-kappaB at the crossroads of life and death, Nat.Immunol., 3: 221-227, 2002. production of Th1 cytokines, Blood, 107: 3575-3583, 2006. 7 Elliott,P.J., Zollner,T.M. and Boehncke,W.H. Proteasome inhibition: a new anti-inflammatory 24 Van der Heijden,J.W., Dijkmans, B.A.C., Scheper,R.J. and Jansen,G. Drug Insight: resistance to strategy, J.Mol.Med., 81: 235-245, 2003. methotrexate and other disease-modifying antirheumatic drugs-from bench to bedside, Nat.Clin. 8 Reinstein,E. and Ciechanover,A. Narrative review: protein degradation and human diseases: the Pract.Rheumatol., 3: 26-34, 2007. ubiquitin connection, Ann.Intern.Med., 145: 676-684, 2006. 25 Egerer,K., Kuckelkorn,U., Rudolph,P.E., Ruckert,J.C., Dorner,T., Burmester,G.R., Kloetzel,P.M. 9 Rajkumar,S.V., Richardson,P.G., Hideshima,T. and Anderson,K.C. Proteasome inhibition as a novel and Feist,E. Circulating proteasomes are markers of cell damage and immunologic activity in therapeutic target in human cancer, J.Clin.Oncol., 23: 630-639, 2005. autoimmune diseases, J.Rheumatol., 29: 2045-2052, 2002. 10 Nencioni,A., Grunebach,F., Patrone,F., Ballestrero,A. and Brossart,P. Proteasome inhibitors: 26 Egerer,T., Martinez-Gamboa,L., Dankof,A., Stuhlmuller,B., Dorner,T., Krenn,V., Egerer,K., Rudolph,P. antitumor effects and beyond, Leukemia, 21: 30-36, 2007. E., Burmester,G.R. and Feist,E. Tissue-specific up-regulation of the proteasome subunit beta5i 11 Shen,J., Reis,J., Morrison,D.C., Papasian,C., Raghavakaimal,S., Kolbert,C., Qureshi,A.A., Vogel,S.N. (LMP7) in Sjogren’s syndrome, Arthritis Rheum., 54: 1501-1508, 2006. and Qureshi,N. Key inflammatory signaling pathways are regulated by the proteasome, Shock, 25: 27 Ponnappan,S., Ovaa,H. and Ponnappan,U. Lower expression of catalytic and structural subunits 472-484, 2006. of the proteasome contributes to decreased proteolysis in peripheral blood T lymphocytes during 12 Paramore,A. and Frantz,S. Bortezomib, Nat.Rev.Drug Discov., 2: 611-612, 2003. aging, Int.J.Biochem.Cell Biol., 39: 799-809, 2007. 13 Richardson,P.G., Mitsiades,C., Hideshima,T. and Anderson,K.C. Bortezomib: proteasome inhibition 28 Wheat,L.M., Kohlhaas,S.L., Monbaliu,J., De,C.R., Majid,A., Walewska,R.J. and Dyer,M.J. Inhibition as an effective anticancer therapy, Annu.Rev.Med., 57: 33-47, 2006. of bortezomib-induced apoptosis by red blood cell uptake, Leukemia, 20: 1646-1649, 2006. 14 Mitsiades,C.S., Treon,S.P., Mitsiades,N., Shima,Y., Richardson,P., Schlossman,R., Hideshima,T. and 29 Papandreou,C.N., Daliani,D.D., Nix,D., Yang,H., Madden,T., Wang,X., Pien,C.S., Millikan,R.E., Tu,S. Anderson,K.C. TRAIL/Apo2L ligand selectively induces apoptosis and overcomes drug resistance in M., Pagliaro,L., Kim,J., Adams,J., Elliott,P., Esseltine,D., Petrusich,A., Dieringer,P., Perez,C. and multiple myeloma: therapeutic applications, Blood, 98: 795-804, 2001. Logothetis,C.J. Phase I trial of the proteasome inhibitor bortezomib in patients with advanced solid 15 Strauss,S.J., Maharaj,L., Hoare,S., Johnson,P.W., Radford,J.A., Vinnecombe,S., Millard,L., tumors with observations in androgen-independent prostate cancer, J.Clin.Oncol., 22: 2108-2121, Rohatiner,A., Boral,A., Trehu,E., Schenkein,D., Balkwill,F., Joel,S.P. and Lister,T.A. Bortezomib 2004. therapy in patients with relapsed or refractory lymphoma: potential correlation of in vitro 30 Leveque,D., Carvalho,M.C. and Maloisel,F. Review. Clinical pharmacokinetics of bortezomib, In sensitivity and tumor necrosis factor alpha response with clinical activity, J.Clin.Oncol., 24: 2105- Vivo, 21: 273-278, 2007. 2112, 2006. 31 Berenson,J.R., Jagannath,S., Barlogie,B., Siegel,D.T., Alexanian,R., Richardson,P.G., Irwin,D., 16 Zhang,N., Ahsan,M.H., Purchio,A.F. and West,D.B. Serum amyloid A-luciferase transgenic mice: Alsina,M., Rajkumar,S.V., Srkalovic,G., Singhal,S., Limentani,S., Niesvizky,R., Esseltine,D.L., response to sepsis, acute arthritis, and contact hypersensitivity and the effects of proteasome Trehu,E., Schenkein,D.P. and Anderson,K. Safety of prolonged therapy with bortezomib in relapsed inhibition, J.Immunol., 174: 8125-8134, 2005. or refractory multiple myeloma, Cancer, 104: 2141-2148, 2005. 17 Palombella,V.J., Conner,E.M., Fuseler,J.W., Destree,A., Davis,J.M., Laroux,F.S., Wolf,R.E., Huang,J., 32 Bang,H., Egerer,K., Gauliard,A., Luthke,K., Rudolph,P.E., Fredenhagen,G., Berg,W., Feist,E. Brand,S., Elliott,P.J., Lazarus,D., McCormack,T., Parent,L., Stein,R., Adams,J. and Grisham,M.B. Role and Burmester,G.R. Mutation and citrullination modifies vimentin to a novel autoantigen for of the proteasome and NF-kappaB in streptococcal cell wall-induced polyarthritis, Proc.Natl.Acad. rheumatoid arthritis, Arthritis Rheum., 56: 2503-2511, 2007. Sci.U.S.A, 95: 15671-15676, 1998. 33 Saleem,B., Nizam,S. and Emery,P. Can remission be maintained with or without further drug 18 Neubert,K., Meister,S., Moser,K., Weisel,F., Maseda,D., Amann,K., Wiethe,C., Winkler,T.H., therapy in rheumatoid arthritis? Clin.Exp.Rheumatol., 24: S-6, 2006. Kalden,J.R., Manz,R.A. and Voll,R.E. The proteasome inhibitor bortezomib depletes plasma cells 34 Kisselev,A.F. and Goldberg,A.L. Proteasome inhibitors: from research tools to drug candidates, and protects mice with lupus-like disease from nephritis, Nat.Med., 14: 748-755, 2008. Chem.Biol., 8: 739-758, 2001. 19 Gerards,A.H., de Lafthouder, S., De Groot,E.R., Dijkmans,B.A.C. and Aarden,L.A. Inhibition of 35 Nencioni,A., Grunebach,F., Patrone,F., Ballestrero,A. and Brossart,P. The proteasome and its cytokine production by methotrexate. Studies in healthy volunteers and patients with rheumatoid inhibitors in immune regulation and immune disorders, Crit Rev.Immunol., 26: 487-498, 2006. arthritis, Rheumatology.(Oxford), 42: 1189-1196, 2003. 20 McInnes,I.B. and Schett,G. Cytokines in the pathogenesis of rheumatoid arthritis, Nat.Rev.

180 181 chapter 11 General discussion and future perspectives chapter 11 General discussion and future perspectives

It is generally accepted that the onset of drug resistance is a major limiting factor in drug exposure, but also by environmental factors. Another example of DMARD-induced sustaining a long term therapeutic effect by drugs, regardless whether it concerns MDR protein expression is MRP1, that appeared to be dominantly involved in conferring treatment of patients with infectious diseases, neoplastic diseases or chronic inflammatory resistance to chloroquine (8). In contrast, in sulfasalazine resistant T-cells a decreased diseases. Also, multiple mechanisms can underlie the resistant phenotype of various types MRP1 expression was observed and subsequently enhanced sensitivity to chloroquine. of therapeutic drugs, including disease modifying anti-rheumatic drugs (1, 2). In this Chloroquine resistant T-cells were highly cross-resistant to glucocorticoids, in contrast thesis, we focussed primarily on a major mechanism of drug resistance associated with to sulfasalazine-resistant cells that gained sensitivity to glucocorticoids due to increased drug efflux transporters from the ABC transporter family, being aware that other possible stability of the glucocorticoid receptor α (9). In sulfasalazine resistant T-cells, cross- mechanisms of resistance to DMARDs should not be excluded when a MDR phenotype can resistance to MTX was observed due to inhibition of cellular entry of MTX by sulfasalazine. not fully explain the molecular basis of resistance. These observations underscore that a detailed characterization of resistant cells may disclose specific drug interactions, either adverse or favourable, which may be exploited clinically (10). Specifically, the above-described effects for the drugs used clinically in MDR proteins & DMARD resistance the COBRA and O’Dell treatment protocols for RA (11, 12), may warrant further in depth investigations to provide a better mechanistic basis for its efficacy. Table 1 summarizes the Cumulative data from the current project and studies by others have now established a clear picture of which DMARDs can be substrates for specific drug efflux transporters, Table 1: Possible DMARD interactions in combination schedules for RA. notably P-glycoprotein (Pgp), Multidrug Resistance associated Protein 1-5 (MRP1-5, ABCC1- 5) and Breast Cancer Resistance Protein (BCRP, ABCG2) and how upregulation of drug Drug combinations Type of interaction Mechanism of interaction

transporters may contribute to DMARD resistance (see chapter 2 and Table 1; summary SSZ + glucocorticoids Synergistic SSZ stabilizes the GRα, thereby section). increasing the expression of GRα and enhancing efficacy of glucocoticoids

BCRP expression on synovial tissue macrophages SSZ + CHQ Synergistic SSZ downregulates MRP1 for which Induction of the drug efflux transporter BCRP appeared to play a key role in the onset of CHQ is a substrate, thereby enhancing CHQ efficacy acquired resistance of T-cells to the DMARD sulfasalazine in vitro. Interestingly, rather than a DMARD-provoked induction of BCRP expression, we also observed abundant expression SSZ + MTX Negative interaction (1) SSZ is a potent inhibitor of the RFC, the dominant transporter for of BCRP on macrophages in the intimal lining layer and the synovial sublining of synovial cellular uptake of MTX, thereby biopsies from RA patients even prior to receiving DMARD therapy. These observations may inducing MTX resistance comply with at least two mechanisms implicated in the induction of BCRP expression. First, (2) SSZ induces BCRP expression upon prolonged exposure, thereby evidence has been presented for an epigenetic regulation of BCRP expression by diminished inducing MTX resistance since MTX BCRP promoter methylation (3, 4). Consistent with this possibility we showed (see chapter is a BCRP substrate 4) that sulfasalazine is a potent inhibitor of the cell membrane carrier for reduced folate CHQ + glucocorticoids Negative interaction CHQ induces abberant analogues, utilized intracellularly for DNA-methylation processes. Thus, (partial) blocking signaling via the cAMP/PKA pathway of cellular folate uptake by increasing concentrations of sulfasalazine during development downstream of the GRα, thereby inducing glucocorticoid resistance of resistance may have resulted in enhanced BCRP expression due to diminished

methylation of a potential CpG island in the BCRP promoter. Second, BCRP expression is MTX + CHQ Negative interaction CHQ induces MRP1 expression also induced under hypoxic conditions, which may apply to RA synovium, by the action of for which MTX is a substrate, thereby inducing MTX resistance the transcription factor HIF1α (5). In this context, it has also been reported that specific RA pathophysiological conditions of aberrant production of pro-inflammatory cytokines can Abbreviations: SSZ: sulfasalazine; MTX: methotrexate; CHQ: chloroquine; MRP1: multidrug resistance protein 1, GR: glucocorticoid receptor; cAMP: cyclic adenine monophosphate; upregulate the expression of other MDR transporters, notably Pgp and MRP1 (6, 7). Hence, PKA: protein kinase A; RFC: reduced folate carrier. alterations in expression levels of MDR transporters can be controlled not only by chronic

184 185 chapter 11 General discussion and future perspectives

possible DMARD interactions in combination therapies for RA treatment according to our Reversal of multiple drug resistance in vitro studies on T-cells. Conceptually, specific blockers of MDR proteins could be exploited to reverse a MDR phenotype. Indeed, in cancer chemotherapeutic model systems, specific blockers of MDR MDR-protein expression on RA peripheral blood lymphocytes transporters could reverse resistance to MDR-related drugs. Unfortunately, in a clinical Beyond studies with in vitro experimental model systems and synovial tissue (chapter setting this approach proved to be far less successful (24) (25) for reasons discussed in 3,4 and 5), screening of peripheral blood-derived cells showed protein and/or functional chapter 2. Limitations in effective reversal of Pgp-mediated drug resistance by blocking expression of several MDR transporters in T cells (Pgp, MRPs), monocytes (Pgp, MRPs), agents included for example; (a) effective plasma levels of blockers could not be reached, (b) dendritic cells (Pgp, MRPs, BCRP) and macrophages (MRPs, BCRP), some of which at enhanced metabolism of the blocking agent, (c) toxic side effects of the blockers, (d) drug higher expression levels in RA patients as compared to healthy controls (R. Oerlemans, interaction phenomena, (e) presence of multiple MDR transporters, and (f) interference Ph.D. thesis in preparation) (13). However, it should be taken into account that these with physiological functions of Pgp. However, it still remains to be established whether indicated transporters still comprise a fraction (∼20%) of the 49 members of the ABC family blockers of MRPs or BCRP face similar limitations as Pgp blockers. of drug transporters for which a function has been revealed. Interestingly, some of the One interesting aspect of blocking BCRP activity in relation to DMARD bioavailability other members of the ABC transporter family have recently be identified to be involved was indicated by a study by Zaher et al (26) who showed that blocking of intestinal BCRP in processes that can be of direct and indirect relevance for pathophysiological processes markedly elevated plasma levels of sulfasalazine, which is a substrate for BCRP (see in RA; e.g. involvement of ABCG5 in cholesterol transport (possible implications for chapter 3). Besides enhancing sulfasalazine uptake by blocking intestinal BCRP activity, cardiovascular disease) (14) or involvement of ABCA1 in cellular extrusion of interleukin-1β co-administration of selected BCRP blockers could also increase intracellular sulfasalazine (15). From this perspective, it will be worthwhile to further pursue unravelling the role of levels in BCRP expressing synovial tissue macrophages and subsequently improve specific ABC transporters in RA. For this purpose, customized micro-arrays have become therapeutic efficacy. available that may allow detection of mRNA levels of most of the known ABC-transporters in one assay (16). In this manner, MDR expression in patient samples (synovial tissue biopsies; immune-effector cells from peripheral blood) can be assessed before and during therapy. Methotrexate resistance MDR-proteins that are differentially expressed can then be analyzed for functionality in terms of efflux properties and correlated to the clinical response to specific DMARDs. There is consensus that drug efflux via MDR proteins MRP1-5 and BCRP can provide a mechanistic basis for conferring resistance to the anchor drug in RA treatment, the antifolate Pharmacological & physiological roles of MDR proteins: balanced dual functions MTX (see also chapter 2) (27). The relative importance of MDR proteins for MTX resistance Expression of several specific MDR transporters has been reported in almost all cell types in RA however is not readily clear, as other modes of resistance to MTX (discussed in chapter of the haematopoietic lineage, from stems cells to fully differentiated cells (17). Although 2) (28-31) may be operative separately or alongside a MDR mechanism. The contribution by these MDR proteins constitute an efficient mode of cellular defence to therapeutic MDR proteins to MTX resistance may depend on several factors: type of MDR transporter(s) drugs and xenobiotic compounds, this is unlikely to represent their sole function. MDR being expressed in various types of immune cells, dosing of MTX, efficiency of intracellular transporters such as Pgp, several MRPs and BCRP can transport compounds such as metabolism to polyglutamate forms and cellular folate status. These latter two conditions bioactive lipids (prostaglandins, leucotrienes) and steroid hormones (18-21) through which determine whether MTX is converted to long-chain polyglutamate moieties that are they may regulate functional properties of immune-competent cells. Prototypically, no longer substrates for MDR proteins. It may be anticipated that in immune cells with this is illustrated for antigen presenting dendritic cells whose migration depends on either low levels of polyglutamylation capacity and/or high intracellular folate levels that functional Pgp and MRP1 activity (22, 23). Consequently, interference with Pgp or MRP1 compete with MTX polyglutamylation, MTX will become poorly polyglutamylated and activity may potentially attenuate an appropriate immune response; for (RA) patients thus becomes available for cellular extrusion by either one of the MRPs or BCRP. Regarding with an augmented immune-response this may be therapeutically beneficial, but in intracellular folate status, this may be determined by several factors, of which dietary immune-compromised patients, it may evoke adverse effects. Further assessment of the intake and/or vitamin supplementations are most relevant, especially since food and physiological roles of MDR transporters in patients with chronic inflammatory diseases drug administrations in the US and most recently also in the Netherlands recommended therefore deserves further attention in future studies. folate food fortification to reduce the incidence of neural tube defects in neonates (32)

186 187 chapter 11 General discussion and future perspectives

(‘Towards an optimal use of folic acid’: Health Council of the Netherlands, The Hague, The Netherlands, 2008; publication no. 2008/2). A downside of this arrangement may be an Figure 1 enhancement of MDR mediated efflux of MTX and thereby a reduced efficacy (33, 34). Recently, several attempts have been initiated to generate predictive models for MTX toxicity and MTX responses in RA treatment, based on composites of common single nucleotide polymorphisms (SNPs) of genes coding for folate or methotrexate transporters (RFC) and key enzymes in folate or purine metabolism (35-39). It is intriguing to note that these models have a marked predictive power, even when it has not been established what Figure 1: Routes of cellular entry for MTX and folic acid. MTX has a high affinity for the Reduced Folate Carrier (RFC) and a relatively low affinity for the Folate Receptor specific impact polymorphic variations have on functional properties of the transporters (FR), so uptake of MTX occurs mainly via the RFC. Folic acid however, is mainly taken up by the FR, due to and enzymes included in the model. A limitation of these models could be that patients a very high affinity for this receptor and a concomitant low affinity for the RFC. The RFC is constitutively from different ethnic background do not have the same polymorphisms in the studied expressed on all somatic cells, whereas the expression of the folate receptors (α, β and γ isoform) is restricted to certain tissues/cell types. Given the notion that the β-isoform of the FR is specifically expressed on activated genes (40). Finally, these models do not incorporate other important parameters such macrophages in synovial tissue, this receptor may be exploited as selective target in RA treatment. as functional expression and regulation of different folate/MTX transporters and (feed back) regulation of folate enzymes, contribution of various MDR transporter, intracellular folate status along with folate compartmentalization (2, 41, 42). In conjunction with this Targeting of synovial macrophages via the folate receptor-β knowledge, predictive models for MTX responsiveness and toxicity may be even more Activated macrophages in the synovial membrane play a central role in the chronic course reliable. Thus, before clinicians can predict the response on MTX treatment, these models of RA, by facilitating a persistent pro-inflammatory state of other inflammatory cells and have to be fine-tuned and further tested in appropriate study populations. synovial fibroblasts and the bone-destructive activity of osteoclasts. Numbers of synovial tissue macrophages are associated with disease activity and recently Haringman et al. showed that change in the number of synovial tissue macrophages is a highly sensitive Novel folate antagonists biomarker for response to treatment in RA patients (44). Therefore, selective targeting of synovial tissue macrophages via the FR-β might be an attractive strategy (figure 1). We MTX is the anchor drug in RA treatment, exhibiting a relatively long-lasting activity demonstrated a high expression of FR-β on macrophages in RA-synovial tissue. We also as single agent or in combination schedules. A second generation of folate antagonists identified an antifolate drug candidate for selective targeting of this receptor, notably the was rationally developed to overcome various modes of cellular resistance to MTX by TS inhibitor BGC945. However, an important issue in FR-β targeting to take into account harboring properties of either: more efficient drug uptake via the Reduced Folate Carrier is again a current nutritional policy of folate food fortification (32), which may reduce than MTX (e.g. PT523, PT644); better polyglutamylation than MTX (e.g. pemetrexed, the activity of folate antagonists by receptor occupancy/competition (45). For example, raltitrexed); being polyglutamylation-independent (e.g. BGC9331), targeting other key Arabelovic et al. recently showed that folic acid fortification is associated with higher enzymes in folate metabolism (Pemetrexed, Raltitrexed); or retaining activity regardless MTX dosing in RA patients (46). Furthermore, high levels of circulating folates in synovial of intracellular folate status. Several of these novel generation drugs have been evaluated tissue/plasma can result in down regulation of FR-β expression (47). Thus, it is anticipated in clinical trials for cancer treatment and some have recently been registered as anti- that dietary folate restrictions would facilitate optimal FR-β targeting of synovial tissue cancer drugs (43). We demonstrated that these compounds also harbor promising anti- macrophages in RA patients. inflammatory properties. Although it may be hard to replace MTX as golden standard by one of these novel generation folate antagonists, their use may be considered for those RA Expectations for future research patients that are refractory to MTX treatment. Since all these drug are preferentially taken Interestingly, all-trans retinoic acid (ATRA) was reported to enhance the expression of up by the Reduced Folate Carrier, which is constitutively expressed in most somatic cells, FR-β on myeloid cells and to render these cells more susceptible to selective therapeutic these drugs may have a broad spectrum of immune-competent cells for targeting. The one interventions (48-50). Recently it was also shown that, at non-toxic concentrations, exception may be activated macrophages, in which a folate receptor-β mediated route of histone deacetylase (HDAC) inhibitors (e.g. valproic acid and trichostatin A) may be entry is operative, which may require specific targeting with other folate antagonists. capable of inducing FR-β expression on myeloid cells, consistent with the notion that

188 189 chapter 11 General discussion and future perspectives

histone deacetylation regulates the transcription of the FR-β gene (51). For this reason, in salivary glands of patients with Sjögren syndrome (61). Whether this implicates a role ATRA and HDAC inhibitors warrant further evaluation as potential positive modulators of for proteasomes and altered protein/peptide degradation in the pathophysiology of FR-β expression on macrophages. This approach may facilitate a selective macrophage- auto-immune diseases or just reflects disease activity, remains to be established. Finally, targeted therapy in RA, with greater efficacy and low systemic toxicity (because FR-β is Yoshimura et al showed that activated NFκB is required for antigen presentation by dominantly expressed on activated macrophages). Dendritic Cells (DCs), and that NFκB inhibition with the proteasome inhibitor bortezomib resulted in down regulation of MHC class II molecules and CD86 on DCs along with Beyond exploitation of the FR-β on synovial macrophages for targeting with small molecule inhibition of the production of immunostimulatory cytokines like IL-12 and TNFα (62). folate antagonists, the high affinity binding of the receptor for any folate conjugated Based on these studies, it may be speculated that inhibition of the proteasome could (toxic) macromolecule/protein or tracer allows additional therapeutic targeting options evoke dampening of the over-active immune response in RA. as well as opportunities for imaging of RA disease activity, e.g. by folate-conjugated Positron Emission Tomography (PET) tracers. (52-55). In fact, these latter types of studies The proteasome and ageing are currently being initiated in our department. The proteasome is also thought to play a central role in the process of ageing. Several studies showed a decrease in the constitutive proteasome content in different tissues and a concomitant accumulation of oxidized and ubiquinated proteins upon ageing (63-65). Novel experimental drugs: proteasome inhibitors Conversely, the cellular content of immunoproteasomes remains constant, even though qualitative or quantitative changes in immunoproteasome activity in elderly might lead Proteasome inhibitors represent a novel generation of therapeutic drugs that may be of to alterations of presented epitopes (66). This impaired processing of proteins/peptides interest to bypass MDR-associated drug resistance; the proteasome inhibitor bortezomib, and altered antigen presentation, may render susceptibility for an auto-immune response which is registered for therapy-refractory multiple myeloma, has been identified to be a in ageing people. poor substrate for most MDR transporters. We demonstrated that bortezomib exhibited potent anti-inflammatory properties by blocking TNFα production from ex-vivo activated Expectations for future research T-cells of RA patients, tentatively through inhibition of the activation of the transcription Given the above considerations, it would be of interest to investigate whether either factor NFκB (56). In addition, other studies have reported that bortezomib effectively short or chronic-exposure of immune effector cells to bortezomib or other experimental reduced inflammation in animal models of streptococcal cell wall-induced polyarthritis proteasome inhibitors may elicit beneficial therapeutic effects. Obviously, any design (57) and systemic lupus erythematosus (58). of a trial for these group of drugs in RA treatment should address a number of issues including (a) assessment of optimal dosing/therapeutic window for RA treatment, and The proteasome and auto-immunity (b) special precautions with respect to long term drug safety given the anticipated Apart from blocking of NFκB activation in RA inflammatory cells, bortezomib could chronic administration (67). When these issues can not be met for bortezomib, second have more potentially benefits in RA treatment. It is well known that the proteasome is generation of proteasome inhibitors (68-70) may meet this profile as, unlike bortezomib, engaged in the process of antigen presentation via MHC class I molecules and therefore these are irreversible inhibitors which may provoke less off-target (toxic) effects than plays an important role in the initiation and prolongation of the immune response bortezomib. Finally, proteasome inhibitors may also serve, at low doses, as potentiators by determination of the difference between self and non-self antigens (59). In fact, of glucocorticoid activity (71) which may also be of interest from an RA therapeutic alterations in the functionality or expression of the proteasome may be involved in the perspective. onset of auto-immune diseases. In this context, Hayashi et al showed that downregulation of one of the proteasome subunits, and thereby impaired antigen presentation and T-cell education towards self, has been identified as the cause for the onset of insulinitis in type - 1 diabetes in the Non Obese Diabetes (NOD) mouse model (60). On the other hand, Egerer et al observed that the catalytic subunit LMP7 of the immunoproteasome (differing from constitutive proteasomes by 3 IFNγ-inducible β-subunits) was specifically up regulated

190 191 References References

References secretion is impaired by inhibitors of the Atp binding cassette transporter, ABC1, Blood, 90: 2911- 2915, 1997. 1 Jansen,G., Scheper,R.J. and Dijkmans,B.A.C. Multidrug resistance proteins in rheumatoid 16 Gillet,J.P., Efferth,T., Steinbach,D., Hamels,J., de Longueville,F., Bertholet,V. and Remacle,J. arthritis, role in disease-modifying antirheumatic drug efficacy and inflammatory processes: an Microarray-based detection of multidrug resistance in human tumor cells by expression profiling overview, Scand.J.Rheumatol., 32: 325-336, 2003. of ATP-binding cassette transporter genes, Cancer Res., 64: 8987-8993, 2004. 2 Van der Heijden,J.W., Dijkmans,B.A.C., Scheper,R.J. and Jansen,G. Drug Insight: resistance to 17 Kock,K., Grube,M., Jedlitschky,G., Oevermann,L., Siegmund,W., Ritter,C.A. and Kroemer,H.K. methotrexate and other disease-modifying antirheumatic drugs-from bench to bedside, Nat. Expression of adenosine triphosphate-binding cassette (ABC) drug transporters in peripheral Clin.Pract.Rheumatol., 3: 26-34, 2007. blood cells: relevance for physiology and pharmacotherapy, Clin.Pharmacokinet., 46: 449-470, 3 To,K.K., Zhan,Z. and Bates,S.E. Aberrant promoter methylation of the ABCG2 gene in renal 2007. carcinoma, Mol.Cell Biol., 26: 8572-8585, 2006. 18 Deeley,R.G., Westlake,C. and Cole,S.P. Transmembrane transport of endo- and xenobiotics by 4 Turner,J.G., Gump,J.L., Zhang,C., Cook,J.M., Marchion,D., Hazlehurst,L., Munster,P., Schell,M.J., mammalian ATP-binding cassette multidrug resistance proteins, Physiol Rev., 86: 849-899, Dalton,W.S. and Sullivan,D.M. ABCG2 expression, function, and promoter methylation in human 2006. multiple myeloma, Blood, 108: 3881-3889, 2006. 19 Sarkadi,B., Homolya,L., Szakacs,G. and Varadi,A. Human multidrug resistance ABCB and ABCG 5 Krishnamurthy,P., Ross,D.D., Nakanishi,T., Bailey-Dell,K., Zhou,S., Mercer,K.E., Sarkadi,B., transporters: participation in a chemoimmunity defense system, Physiol Rev., 86: 1179-1236, Sorrentino,B.P. and Schuetz,J.D. The stem cell marker Bcrp/ABCG2 enhances hypoxic cell survival 2006. through interactions with heme, J.Biol.Chem., 279: 24218-24225, 2004. 20 Gottesman,M.M. and Ling,V. The molecular basis of multidrug resistance in cancer: the early 6 Stein,U., Walther,W., Laurencot,C.M., Scheffer,G.L., Scheper,R.J. and Shoemaker,R.H. Tumor years of P-glycoprotein research, FEBS Lett., 580: 998-1009, 2006. necrosis factor-alpha and expression of the multidrug resistance- associated genes LRP and MRP, 21 Borst,P. and Oude Elferink,R.P. Mammalian ABC transporters in health and disease, Annu.Rev. J.Natl.Cancer Inst., 89: 807-813, 1997. Biochem., 71: 537-592, 2002. 7 Ho,E.A. and Piquette-Miller,M. Regulation of multidrug resistance by pro-inflammatory 22 Robbiani,D.F., Finch,R.A., Jager,D., Muller,W.A., Sartorelli,A.C. and Randolph,G.J. The leukotriene cytokines, Curr.Cancer Drug Targets., 6: 295-311, 2006. C(4) transporter MRP1 regulates CCL19 (MIP-3beta, ELC)- dependent mobilization of dendritic 8 Oerlemans,R., van der Heijden J., Vink,J., Dijkmans,B.A.C., Kaspers,G.J., Lems,W.F., Scheffer,G.L., cells to lymph nodes, Cell, 103: 757-768, 2000. Ifergan,I., Scheper,R.J., Cloos,J., Assaraf,Y.G. and Jansen,G. Acquired resistance to chloroquine in 23 van der Ven,R., de Jong,M.C., Reurs,A.W., Schoonderwoerd,A.J., Jansen,G., Hooijberg,J.H., human CEM T cells is mediated by multidrug resistance-associated protein 1 and provokes high Scheffer,G.L., de Gruijl,T.D. and Scheper,R.J. Dendritic cells require multidrug resistance protein levels of cross-resistance to glucocorticoids, Arthritis Rheum., 54: 557-568, 2006. 1 (ABCC1) transporter activity for differentiation, J.Immunol., 176: 5191-5198, 2006. 9 Oerlemans,R., Vink,J., Dijkmans,B.A.C., Assaraf,Y.G., van Miltenburg,M., Van der Heijden,J.W., 24 Szakacs,G., Paterson,J.K., Ludwig,J.A., Booth-Genthe,C. and Gottesman,M.M. Targeting Ifergan,I., Lems,W.F., Scheper,R.J., Kaspers,G.J., Cloos,J. and Jansen,G. Sulfasalazine sensitises multidrug resistance in cancer, Nat.Rev.Drug Discov., 5: 219-234, 2006. human monocytic/macrophage cells for glucocorticoids by upregulation of glucocorticoid 25 Modok,S., Mellor,H.R. and Callaghan,R. Modulation of multidrug resistance efflux pump activity receptor alpha and glucocorticoid induced apoptosis, Ann.Rheum.Dis., 66: 1289-1295, 2007. to overcome chemoresistance in cancer, Curr.Opin.Pharmacol., 6: 350-354, 2006. 10 Smolen,J.S., Aletaha,D. and Keystone,E. Superior efficacy of combination therapy for rheumatoid 26 Zaher,H., Khan,A.A., Palandra,J., Brayman,T.G., Yu,L. and Ware,J.A. Breast Cancer Resistance arthritis: fact or fiction?, Arthritis Rheum., 52: 2975-2983, 2005. Protein (Bcrp/abcg2) is a major determinant of sulfasalazine absorption and elimination in 11 Landewe,R.B., Boers,M., Verhoeven,A.C., Westhovens,R., van de Laar,M.A., Markusse,H.M., mouse, Molecular Pharmaceutics, 3: 55-61, 2006. van Denderen,J.C., Westedt,M.L., Peeters,A.J., Dijkmans,B.A.C., Jacobs,P., Boonen,A., van der 27 Assaraf,Y.G. Molecular basis of antifolate resistance, Cancer Metastasis Rev., 26: 153-181, 2007. Heijde,D.M. and Van der Linden,S. COBRA combination therapy in patients with early rheumatoid 28 Rots,M.G., Pieters,R., Peters,G.J., Noordhuis,P., Van Zantwijk,C.H., Henze,G., Janka-Schaub,G.E., arthritis: Long-term structural benefits of a brief intervention, Arthritis Rheum., 46: 347-356, Veerman,A.J. and Jansen,G. Methotrexate resistance in relapsed childhood acute lymphoblastic 2002. leukaemia, Br.J.Haematol., 109: 629-634, 2000. 12 Goekoop-Ruiterman,Y.P., De Vries-Bouwstra,J.K., Allaart,C.F., van Zeben,D., Kerstens,P. 29 Kager,L., Cheok,M., Yang,W., Zaza,G., Cheng,Q., Panetta,J.C., Pui,C.H., Downing,J.R., Relling,M. J., Hazes,J.M., Zwinderman,A.H., Ronday,H.K., Han,K.H., Westedt,M.L., Gerards,A.H., van V. and Evans,W.E. Folate pathway gene expression differs in subtypes of acute lymphoblastic Groenendael,J.H., Lems,W.F., van Krugten,M.V., Breedveld,F.C. and Dijkmans,B.A.C. Clinical and leukemia and influences methotrexate pharmacodynamics, J.Clin.Invest, 115: 110-117, 2005. radiographic outcomes of four different treatment strategies in patients with early rheumatoid 30 Gorlick,R., Goker,E., Trippett,T., Waltham,M., Banerjee,D. and Bertino,J.R. Intrinsic and acquired arthritis (the BeSt study): a randomized, controlled trial, Arthritis Rheum., 52: 3381-3390, 2005. resistance to methotrexate in acute leukemia, N.Engl.J.Med., 335: 1041-1048, 1996. 13 Oerlemans,R., Dijkmans,B.A.C., Van der Heijden,J.W., Lems,W.F., Reurs,A.W., Scheffer,G.L., 31 Kremer,J.M. Toward a better understanding of methotrexate, Arthritis Rheum., 50: 1370-1382, Scheper,R.J. and Jansen,G. Differential expression of multidrug resistance-related proteins 2004. on monocyte-derived macrophages from rheumatoid arthritis patients, Arthritis Research & 32 Smith,A.D., Kim,Y.I. and Refsum,H. Is folic acid good for everyone? Am.J.Clin.Nutr., 87: 517-533, Therapy, 7: S32, 2005. 2008. 14 Langmann,T., Mauerer,R. and Schmitz,G. Human ATP-binding cassette transporter TaqMan low- 33 Hooijberg,J.H., Jansen,G., Assaraf,Y.G., Kathmann,I., Pieters,R., Laan,A.C., Veerman,A.J., density array: analysis of macrophage differentiation and foam cell formation, Clin.Chem., 52: Kaspers,G.J. and Peters,G.J. Folate concentration dependent transport activity of the Multidrug 310-313, 2006. Resistance Protein 1 (ABCC1), Biochem.Pharmacol., 67: 1541-1548, 2004. 15 Hamon,Y., Luciani,M.F., Becq,F., Verrier,B., Rubartelli,A. and Chimini,G. Interleukin-1beta 34 Hooijberg,J.H., de Vries,N.A., Kaspers,G.J., Pieters,R., Jansen,G. and Peters,G.J. Multidrug

192 193 References References

resistance proteins and folate supplementation: therapeutic implications for antifolates and 50 Elnakat,H. and Ratnam,M. Distribution, functionality and gene regulation of folate receptor other classes of drugs in cancer treatment, Cancer Chemother.Pharmacol., 58: 1-12, 2006. isoforms: implications in targeted therapy, Adv.Drug Deliv.Rev., 56: 1067-1084, 2004. 35 Ranganathan,P., Eisen,S., Yokoyama,W.M. and McLeod,H.L. Will pharmacogenetics allow better 51 Qi,H. and Ratnam,M. Synergistic induction of folate receptor beta by all-trans retinoic acid and prediction of methotrexate toxicity and efficacy in patients with rheumatoid arthritis?, Ann. histone deacetylase inhibitors in acute myelogenous leukemia cells: mechanism and utility in Rheum.Dis., 62: 4-9, 2003. enhancing selective growth inhibition by antifolates, Cancer Res., 66: 5875-5882, 2006. 36 Dervieux,T., Furst,D., Lein,D.O., Capps,R., Smith,K., Walsh,M. and Kremer,J. Polyglutamation 52 Turk,M.J., Breur,G.J., Widmer,W.R., Paulos,C.M., Xu,L.C., Grote,L.A. and Low,P.S. Folate-targeted of methotrexate with common polymorphisms in reduced folate carrier, aminoimidazole imaging of activated macrophages in rats with adjuvant- induced arthritis, Arthritis Rheum., 46: carboxamide ribonucleotide transformylase, and thymidylate synthase are associated with 1947-1955, 2002. methotrexate effects in rheumatoid arthritis, Arthritis Rheum., 50: 2766-2774, 2004 53 Paulos,C.M., Turk,M.J., Breur,G.J. and Low,P.S. Folate receptor-mediated targeting of therapeutic 37 Wessels,J.A., De Vries-Bouwstra,J.K., Heijmans,B.T., Slagboom,P.E., Goekoop-Ruiterman,Y. and imaging agents to activated macrophages in rheumatoid arthritis, Adv.Drug Deliv.Rev., 56: P., Allaart,C.F., Kerstens,P.J., van Zeben,D., Breedveld,F.C., Dijkmans,B.A.C., Huizinga,T.W. and 1205-1217, 2004. Guchelaar,H.J. Efficacy and toxicity of methotrexate in early rheumatoid arthritis are associated 54 Low,P.S., Henne,W.A. and Doorneweerd,D.D. Discovery and development of folic-Acid-based with single-nucleotide polymorphisms in genes coding for folate pathway enzymes, Arthritis receptor targeting for imaging and therapy of cancer and inflammatory diseases, Acc.Chem. Rheum., 54: 1087-1095, 2006. Res., 41: 120-129, 2008. 38 Wessels,J.A., van der Kooij,S.M., le Cessie S., Kievit,W., Barerra,P., Allaart,C.F., Huizinga,T.W. 55 Salazar,M.D. and Ratnam,M. The folate receptor: what does it promise in tissue-targeted and Guchelaar,H.J. A clinical pharmacogenetic model to predict the efficacy of methotrexate therapeutics? Cancer Metastasis Rev., 26: 141-152, 2007. monotherapy in recent-onset rheumatoid arthritis, Arthritis Rheum., 56: 1765-1775, 2007. 56 Rajkumar,S.V., Richardson,P.G., Hideshima,T. and Anderson,K.C. Proteasome inhibition as a 39 Dervieux,T., Greenstein,N. and Kremer,J. Pharmacogenomic and metabolic biomarkers in the novel therapeutic target in human cancer, J.Clin.Oncol., 23: 630-639, 2005. folate pathway and their association with methotrexate effects during dosage escalation in 57 Palombella,V.J., Conner,E.M., Fuseler,J.W., Destree,A., Davis,J.M., Laroux,F.S., Wolf,R.E., Huang,J., rheumatoid arthritis, Arthritis Rheum., 54: 3095-3103, 2006. Brand,S., Elliott,P.J., Lazarus,D., McCormack,T., Parent,L., Stein,R., Adams,J. and Grisham,M.B. 40 Cronstein,B.N. Do genetic variations in the adenosine pathway affect patient response to Role of the proteasome and NF-kappaB in streptococcal cell wall-induced polyarthritis, Proc. methotrexate? Nat.Clin.Pract.Rheumatol., 2: 648-649, 2006. Natl.Acad.Sci.U.S.A, 95: 15671-15676, 1998. 41 Assaraf,Y.G. The role of multidrug resistance efflux transporters in antifolate resistance and 58 Neubert,K., Meister,S., Moser,K., Weisel,F., Maseda,D., Amann,K., Wiethe,C., Winkler,T.H., folate homeostasis, Drug Resist.Updat., 9: 227-246, 2006. Kalden,J.R., Manz,R.A. and Voll,R.E. The proteasome inhibitor bortezomib depletes plasma cells 42 Jansen,G., Mauritz,R.M., Assaraf,Y.G., Sprecher,H., Drori,S., Kathmann,I., Westerhof,G.R., and protects mice with lupus-like disease from nephritis, Nat.Med., 14: 748-755, 2008. Priest,D.G., Bunni,M., Pinedo,H.M., Schornagel,J.H. and Peters,G.J. Regulation of carrier- 59 Kloetzel,P.M. and Ossendorp,F. Proteasome and peptidase function in MHC-class-I-mediated mediated transport of folates and antifolates in methotrexate-sensitive and-resistant leukemia antigen presentation, Curr.Opin.Immunol., 16: 76-81, 2004. cells, Adv.Enzyme Regul., 37: 59-76, 1997. 60 Hayashi,T. and Faustman,D. The role of the proteasome in autoimmunity, Diabetes Metab Res. 43 Walling,J. From methotrexate to pemetrexed and beyond. A review of the pharmacodynamic Rev., 16: 325-337, 2000. and clinical properties of antifolates, Invest New Drugs, 24: 37-77, 2006. 61 Egerer,T., Martinez-Gamboa,L., Dankof,A., Stuhlmuller,B., Dorner,T., Krenn,V., Egerer,K., 44 Haringman,J.J., Gerlag,D.M., Zwinderman,A.H., Smeets,T.J., Kraan,M.C., Baeten,D., McInnes,I. Rudolph,P.E., Burmester,G.R. and Feist,E. Tissue-specific up-regulation of the proteasome B., Bresnihan,B. and Tak,P.P. Synovial tissue macrophages: a sensitive biomarker for response to subunit beta5i (LMP7) in Sjogren’s syndrome, Arthritis Rheum., 54: 1501-1508, 2006 treatment in patients with rheumatoid arthritis, Ann Rheum.Dis., 64: 834-838, 2005. 62 Yoshimura,S., Bondeson,J., Brennan,F.M., Foxwell,B.M. and Feldmann,M. Role of NFkappaB in 45 Mauritz,R., Peters,G.J., Kathmann,I., Teshale,H., Noordhuis,P., Comijn,E.M., Pinedo,H.M. and antigen presentation and development of regulatory T cells elucidated by treatment of dendritic Jansen,G. Dynamics of antifolate transport via the reduced folate carrier and the membrane cells with the proteasome inhibitor PSI, Eur.J.Immunol., 31: 1883-1893, 2001. folate receptor in murine leukaemia cells in vitro and in vivo, Cancer Chemother.Pharmacol., 63 Egerer,K., Kuckelkorn,U., Rudolph,P.E., Ruckert,J.C., Dorner,T., Burmester,G.R., Kloetzel,P.M. 2008. and Feist,E. Circulating proteasomes are markers of cell damage and immunologic activity in 46 Arabelovic,S., Sam,G., Dallal,G.E., Jacques,P.F., Selhub,J., Rosenberg,I.H. and Roubenoff,R. autoimmune diseases, J.Rheumatol., 29: 2045-2052, 2002. Preliminary evidence shows that folic acid fortification of the food supply is associated with 64 Petropoulos,I., Conconi,M., Wang,X., Hoenel,B., Bregegere,F., Milner,Y. and Friguet,B. Increase higher methotrexate dosing in patients with rheumatoid arthritis, J.Am.Coll.Nutr., 26: 453-455, of oxidatively modified protein is associated with a decrease of proteasome activity and content 2007. in aging epidermal cells, J.Gerontol.A Biol.Sci.Med.Sci., 55: B220-B227, 2000. 47 Lucock,M. and Yates,Z. Folic acid - vitamin and panacea or genetic time bomb?, Nat.Rev.Genet., 65 Hwang,J.S., Hwang,J.S., Chang,I. and Kim,S. Age-associated decrease in proteasome content 6: 235-240, 2005. and activities in human dermal fibroblasts: restoration of normal level of proteasome subunits 48 Wang,H., Zheng,X., Behm,F.G. and Ratnam,M. Differentiation-independent retinoid induction reduces aging markers in fibroblasts from elderly persons, J.Gerontol.A Biol.Sci.Med.Sci., 62: of folate receptor type beta, a potential tumor target in myeloid leukemia, Blood, 96: 3529- 490-499, 2007. 3536, 2000. 66 Mishto,M., Santoro,A., Bellavista,E., Bonafe,M., Monti,D. and Franceschi,C. Immunoproteasomes 49 Pan,X.Q., Zheng,X., Shi,G., Wang,H., Ratnam,M. and Lee,R.J. Strategy for the treatment of acute and immunosenescence, Ageing Res.Rev., 2: 419-432, 2003. myelogenous leukemia based on folate receptor beta-targeted liposomal doxorubicin combined 67 Berenson,J.R., Jagannath,S., Barlogie,B., Siegel,D.T., Alexanian,R., Richardson,P.G., Irwin,D., with receptor induction using all-trans retinoic acid, Blood, 100: 594-602, 2002. Alsina,M., Rajkumar,S.V., Srkalovic,G., Singhal,S., Limentani,S., Niesvizky,R., Esseltine,D.L.,

194 195 References References

Trehu,E., Schenkein,D.P. and Anderson,K. Safety of prolonged therapy with bortezomib in relapsed or refractory multiple myeloma, Cancer, 104: 2141-2148, 2005. 68 Chauhan,D., Catley,L., Li,G., Podar,K., Hideshima,T., Velankar,M., Mitsiades,C., Mitsiades,N., Yasui,H., Letai,A., Ovaa,H., Berkers,C., Nicholson,B., Chao,T.H., Neuteboom,S.T., Richardson,P., Palladino,M.A. and Anderson,K.C. A novel orally active proteasome inhibitor induces apoptosis in multiple myeloma cells with mechanisms distinct from Bortezomib, Cancer Cell, 8: 407-419, 2005. 69 Kuhn,D.J., Chen,Q., Voorhees,P.M., Strader,J.S., Shenk,K.D., Sun,C.M., Demo,S.D., Bennett,M. K., van Leeuwen,F.W., Chanan-Khan,A.A. and Orlowski,R.Z. Potent activity of carfilzomib, a novel, irreversible inhibitor of the ubiquitin-proteasome pathway, against preclinical models of multiple myeloma, Blood, 110: 3281-3290, 2007. 70 Demo,S.D., Kirk,C.J., Aujay,M.A., Buchholz,T.J., Dajee,M., Ho,M.N., Jiang,J., Laidig,G.J., Lewis,E. R., Parlati,F., Shenk,K.D., Smyth,M.S., Sun,C.M., Vallone,M.K., Woo,T.M., Molineaux,C.J. and Bennett,M.K. Antitumor activity of PR-171, a novel irreversible inhibitor of the proteasome, Cancer Res., 67: 6383-6391, 2007. 71 Hideshima,T., Richardson,P., Chauhan,D., Palombella,V.J., Elliott,P.J., Adams,J. and Anderson,K. C. The proteasome inhibitor PS-341 inhibits growth, induces apoptosis, and overcomes drug resistance in human multiple myeloma cells, Cancer Res., 61: 3071-3076, 2001.

196 197 chapter 12 Summary chapter 12 Summary

Summary In chapter three we determined whether overexpression of MDR proteins contributed to a diminished efficacy of sulfasalazine in a human T cell line after prolonged cellular exposure Rheumatoid arthritis (RA) requires chronic treatment with anti-inflammatory drugs. to this drug. The rationale to select sulfasalazine for these studies was that this DMARD Unfortunately, upon prolonged treatment with potent disease modifying anti-rheumatic displayed a relatively rapid onset of loss of efficacy following prolonged administration. We drugs (DMARDs), many patients sooner or later experience loss of efficacy of these drugs found that after a period of 4-6 months of sulfasalazine exposure, human T-cells became which could be related to the onset of acquired drug resistance in the target inflammatory 3.5-6.0 fold resistant to this drug. Characterization of the resistant cell line revealed cells. Obviously, a phenomenon of drug resistance is not unique to DMARDs in RA; it is a marked induction of the MDR protein ‘breast cancer resistance protein’ (BCRP), no generally accepted that the onset of drug resistance is a common mechanism of treatment involvement of P-glycoprotein (Pgp) and even a down regulated expression of multi-drug failure for many types of drugs, for example antibiotic drugs, antimalarial drugs and resistance protein 1 (MRP1). Sulfasalazine sensitivity could be largely restored by adding anti-cancer drugs. We started our research to address this clinically relevant problem to a specific BCRP blocking agent to the culture medium, proving the functional relation of explore mechanisms that could confer cellular resistance against methotrexate (MTX) BCRP expression and sulfasalzine resistance. In sulfasalazine resistant T-cells, basal levels of and sulfasalazine, two widely applied DMARDs in RA. In addition we assessed whether TNFα production after stimulation were markedly increased compared to parental T-cells, rationally designed therapeutic drugs or drug targets may be exploited to circumvent drug pointing to an enhanced inflammatory state of these cells. Subsequently TNFα production resistance related mechanisms. This issue is described in the general introduction (chapter was less efficiently blocked by sulfasalazine, consistent with BCRP-mediated efflux of this one), followed by a literature review that outlines molecular mechanisms involved in drug. DMARD resistance in chapter two. In chapter four we determined the stability of the sulfasalazine resistant phenotype in In oncological research, increased drug efflux by multi-drug resistance (MDR) proteins human T-cells. We noted that BCRP expression in these cells was stable for at least 4 weeks has been recognized as an important mechanism of resistance to anti-cancer agents. after withdrawal of sulfasalazine but gradually declined, along with sulfasalazine resistance MDR proteins belong to the family of ATP-binding (ABC) transporters. A wide range levels, to non-detectable levels of BCRP after 6 months. Strikingly, rechallenging with of structurally and functionally different drugs can be pumped out of the cells by these sulfasalazine led to a rapid resumption of sulfasalazine resistance and BCRP expression proteins, resulting in a multi-drug resistance phenotype. There is now accumulating within 2 weeks. In this chapter we also assessed the impact of sulfasalazine resistance evidence that several DMARDs may be among the substrates of distinct MDR proteins and on responsiveness to other, non-related, DMARDs. Sulfasalazine resistant cells displayed thereby cause a diminished efficacy of DMARDs. Table 1 summarizes the MDR proteins that diminished sensitivity to leflunomide (5.1-fold), and MTX (1.8-fold), were moderately more can be involved in drug efflux of selected DMARDs. sensitive (1.8-fold) to cyclosporine A and chloroquine, and markedly more sensitive (13- fold) to dexamethasone as compared with parental T-cells. This observation may provide a further rationale for sequential mono- or combination therapies with distinct DMARDs Table 1: MDR transporters and their pharmacological (DMARD) substrates. upon decreased efficacy of sulfasalazine.

MDR transporter DMARD substrate Sulfasalazine is often combined with MTX and other DMARDs in RA combination-therapy schedules. However, the combination of sulfasalazine and MTX does not display clinical ABCB1 (Pgp) chloroquine, glucocorticoids additive effects compared to monotherapy with either sulfasalazine or MTX. Moreover, we ABCC1 (MRP1) methotrexate, chloroquine observed that sulfasalazine-resistant human T cells were cross-resistant to MTX (described ABCC2 (MRP2) methotrexate in chapter four) by a mechanism that does not involve enhanced cellular efflux of MTX by ABCC3 (MRP3) methotrexate BCRP. To further unravel interaction(s) of sulfasalazine and MTX we investigated in chapter ABCC4 (MRP4) methotrexate, azathioprin five whether exposure of cells to sulfasalazine interferes with the cellular pharmacology ABCC5 (MRP5) methotrexate, azathioprin of MTX. For this purpose human T-cells were used to analyse the effect of sulfasalazine on BCG2 (BCRP) methotrexate,sulfasalazine, leflunomide the cellular uptake of radiolabelled MTX and the natural folate leucovorin by the reduced folate carrier (RFC). Moreover T-cells with and without acquired resistance to sulfasalazine

200 201

chapter 12 Summary

were used to assess the anti-proliferative effects of MTX in combination with sulfasalazine. to our laboratory-directed studies on novel experimental antifolate drugs, described in Transport kinetic analysis revealed that sulfasalazine is a potent, non-competitive inhibitor chapter eight and nine. Publications on efficacy of MTX and leflunomide show that both of RFC mediated cellular uptake of MTX and the natural reduced folate analogue leucovorin. drugs are very potent DMARDs in RA treatment. Long-term observational studies of MTX, Consistent with this proven interaction, a marked loss of MTX efficacy was observed when with follow-up periods of more than 10 years, showed long-lasting effectiveness with low MTX was co-administered with sulfasalazine: up to 3.5 fold for parental T-cells in the discontinuation rates and drug survival rates of more than 5 years in approximately 50% of presence of 0.25 mM sulfasalazine and >400-fold for sulfasalazine-resistant cells in the patients, which compares favourably with 25% reported for other DMARDs. The efficacy of presence of 2.5 mM sulfasalazine. Along with the diminished efficacy of MTX, a sulfasalazine low dose MTX also includes a reduction in joint damage progression in RA patients, as seen dose-dependent decrease in leucovorin accumulation was observed, suggesting the onset on X-rays of hands and feet. Moreover, it has been proven that the effective TNFα blocking of cellular folate depletion. Thus, when considering use of these drugs in combination biologic agents are even more powerful if concomitantly MTX is prescribed. This might be therapies, these results provide a rationale for both the use of folate supplementation partly due to suppression of the production of antibodies against these agents by plasma and for spacing administration of sulfasalazine and MTX over time, because the inhibitory cells. effects of sulfasalazine on RFC dependent uptake of MTX are only transient. Despite an initially good response to MTX and relatively long-lasting effectiveness in Chapter six described an inventory study of expression of MDR transporters on approximately 50% of RA patients, many patients ultimately experience loss of efficacy upon inflammatory cells in synovial tissue of RA patients. In this study we determined the prolonged treatment. Beyond the mechanism of MTX efflux by specific MDR transporters expression of Pgp, MRP1-5, MRP8, MRP9 and BCRP by immunohistochemical techniques (e.g. BCRP and MRP1), several other causes for impaired responsiveness to MTX have been in synovial tissue of RA patients with active disease before and after treatment with revealed in cancer chemotherapy. Some of these mechanisms may also be of relevance MTX (7.5-15 mg/week) or leflunomide (20mg/day) and non-inflammatory synovial tissue for RA treatment, e.g. (a) impaired cellular uptake of MTX via the Reduced Folate Carrier from orthopaedic patients suffering from mechanical joint injury. We found abundant (RFC), (b) reduced conversion to polyglutamate forms of MTX due to decreased activity expression of BCRP in all RA synovial biopsies, both prior to treatment and after 4 months of folylpolyglutamate synthetase (FPGS), (c) increased expression of the target enzyme of MTX treatment, in the intimal lining layer as well as on macrophages and endothelial of MTX, dihydrofolate reductase (DHFR) or (d) polymorphism in genes coding for folate- cells in the synovial sublining. Statistical analysis showed that there was a trend towards depended enzymes. more abundant BCRP expression at higher disease activity (defined by the disease activity We therefore tested in chapter eight the anti-inflammatory effects of eight novel score of 28 joints/DAS28). Furthermore, median BCRP expression was 4-8 fold higher for antifolate drugs, rationally designed to overcome one or more of these mechanisms of MTX-non-responders compared to MTX-responders. The same trend was observed for MTX resistance. These drugs, for example, exhibit higher affinity for the RFC and FPGS RA patients treated with leflunomide: a 2.5-fold higher BCRP expression was observed in compared to MTX, and/or target other enzymes in the folate metabolism than DHFR; such synovial biopsies from ‘leflunomide-failures’ compared to biopsies from patients with a as thymidylate synthase (TS) and glycinamide ribonucleotide transformylase (GARTFase). good respons on leflunomide. For other MDR proteins, moderate expression of MRP1 was The ability of these antifolate drugs to inhibit TNFα production was analyzed for ex vivo observed in T-cell areas of some synovial biopsies, while expression of Pgp, MRP2-5, MRP8 stimulated T-cells in whole-blood from RA patients. Two novel DHFR inhibitors (PT523 and and MRP9 were below the immunohistochemical detection levels. In control synovial PT644) and two novel TS inhibitors (Raltitrexed and GW1843), all harbouring high substrate tissue, along with very low levels of infiltrated macrophages, only a few BCRP positive affinities for RFC and FPGS, turned out to be very effective in abrogating TNFα release by T- cells were observed, while staining for the other MDR-proteins was negative. Since we cells of RA patients (at nanomolar concentrations). Median concentrations of these drugs found positive staining on macrophages in all RA synovial biopsies prior to therapeutic to inhibit TNFα production by 50% (IC-50 values) were 6.9-10.5 lower compared to MTX, interventions, expression of BCRP seems to be inflammation dependent rather than drug also in RA patients who were clinically unresponsive to MTX. Conceivably, experimental induced. Because MTX, sulfasalazine and leflunomide are identified as substrates for BCRP, therapies with one of these novel antifolate drugs deserve further consideration for this transporter may contribute to a reduced therapeutic effect of these drugs. patients with MTX-refractory RA.

In chapter seven a clinically-oriented review on the status of MTX and leflunomide In chapter nine we explored the folate receptor-β (FR-β) as a novel target for selective (monotherapy and in combination schedules) in RA treatment served as an introduction macrophage directed RA treatment with folate antagonist drugs. Folate receptors (FR)

202 203 chapter 12 Summary

are membrane-associated proteins consisting of at least three isoforms (α,β,γ). These of their clinical response to MTX. Along with a reduction in TNFα release, a marked receptors have a high affinity for folic acid and a relatively low affinity for MTX, and cell induction of apoptosis was observed in peripheral blood lymphocytes of RA patients entrance of these agents occurs via endocytosis. Given the notion that the β isoform is following 48 hours of bortezomib exposure. Furthermore we found that bortezomib specifically expressed on synovial macrophages, this receptor may be exploited as a inhibits T-cell activation by CD3/CD28 as defined by a (apoptosis unrelated) concentration selective target in RA treatment. For this reason we determined the expression of FR-β dependent decrease in CD25 expression. Along with these effects, it is also appreciated on macrophages in cryopreserved synovial tissue from RA patients with active disease, that proteasome inhibitors may retain therapeutic activity against drug resistant cells. along with expression on peripheral blood cells of RA patients (lymphocytes, monocytes, Therefore, this class of drugs with a novel mode of action would be worthy for further ex-vivo cultured macrophages and ex-vivo activated T-cells). We also determined FR-β (pre) clinical evaluation in a RA setting. binding affinities for 10 novel antifolate drugs by competition experiments with [3H]-folic acid on FR-β transfected cells. As hypothesized, immunohistochemical staining of RA synovial tissue showed high expression of FR-β on macrophages in the synovial (sub)lining, while no staining was Key points of this thesis: observed in T-cell areas or control synovial tissue. Consistently, FR-β mRNA levels were highest in synovial tissue extracts and monocyte-derived macrophages, but low in • Chronic exposure of inflammatory cells to sulfasalazine induces expression of the multi- peripheral blood T-cells and monocytes. Screening of 10 novel antifolate drugs revealed 4 drug resistance protein BCRP on the cell membrane, causing sulfasalazine resistance compounds for which FR-β had a high binding affinity (20-77 folds higher than for MTX). One of these compounds, the thymidylate synthase inhibitor BCG945, displayed selective • BCRP is highly expressed on the cell membrane of synovial tissue macrophages and may targeting against FR-β transfected cells because of a concomitant low affinity for the therefore cause diminished efficacy of the DMARDs sulfasalazine, methotrexate and RFC. This drug therefore deserves further exploration for selective macrophage directed leflunomide (BCRP substrates) therapy in RA. • Sulfasalazine is a potent inhibitor of methotrexate uptake via the Reduced Folate A potential novel target in the treatment of RA is NFκB, because activation of this Carrier; this finding may explain the lack of additive clinical effects of the combination transcription factor is thought to play a central role in the onset and progression of of methotrexate and sulfasalazine in therapeutic regimens for RA inflammation in RA. From this perspective, experimental and therapeutic strategies aiming at inhibition of the activation of NFkB have been developed and tested in preclinical • Novel generation antifolate drugs, designed to overcome methotrexate resistance in and clinical stages of RA treatment. These strategies include the use of (a) direct NFκB oncology, are very potent anti-inflammatory drugs in ex-vivo activated T-cells of RA inhibitors; (b) NFκB decoy oligonucleotides to prevent NFkB binding to its promotor site patients. These drugs might therefore be beneficial for RA patients who do not respond and (c) inhibitors of IκB kinase. Another approach to interfere with NFkB activation, which to treatment with methotrexate is relatively unexplored, is by blocking the 26S proteasome-mediated breakdown of the IkBα protein, the natural inhibitor of NFkB. Cell activation initiates both phosphorylation • The high folate receptor-β expression on synovial tissue macrophages may serve as a and ubiquitination of IkBα. The latter process triggers breakdown of IkBα protein by distinctive target for therapy with novel generation antifolate drugs and imaging of the 26S proteasome system. Upon loss of binding of its natural inhibitor protein, NFkB (sub-clinical) RA disease activity can translocate to the nucleus to drive the transcription of pro-inflammatory cytokines (like TNFα and IL1β) and anti-apoptotic genes. Thus, it is anticipated that inhibitors of • Proteasome inhibition by bortezomib is highly effective in abrogating cytokine the proteasome may counteract this process and elicit a potential anti-inflammatory production in activated RA T-cells response. We therefore tested in chapter ten the anti-inflammatory properties of the proteasome inhibitor bortezomib, a boronic acid dipeptide, currently used in the treatment of advanced multiple myeloma. We found that bortezomib conveys potent inhibition of TNFα production upon activation of T-cells from RA patients, regardless

204 205 Nederlandse samenvatting Dankwoord/Acknowledgements Curriculum vitae Curriculum vitae (english) List of publications Nederlandse samenvatting Nederlandse samenvatting

Samenvatting eiwitten betrokken zijn bij resistentie tegen sulfasalazine in een humane T-cellijn, wanneer deze cellen langdurig blootgesteld worden aan deze DMARD. Na 4-6 maanden Patiënten met reumatoïde artritis (RA) worden langdurig behandeld met ontstekings- blootstelling aan sulfasalazine waren deze cellen een factor 3.5-6.0 resistent geworden. In remmende medicijnen, waaronder de zogenaamde Disease Modifying Anti-rheumatic Drugs de resistente cellen bleek een duidelijke opregulatie te zijn van het MDR-eiwit BCRP en een (DMARDs). Helaas worden veel patiënten in de loop van de behandeling resistent tegen deze verminderde expressie van MRP1. Door het blokkeren van BCRP, werden de resistente cellen DMARDs. Resistentie tegen antibiotica bij infectieziekten en resistentie tegen cytostatica bij weer gevoelig voor sulfasalazine, waarmee een directe relatie tussen BCRP expressie en kanker is een bekend fenomeen en daar is veel onderzoek naar gedaan; naar resistentie tegen sulfasalazine-resistentie werd aangetoond. Verder bleek dat sulfasalazine-resistente cellen DMARDs is nog relatief weinig onderzoek verricht. na stimulatie meer TNFα produceerden dan controle cellen, wat duidt op een verhoogde In dit proefschrift beschrijven wij mogelijke mechanismen van resistentie tegen ontstekingsactiviteit van deze cellen. De TNFα productie in de resistente cellen kon ook methotrexaat (MTX) en sulfasalazine, twee veel gebruikte DMARDs bij de behandeling van moeilijker worden geremd door sulfasalazine, samenhangend met de verhoogde expressie patiënten met reumatoïde artritis. Daarnaast hebben we onderzoek gedaan naar nieuwe van BCRP. generatie geneesmiddelen die deze mechanismen van resistentie mogelijk kunnen omzeilen. In hoofdstuk 1 en 2 wordt een overzicht gegeven van de tot nu toe bekende moleculaire In hoofdstuk 4 werd onderzocht of sulfasalazine-resistentie in de humane T-cellijn reversibel mechanismen van resistentie tegen DMARDs. was als de cellen werden gekweekt in afwezigheid van sulfasalazine. We zagen dat de BCRP Vanuit de oncologie is bekend dat een verhoogde expressie van multi-drug resistentie (MDR) expressie een maand stabiel verhoogd bleef, daarna geleidelijk afnam (evenals de resistentie- eiwitten op de celmembraan van kankercellen kan leiden tot resistentie tegen cytostatica. factor) en na 6 maanden niet meer detecteerbaar was. Echter, wanneer sulfasalazine opnieuw MDR eiwitten behoren tot de familie van de ATP afhankelijke transporteiwitten, waarvan er aan het kweekmedium werd toegevoegd, was er weer een snelle inductie van BCRP-expressie 49 bekend zijn. Deze eiwitten zijn in staat chemisch- en functioneel verschillende medicijnen waar te nemen en werden de cellen dientengevolge binnen 2 weken opnieuw resistent tegen de kankercellen uit te pompen, wat aanleiding geeft tot zogenaamde multipele-drug sulfasalazine. In dit hoofdstuk werd ook onderzocht of er in de sulfasalazine-resistente resistentie. Het is bekend dat verschillende DMARDs (onder andere MTX) substraat zijn voor cellen kruisresistentie bestaat tegen andere DMARDs. Er bleek sprake van kruisresistentie een of meerdere van deze MDR eiwitten (zie tabel 1). Het is echter onbekend welke MDR- voor leflunomide (factor 5.1) en MTX (factor 1.8), maar juist een verhoogde gevoeligheid eiwitten voorkomen op perifere bloedcellen of ontstekingscellen in synoviaal weefsel van voor cyclopsorine A, chloroquine (factor 1.8) en dexamethason (factor 13), vergeleken met RA-patiënten, en of dit aanleiding geeft tot DMARD resistentie. controle cellen. Vanuit dit oogpunt zou het een goede keuze kunnen zijn om deze laatste Meta-analyses hebben aangetoond dat RA-patiënten betrekkelijk snel resistent kunnen middelen voor te schrijven aan patiënten die gefaald hebben op sulfasalazine. worden tegen de DMARD sulfasalazine. In hoofdstuk 3 hebben we onderzocht of MDR- RA-patiënten worden vaak behandeld met een combinatie van DMARDs. Een veel gebruikte combinatie is sulfasalazine met MTX. Uit literatuurgegevens blijkt echter dat de combinatie Table 1: Overzicht van de MDR eiwitten die DMARDs kunnen verpompen. van sulfasalazine en MTX niet effectiever is dan mono-therapie met deze middelen, tenzij een derde DMARD zoals chloroquine of prednison wordt toegevoegd. Ook in onze MDR eiwit DMARD substraat laboratoriumstudies zagen we dat sulfasalazine resistente T-cellen minder gevoelig waren voor MTX (hoofdstuk 4), wat niet werd verklaard door verhoogde expressie van het MDR- ABCB1 (Pgp) chloroquine, glucocorticoiden eiwit BCRP. In hoofdstuk 5 onderzochten we daarom de farmacologische interactie tussen ABCC1 (MRP1) methotrexaat, chloroquine sulfasalazine en MTX. Dezelfde humane T-cellijn die beschreven werd in hoofdstuk 3 en ABCC2 (MRP2) methotrexaat 4, werd nu gebruikt om het effect van sulfasalazine op de cellulaire opname van MTX en ABCC3 (MRP3) methotrexaat natuurlijke folaten (leucovorin) door de Reduced Folate Carrier (RFC) (het eiwit dat betrokken ABCC4 (MRP4) methotrexaat, azathioprine is bij de opname van MTX en natuurlijke folaten in de cel) te bestuderen. Analyse van de ABCC5 (MRP5) methotrexaat, azathioprine transportkinetiek van de RFC toonde aan dat sulfasalazine een potente (reversibele), niet- ABCG2 (BCRP) methotrexaat, sulfasalazine, leflunomide competitieve remmer is van de cellulaire opnamecapaciteit voor MTX en leucovorin. In overeenstemming met deze bevinding was dat er MTX resistentie optrad in sulfasalazine-

208 209

Nederlandse samenvatting Nederlandse samenvatting

resistente T-cellen in aanwezigheid van 0.25 mM sulfasalazine (resistentiefactor: 3.5) en 2.5 studies tonen effectiviteit aan van MTX (op vermindering van ziekte-activiteit en mM sulfasalazine (resistentiefactor >400). Naast MTX-resistentie werd een dosis-afhankelijk gewrichtsschade) gedurende meer dan 5 jaar bij ongeveer 50% van de RA-patiënten. remmend effect gezien van sulfasalazine op de opname van leucovorin, wat aanleiding kan Daarnaast is bewezen dat MTX de effectiviteit van TNFα blokkers (antilichamen tegen TNFα) geven tot intracellulaire folaat-depletie. Dit onderzoek pleit ervoor dat sulfasalazine en MTX versterkt, waarschijnlijk via remming van de productie van antistoffen tegen deze middelen niet gelijktijdig (op dezelfde dag) moeten worden ingenomen maar juist gespreid, om een door plasmacellen. maximaal effect te kunnen bereiken. Daarnaast moet er bij deze combinatie in ieder geval Ondanks een aanvankelijk goede respons op behandeling met MTX en een relatief lange foliumzuur worden voorgeschreven om ‘gezonde cellen’ te beschermen tegen mogelijke gebruiksduur bij ongeveer 50% van de RA patiënten, worden veel patiënten geconfronteerd folaat-depletie. met verlies van MTX-effectiviteit bij langdurig gebruik (klinische resistentie). Naast het In hoofdstuk 6 werd de expressie van MDR-eiwitten onderzocht op ontstekingscellen in mechanisme van efflux van MTX via MDR-eiwitten (zoals BCRP en MRP1) kunnen er ook synoviaal weefsel van RA-patiënten met actieve ziekte, voor en na behandeling met MTX (7.5- andere oorzaken zijn voor de verminderde effectiviteit van MTX, zoals eerder is aangetoond 15 mg/week) of leflunomide (20mg/dag) en ‘gezond’ synoviaal weefsel van patiënten die een voor resistentie van kankercellen tegen MTX. Sommige van deze mechanismen zouden orthopedische operatie ondergingen. Met behulp van immunohistochemische technieken ook een rol kunnen spelen bij de behandeling van RA-patiënten, zoals: (a) verminderde werd de expressie bepaald van Pgp, MRP1-5, MRP8, MRP9 en BCRP. Er werd een hoge cellulaire opname van MTX via de Reduced Folate Carrier (RFC), (b) verminderde conversie expressie gevonden van BCRP in de synoviale lining en op macrofagen en endotheelcellen in naar polyglutamaat metabolieten van MTX door een verminderde activiteit van het enzym de synoviale sublining bij alle patiënten. Statistische analyse liet een trend zien van hogere folylpolyglutamaat synthetase (FPGS), (c) verhoogde expressie van het target-enzym van BCRP expressie in biopten van patiënten met een hoge ziekte-activiteit (gedefinieerd door MTX, dihydrofolate reductase (DHFR) of (d) polymorfismen in genen die coderen voor een hoge DAS28 score). Daarnaast bleek dat de mediane BCRP expressie 4-8 keer zo hoog enzymen in het foliumzuurmetabolisme. Om dit te onderzoeken werd in hoofdstuk 8 de was in biopten van ‘MTX-falers’ vergeleken met biopten van patiënten met een goede ontstekingsremmende capaciteit van 8 nieuwe generatie anti-folaten bepaald, die zijn respons op MTX na 4 maanden. Dezelfde trend werd gezien voor RA-patiënten die werden ontworpen om MTX resistentie bij leukemie-patiënten te omzeilen. Deze geneesmiddelen behandeld met leflunomide: een 2.5 keer hogere BCRP expressie werd waargenomen in hebben bijvoorbeeld een hogere affiniteit voor de RFC en/of FPGS in vergelijking met MTX synoviale biopten van ‘leflunomide-falers’ in vergelijking met patiënten met een goede of hebben een ander target in het foliumzuurmetabolisme dan DHFR, zoals thymidylate respons op leflunomide. Naast BCRP werd een duidelijke expressie gevonden van MRP1 synthase (TS) en glycinamide ribonucleotide transformylase (GARTFase). De effectiviteit op T-cellen (gelegen in aggregaten) en een lage expressie op macrofagen. De expressie van deze geneesmiddelen om TNFα productie door geactiveerde T-cellen (na αCD3/CD28 van Pgp, MRP2-5, MRP8 en MRP9 was te laag om met immunohistochemische technieken stimulatie) te remmen werd onderzocht in volbloed van RA-patiënten. Twee nieuwe aan te tonen. In ‘gezond’ synoviaal weefsel, met zeer weinig ontstekingscellen, werden generatie DHFR remmers (PT523 en PT644) en twee nieuwe generatie TS remmers slechts enkele BCRP positieve cellen aangetoond. In dit weefsel werd ook geen expressie (Raltitrexed en GW1843), welke een hoge affiniteit hebben voor zowel RFC en FPGS bleken bij gevonden van de andere MDR-eiwitten. Omdat hoge expressie van BCRP werd aangetoond lage concentraties effectief in het remmen van TNFα productie door geactiveerde T-cellen. in synoviale biopten van alle patiënten reeds vóór aanvang van de therapie met MTX, is het De mediane concentraties van deze medicijnen om 50% van de TNFα productie te remmen waarschijnlijker dat de expressie wordt geïnduceerd door het ontstekingsproces dan door (IC-50 waarden) waren 6.9-10.5 keer lager in vergelijking met MTX, ook in volbloed van RA- blootstelling aan DMARDs. Omdat aangetoond is dat MTX, sulfasalazine en leflunomide patiënten die klinisch hadden gefaald op MTX. Deze data tonen aan dat behandeling met substraten zijn voor BCRP, kan expressie van dit pomp-eiwit op macrofagen echter wel leiden deze nieuwe generatie anti-folaten te overwegen is bij RA-patiënten die falen op MTX. tot resistentie tegen deze geneesmiddelen. In hoofdstuk 9 hebben we de folaatreceptor-β (FR-β) onderzocht als potentieel nieuw Hoofdstuk 7 is een klinisch georiënteerd review-artikel over de status van MTX en target voor de behandeling van RA-patiënten. Folaatreceptoren zijn membraangebonden leflunomide (als monotherapie en in combinatieschema’s) bij de behandeling van patiënten receptoren waarvan drie isotypen bestaan (α,β,γ). Deze receptoren hebben een hoge met RA. Dit hoofdstuk dient als inleiding van hoofdstuk 8, waar ex vivo analyses naar affiniteit voor foliumzuur en een relatief lage affiniteit voor MTX, waarna opname van deze de ontstekingsremmende capaciteit van nieuwe generatie anti-folaten (MTX-analoga) middelen geschiedt via endocytose van het receptor-ligand complex. Het is bekend dat de FR- worden beschreven. Effectiviteitstudies laten zien dat MTX en leflunomide zeer potente β selectief tot expressie komt op geactiveerde synoviale macrofagen en daarom een target geneesmiddelen zijn bij de behandeling van RA-patiënten. Lange termijn observationele voor therapie kan zijn bij patiënten met reumatoïde artritis. Om een voorspelling te kunnen

210 211 Nederlandse samenvatting Nederlandse samenvatting

doen over de potentiële effectiviteit van deze benadering hebben we in eerste instantie resultaten, het unieke werkingsmechanisme van bortezomib, en het feit dat bortezomib de expressie bepaald van de FR-β op ontstekingscellen in intact synoviaal weefsel van RA waarschijnlijk ook effectief is tegen DMARD resistente ontstekingscellen, verdient het patiënten en op perifere bloedlymfocyten. Daarnaast werd de bindingsaffiniteit voor de FR-β aanbeveling om dit geneesmiddel en tweede-generatie proteasoomremmers verder te onderzocht van 10 nieuwe generatie anti-folaten op basis van competitiestudies met [3H]- onderzoeken als potentiële anti-reumatica. foliumzuur op een cellijn die getransfecteerd is met de FR-β. Zoals verwacht kon met behulp De belangrijkste bevindingen uit dit proefschrift kunnen als volgt worden samengevat: van immunohistochemische technieken hoge expressie van de FR-β worden aangetoond op synoviale lining cellen en macrofagen in de synoviale sublining van RA-patiënten, terwijl geen expressie werd gevonden in T-cel gebieden en in ‘gezond’ synoviaal weefsel. In Belangrijkste bevindingen: overeenstemming hiermee werd een hoge mRNA expressie gevonden in synoviaal weefsel van RA-patiënten, een duidelijke expressie werd gezien in ex-vivo gekweekte macrofagen uit • Chronische blootstelling van ontstekingscellen aan sulfasalazine induceert expressie perifeer bloed van RA-patiënten, terwijl geen expressie werd waargenomen in monocyten en van het multi-drug resistentie eiwit BCRP op de celmembraan, wat resulteert in ex-vivo geactiveerde T-cellen. Screening van 10 nieuwe generatie anti-folaten voor affiniteit sulfasalazine resistentie voor de FR-β leverde 4 geneesmiddelen op met een hoge affiniteit voor deze receptor (20- 77 keer hogere affiniteit dan MTX). Een van deze geneesmiddelen, de TS-remmer BCG945, • Hoge expressie niveaus van BCRP werden aangetoond op de celmembraan van bleek een heel hoge affiniteit te hebben voor de FR-β, terwijl bekend is dat dit middel een heel synoviale macrofagen, wat voor verminderde werkzaamheid van sulfasalazine, lage affiniteit heeft voor de RFC. BGC945 verdient daarom nadere evaluatie als potentiële methotrexaat en leflunomide (allen BCRP substraten) kan zorgen selectieve macrofaag-gerichte therapie bij RA. • Sulfasalazine is een potente remmer van de opname van methotrexaat via de Reduced Een ander potentieel nieuw target voor de behandeling van patiënten met reumatoïde Folate Carrier; deze bevinding zou kunnen verklaren waarom er geen synergistich artritis is NFκB. Aangenomen wordt dat activatie van deze transcriptie factor een belangrijke effect wordt gevonden bij de combinatie van sulfasalazine en methotrexaat in rol speelt bij het ontstekingsproces in RA. Om deze reden zijn er diverse therapeutische combinatietherapieën strategieën ontwikkeld om de activiteit van NFκB te remmen, bijvoorbeeld door gebruik te maken van directe NFκB-remmers, NFκB oligonucleotides om binding van NFκB aan de • Nieuwe generatie anti-folaten, ontwikkelt om methotrexaat resistentie bij promotor tegen te gaan, of remmers van IκB-kinase. Een andere benadering is blokkade leukemiepatiënten te omzeilen, blijken zeer effectieve ontstekingsremmers van ex- van de proteasoom-afhankelijke afbraak van IκBα, de natuurlijke remmer van NFkB. Cel- vivo geactiveerde T-cellen van RA-patiënten. Deze geneesmiddelen zouden kunnen activatie resulteert normaliter in fosforylering en ubiquitinering van IκBα. Deze processen worden gebruikt voor de behandeling van RA-patiënten die falen op behandeling met zorgen ervoor dat IκBα kan worden afgebroken door het 26S proteasoom. Nadat IκBα is methotrexaat. afgebroken, kan NFκB naar de kern worden getransporteerd waar het de transcriptie van pro-inflammatoire cytokines en anti-apoptotische genen kan initiëren. Vanuit dit concept • De folaat receptor-β, die hoog tot expressie komt op synoviale macrofagen, kan is dus te verwachten dat remmers van het proteasoom deze processen kunnen remmen en worden gebruikt als een selectieve target bij de behandeling van RA-patiënten daarmee een anti-inflammatoire respons bewerkstelligen. In hoofdstuk 10 werd daarom met nieuwe generatie anti-folaten en voor beeldvorming van (subklinische) ziekte- het ontstekingsremmende effect onderzocht van de proteasoomremmer bortezomib, een activiteit boronzuur bevattend dipeptide, dat geregistreerd is voor de behandeling van (therapie- resistente) multipele myeloom patiënten. We konden aantonen dat bortezomib heel • Het remmen van de cellulaire proteasoomactiviteit met bortezomib veroorzaakt effectief is in remming van de TNFα productie door ex-vivo geactiveerde T-cellen van RA- een effectieve remming van de cytokine-productie door geactiveerde T-cellen van patiënten, onafhankelijk van hun klinische respons op MTX. Naast remming van TNFα RA-patiënten productie werd ook een opvallende inductie van apoptose gezien in geactiveerd T-cellen, 48 uur na blootstelling aan dit geneesmiddel. Ook vonden we een concentratie-afhankelijke, maar apoptose-onafhankelijke, remming van T-cel activatie door αCD3/CD28. Gezien deze

212 213 Dankwoord/acknowledgements Dankwoord/acknowledgements

Dankwoord/acknowledgements Ruud, mijn mede co-piloot op het MDR-project. Ik heb met heel veel plezier met je samengewerkt. Dank dat je mij als ‘simpele dokter’ zo goed hebt geholpen in het De gezagvoerder meldt vanuit de cockpit de te vliegen route. Hij zegt dat het mooi weer is laboratorium. Ons werkbezoek aan de VS zal ik nooit vergeten. Het schept een bijzondere op de plaats van bestemming, maar dat weet je nooit zeker…. band om je samen door 6 kg vlees heen te werken tijdens een BBQ met een om aandacht verlegen hospita. Heel veel succes met het afronden van je promotie en in je nieuwe carrière. Promoveren is als een vliegreis, een onderneming waar veel mensen bij betrokken zijn om Veel dank ook aan mijn andere collega’s van ‘de oude’ verkeerstoren 4A64. De harde kern: het mogelijk te maken. Vanaf deze plaats wil ik graag iedereen bedanken die mij op enige Mirjam, Ernst en Marijn, daarnaast natuurlijk ook Marleen, Vokko, Mike, Danielle, Debby, wijze heeft geholpen bij mijn onderzoek. Mignon en Henny, ik heb een enorm leuke tijd met jullie gehad! Het was een wonder dat we nog aan werken toekwamen als ‘zuurkool met vette jus’ door de speakers schalde. Ik wens Allereerst gezagvoerder dr. Gerrit Jansen, co-promotor. Beste Gerrit, in 2001 ben ik als co- jullie allemaal veel succes met het afronden van de promotie-onderzoeken. piloot begonnen in jouw laboratorium. Je enthousiaste en deskundige begeleiding heeft ervoor gezorgd dat deze stage uiteindelijk is uitgemond in dit promotieonderzoek. Je Ik wil al het ‘vliegend personeel’ (reumatologen, arts-assistenten en arts-onderzoekers literatuurkennis is ongeëvenaard; als er iemand over een fotografisch geheugen beschikt van het VU-Medisch Centrum, het Slotervaart ziekenhuis en het Jan van Breemeninstituut) dan ben jij het. Ik heb zeer prettig met je samengewerkt en heb veel van je geleerd. Dank bedanken voor de hulp bij het selecteren van patiënten voor ons onderzoek: Conny van der voor de leuke jaren! Laken, Irene van der Horst, Irene Bultink, Alexandre Voskuyl, Dirk-Jan van Schaardenburg, Kirsti Steen, Franktien Turkstra, Natalja Basoski en Andreas Gerards (Vlietland ziekenhuis). De overige gezagvoerders: professor Ben Dijkmans en professor Rik Scheper (promotoren) Ook de secretaressen, Marjo Sluiter, Ida Gaspersz en Noortje Vesters, heel hartelijk dank en professor Willem Lems (co-promotor). voor jullie enthousiaste ondersteuning. Michelien Kolff en Ewa Platek, bedankt voor het Beste Ben, ik wil je hartelijk bedanken voor het vertrouwen en de goede begeleiding. Ik scoren van de ziekte-activiteit bij de patiënten in onze studie. De patiënten, de passagiers heb veel van je geleerd over de klinische reumatologie. Je vestigde steeds de aandacht op op deze vlucht, wil ik hartelijk bedanken voor deelname aan de studie. Het Nationaal de klinische implicaties van ons onderzoek, en daar gaat het natuurlijk ook om. Je morele Reumafonds wil ik bedanken voor financiering van de vlucht. steun, bij zowel het onderzoek, mijn klinische carrière als ook op persoonlijk vlak waardeer ik zeer. Van de afdeling pathologie: George Scheffer, van jouw eigen-wijze kijk op de immuno- Beste Rik, jij bracht me in contact met de afdeling reumatologie waar ik ben gaan werken histochemie heb ik enorm veel geleerd en je hulp heeft geleid tot robuuste data op dit vlak, op het RA/MDR-project, een samenwerking tussen de afdelingen reumatologie en waarvoor mijn dank. Tanja de Gruijl, Antoinet Schoonderwoerd, Anneke Reurs, Rieneke pathologie. De wekelijkse MDR-besprekingen hebben mij wetenschappelijk gevormd, en van der Ven en Wouter Bierman: bedankt voor de fijne samenwerking. je scherpe commentaar bij zowel het onderzoek als de manuscripten waren voor mij van grote waarde. Veel dank voor de prettige samenwerking! Speciale herinneringen bewaar ik aan de vriendschap en samenwerking met Mariska Willem, bedankt voor je hulp en begeleiding bij het onderzoek. Tijdens de ‘ronde tafel de Jong, die zo jong overleden is. Haar enthousiaste begeleiding aan het begin van mijn gesprekken’ met Ben, Rik, Gerrit, Ruud en mijzelf was jij van grote waarde voor het bepalen promotieonderzoek zal ik nooit vergeten. van de te volgen strategie, met name waar het onderzoek met patiëntenmateriaal betrof. Steun en toeverlaat in het laboratorium, purser Ietje Kathmann, dank voor je hulp en De leescommissie/promotie-commissie, de douane, bestaande uit prof. dr. L.A. Aarden, begeleiding tijdens de proeven! Alle collega’s van de geneeskundige oncologie (lab van prof. dr. W.B. van der Berg, prof. dr. J.W.J. Bijlsma, prof. dr. G.J.L. Kaspers, prof. dr. R.E. Frits Peters), hematologie/kindergeneeskunde (Niels, dank voor je hulp bij de FACS- Mebius, prof. dr. G.J. Peters, prof. dr. M. Ratnam, prof. dr. P.P. Tak en prof. dr. C.L. Verweij wil experimenten!) en het pathologie-lab binnen het Cancer Centre Amsterdam, dank ik voor ik hartelijk bedanken voor het beoordelen van het manuscript. de prettige samenwerking en gezelligheid.

Sue-Ellen, jij bent net gestart met een mooi project in het reumatologie-lab. Ik wens je daarbij heel veel succes en plezier!

214 215 Dankwoord/acknowledgements Dankwoord/acknowledgements

Zonder de volgende mensen had ik deze vlucht niet kunnen maken: professor Paul-Peter Tak, bedankt dat ik gebruik mocht maken van het mooie panel synoviale biopten waarop twee hoofdstukken uit dit proefschrift zijn gebaseerd. Marjolein Vinkenoog, bedankt voor je hulp bij de ‘digital image analysis’ van synoviale coupes. Professor Lucien Aarden, dank voor uw hulp bij het opzetten van de volbloed assays. Professor Yehuda Assaraf (Haifa, Israel): Your visits to our laboratory were always very inspiring. Thanks for all your help in designing experiments and for giving helpfull feedback on my manuscripts.

Special thanks to professor Manohar Ratnam from the laboratory for Cancer Biology at the Medical University of Toledo, Ohio, USA, for the fruitful collaboration. I really enjoyed visiting your laboratory twice! Thank you very much for your hospitality and for training me in molecular biology. Marcela D’Alincourt Salazar: thank you for your help (and troubleshooting) in designing a model-system of rheumatoid arthritis macrophages and for the nice social program along the side. Mariana and Stoytcho, my Bulgarian friends: Thank you for your help in the lab and making me feel at home in Toledo. I will never forget Christmas Eve ‘Bulgarian style’! The other people in the ‘Ratnam lab’: Hala, Juan, Huiling, Mesfin and Aymen, thanks for all your help and hospitality during my stay in Toledo.

Esther Beekman: hartelijk dank voor de smaakvolle vormgeving van het proefschrift. Het is een plezier om alles weer door te lezen. Maarten van Haaff, dank dat ik je foto mocht gebruiken voor de omslag. Henk Dekker, bedankt voor je hulp bij het aanpassen van de figuren.

Mijn ‘Schiphol’: mama, dank voor al je liefde en steun door de jaren heen! Janneke & Casper & Lucas, Dirk & Lotte, Papa & Helma & Maarten & Laurens, Opa & Oma Offermans, Opa van der Heijden; mijn schoonfamilie: Marijke, Mirjam & Henk-Willem, Judith & Peter & Naud; al mijn vrienden: dank voor jullie niet aflatende belangstelling voor mijn onderzoek en gezelligheid na(ast) het werk!

Frans en Daniel, mijn paranimfen. Jullie brengen, beide op een eigen manier, het beste in mij naar boven. Ik vind het bijzonder om jullie aan mijn zijde te hebben tijdens de promotie.

Lieve Inge, jij bent mijn allessie, mijn ‘baken’. Dank voor je liefde!

Vertrouwend op een zachte landing!

Joost.

216 217 Curriculum vitae Curriculum vitae (english)

Curriculum vitae Curriculum vitae

Joost van der Heijden werd op 6 april 1976 geboren te Gouda. Hij volgde zijn middelbare Joost van der Heijden was born in Gouda, on the 6th of april 1976. In 1995 he obtained schoolonderwijs aan het Rudolf Steiner College (Vrije School) te Rotterdam. In 1995 his VWO diploma at the Gouwe College in Gouda. From 1995-1998 he studied Medical behaalde hij het VWO diploma aan het Gouwe College te Gouda. In de periode 1995-1998 Biology at the Vrije Universiteit in Amsterdam. In 1998 he started his studies in Medicine, studeerde hij medische biologie aan de Vrije Universiteit te Amsterdam (propedeuse obtaining his MSc degree in 2001 and his medical license in January 2004. During his study, behaald in 1996). Van 1998-2001 studeerde hij geneeskunde aan de Vrije Universiteit te he performed a traineeship in the laboratory for Experimental Rheumatology (head: dr. Amsterdam; het arts-examen werd in januari 2004 behaald. Tijdens zijn studie verrichte G. Jansen) at the department of Rheumatology from the VU University Medical Center, hij een wetenschappelijke stage in het laboratorium voor experimentele reumatologie Amsterdam, the Netherlands (head: prof. dr. B.A.C. Dijkmans). Next, a research project on (hoofd: dr. G. Jansen) binnen de afdeling reumatologie van het VU Medisch Centrum te ‘multiple drug-resistance in the treatment of rheumatoid arthritis patients’ was performed Amsterdam (hoofd: prof. dr. B.A.C. Dijkmans). Het stage-onderzoek naar ‘multipele drug- in collaboration with the department of Experimental Pathology (head: prof. dr. R.J. resistentie bij de behandeling van patiënten met reumatoïde artritis’, werd uitgevoerd in Scheper) and with financial support of the Dutch Arthritis Association. After obtaining his samenwerking met de afdeling experimentele pathologie (hoofd: prof. dr. R.J. Scheper). medical licence he continued his research at the department of Rheumatology. One of his Na het arts-examen werkte hij twee jaar als arts-onderzoeker bij de afdeling reumatologie research proposals was rewarded with a Netherlands Organization of Scientific Research om het onderzoek, gefinancierd door het Reumafonds, voort te zetten. In juli 2005 werd ZonMw/NWO-AGIKO stipend. In July 2005 he started his specialization in Internal Medicine hem voor zijn onderzoeksvoorstel een ZonMW/NWO-AGIKO subsidie toegekend, zodat hij at the department of Internal Medicine from the VU University Medical Center (head prof. zijn opleiding tot internist aan het VU Medisch Centrum te Amsterdam (opleider: prof. dr. dr. S.A. Danner, at present prof.dr. M.H.H. Kramer). In this context, in 2006 and 2007 he S.A. Danner, thans prof.dr. M.H.H. Kramer) kon combineren met het promotieonderzoek. worked at the department of Internal Medicine from the Kennemer Gasthuis hospital, In het kader van de perifere opleidingsstage was hij in 2006 en 2007 werkzaam op de Haarlem, the Netherlands (head: prof. dr. R.W. ten Kate). afdeling interne geneeskunde van het Kennemer Gasthuis te Haarlem (opleider: prof. dr. R.W. ten Kate). His research was awarded twice: In April 2002 he won the ‘Arthron Young Investigator Het onderzoek van Joost van der Heijden werd gewaardeerd met een tweetal prijzen. In Award’ for his innovative scientific research in the field of drug resistance in rheumatology. april 2002 won hij de ‘Arthron Young Investigator Award’ voor innovatief wetenschappelijk For his research proposal entitled: ‘Specific macrophage-directed therapy for the onderzoek op het gebied van de reumatologie. Voor zijn onderzoeksvoorstel getiteld: treatment of rheumatoid arthritis patients with novel antifolate drugs’, he received the ‘Specifieke macrofaag gerichte therapie voor de behandeling van RA-patiënten met ‘Rheumatology Grant 2006’ from the Dutch Rheumatology Association. As part of this nieuwe generatie foliumzuur antagonisten’, ontving hij in januari 2006 de ‘Rheumatology grant, he performed a traineeship of three months in the laboratory of prof. dr. M. Ratnam Grant 2006’ van de Nederlandse Vereniging voor Reumatologie (NVR). In het kader van dit (a world leading investigator in the field of folate receptors) at the Medical University of onderzoek werkte hij gedurende 3 maanden in het laboratorium van professor M. Ratnam Toledo, Toledo (OH), USA. (een autoriteit op het gebied van de folaatreceptor) aan de Medical University of Toledo, Toledo (OH), Verenigde Staten. Joost van der Heijden will continue his training in Internal Medicine in November 2008. Vanaf november 2008 zal Joost van der Heijden de opleiding tot internist vervolgen.

218 219

List of publications List of publications

List of publications 7. Oerlemans R, Van der Heijden JW, Vink J, Dijkmans BAC, Kaspers GJL, Lems WF, Scheffer GL, Ifergan I, Scheper RJ, Cloos J, Assaraf YG, Jansen G. Acquired resistance International journals to chloroquine in human CEM (T) cells is mediated by multidrug resistance associated protein 1 (MRP1) and provokes high levels of cross-resistance to glucocorticoids. 1. Van der Heijden JW, Oerlemans R, Dijkmans BAC, Qi H, van der Laken CJ, Lems WF, Arthritis & Rheumatism 54 (2006), 557-568. Jackman AL, Kraan MC, Tak PP, Ratnam M, Jansen G. Folate receptor-β as potential delivery route for novel folate antagonists to 8. Jansen G, Van der Heijden JW, Oerlemans R, Lems WF, Ifergan I, Scheper RJ, Assaraf Macrophages in synovial tissue of rheumatoid arthritis patients. YG, Dijkmans BAC. Accepted for publication in Arthritis & Rheumatism 2008 Sulfasalazine is a potent inhibitor of the reduced folate carrier: implications for combination therapies with methotrexate in rheumatoid arthritis patients. 2. Van der Heijden JW, Oerlemans R, Lems WF, Scheper RJ, Dijkmans BAC, Jansen G. Arthritis & Rheumatism 50 (2004), 2130-2139. The proteasome inhibitor bortezomib inhibits the release of NFκB-inducible Note: A journal invited editorial to this paper was written by BA Cronstein. cytokines and induces apoptosis of activated T cells from rheumatoid arthritis Arthritis & Rheumatism 50 (2004), 2041-2043. patients. Accepted for publication in Clinical & Experimental Rheumatology 2008 9. Van der Heijden JW, De Jong MC, Dijkmans BAC, Lems WF, Oerlemans R, Kathmann I, Scheffer GL, Scheper RJ, Assaraf YG, Jansen, G. 3. Oerlemans R, Franke NE, Assaraf YG, Cloos J, Van Zantwijk I, Berkers CR, Scheffer GL, Acquired resistance of human T cells to sulfasalazine: stability of the resistant Debipersad K, Vojtekova K, Lemos C, Van der Heijden JW, Ylstra B, Peters GJ, Kaspers phenotype and sensitivity to non-related DMARDs. GJL, Dijkmans BAC, Scheper RJ, Jansen G. Annals of the Rheumatic Diseases 63 (2004) 131-137. Molecular basis of bortezomib/Velcade resistance: proteasome subunit beta 5 (PSMB5) gene mutation and overexpression of PSMB5 protein. 10.  Van der Heijden JW, De Jong MC, Dijkmans BAC, Lems WF, Oerlemans R, Kathmann Blood 112 (6) (2008), 2489-2499. I, Schalkwijk CG, Scheffer GL, Scheper RJ, Jansen G. Development of sulfasalazine resistance in human T cells induces expression of the mulltidrug resistance 4. Van der Laken CJ, Elzinga EH, Kropholler MA, Molthoff CFM, Van der Heijden JW, transporter ABCG2 (BCRP) and augmented production of TNFα. Maruyama K, Boellaard R, Dijkmans BAC, Lammertsma AA, Voskuyl AE. Annals of the Rheumatic Diseases 63 (2004) 138-143. Non-invasive imaging of macrophages in rheumatoid synovitis using (R)-[11C]PK11195 Note: A journal invited editorial to this paper was written by DE Furst. and positron emission tomography. Annals of the Rheumatic Diseases 63 (2004), 115-116. Arthritis & Rheumatism (2008), in press.

5. Oerlemans R, Vink J, Dijkmans BAC, Assaraf YG, Van Miltenburg M, Van der Heijden Submitted JW, Ifergan I, Lems WF, Scheper RJ, Kaspers GJ, Cloos J, Jansen G. Sulfasalazine sensitises human monocytic/macrophage cells for glucocorticoids by 1. Van der Heijden JW, Oerlemans R, Tak PP, Assaraf YG, Scheffer GL, Van der Laken CJ, upregulation of glucocorticoid receptor α and glucocorticoid induced apoptosis. Lems WF, Scheper RJ, Dijkmans BAC, Jansen G. Annals of the Rheumatic Diseases 66 (2007), 1289-1295. Involvement of Breast Cancer Resistance Protein expression on RA synovial tissue macrophages in resistance to methotrexate and leflunomide. 6. Van der Heijden JW, Dijkmans BAC, Scheper RJ, Jansen G. Submitted for publication. Drug Insight: resistance to methotrexate and other disease-modifying anti- rheumatic drugs - from bench to bedside. Nature Clinical Practice Rheumatology 3 (2007) 26-34.

220 221 List of publications List of publications

2. Van der Heijden JW, Gerards AH, Oerlemans R, Lems WF, Scheper RJ, Aarden LA, Potential anti-inflammatory properties elicited by the 26-S proteasome inhibitor Dijkmans BAC, Jansen G. bortezomib. Enhanced capacity of selected methotrexate-analogues to inhibit TNF-α production Annals of the Rheumatic Diseases 65 (2006): 457. in whole blood from RA patients. Submitted for publication. 4. Van der Heijden JW, Oerlemans R, Lems WF, Scheper RJ, Dijkmans BAC, Jansen G. The proteasome inhibitor bortezomib induces apoptosis and inhibits TNF-α release by activated T-cells from rheumatoid arthritis (RA) patients. National journals Arthritis & Rheumatism 52 (2005): S261-262.

1. Van der Heijden JW. 5. Van der Heijden JW, Oerlemans R, Tak PP, Smeets TJM, Scheper RJ, Scheffer GL, Toename van een pompeiwit in de celmembraan van CEM (T) cellen geeft aanleiding Assaraf YG, Lems WF, Dijkmans BAC, Jansen G. tot resistentie/ongevoeligheid voor de DMARD sulfasalazine. Expression of the multi-drug resistance protein BCRP in synovial tissue of RA- Nederlands Tijdschrift voor Reumatologie, 5 (2) (2002), 53-55. patients. A marker for inflammation or resistance to MTX? Arthritis & Rheumatism 52 (2005): S540-541. 2. Van der Heijden JW. Macrofaag gerichte therapie voor de behandeling van RA-patienten met nieuwe 6. Van der Heijden JW, Gerards, AH, Oerlemans R, Lems WF, Scheper RJ, Aarden LA, generatie foliumzuur antagonisten. Dijkmans BAC, Jansen G. Nederlands Tijdschrift voor Reumatologie, 9 (1) (2006), 8-11. Inhibition of TNF-α production by activated T-cells of rheumatoid arthritis patients by novel anti-folate drugs: an ex vivo pilot study. 3. Van der Heijden JW. Annals of the Rheumatic Diseases 64 (2005): 417. Waarom worden mensen met RA resistent tegen anti-reumatica? In Beweging 1 (2007), 12-13. 7. Van der Heijden JW, Gerards AH, Oerlemans R, Lems WF, Scheper RJ, Aarden LA, Dijkmans BAC, Jansen G. Inhibition of tumor necrosis factor alpha production by activated T-cells of Abstracts (first author) rheumatoid arthritis patients by novel anti-folate drugs: an ex vivo pilot study. Arthritis Research & Therapy 7 (2005): S261-262. 1. Van der Heijden JW, Oerlemans R, Tak PP, Scheper RJ, Scheffer GL, Assaraf YG, Lems WF, Dijkmans BAC, Jansen G. Expression of lung resistance protein on CD68 positive synovial tissue macrophages Conference proceedings is associated with disease activity in rheumatoid arthritis patients. Arthritis & Rheumatism 54 (2006): S399-400. 1. Van der Heijden JW, Jansen G, Dijkmans BAC. Antifolate drug combinations for inflammatory diseases. 2. Van der Heijden JW, Oerlemans R, Tak PP, Van der Laken CJ, Scheper RJ, Scheffer GL, In “Chemistry and Biology of Pteridines and Folates” (G Jansen & GJ Peters, eds), SHS Assaraf YG, Lems WF, Dijkmans BAC, Jansen G. Publications, Heilbronn, Germany (2007), 373-385. Expression of multi-drug resistance related proteins in synovial tissue of rheumatoid arthritis patients: correlation with disease activity and methotrexate therapy. 2. Oerlemans R, Van der Heijden JW, Dijkmans BAC, Lems WF, Scheper RJ, Assaraf YG, Annals of the Rheumatic Diseases 65 (2006): 340-341. Jansen, G. Sulfasalazine exposure confers MTX resistance in human monocytic/ macrophage cells 3. Van der Heijden JW, Oerlemans R, Lems WF, Scheper RJ, Dijkmans BAC, Jansen G. by differential effects on folate influx and folate efflux systems.

222 223 List of publications

In: “Chemistry and Biology of Pteridines and Folates” (G Jansen & GJ Peters, eds), SHS Publications, Heilbronn, Germany (2007), 177-187.

3. Van der Heijden JW, Dijkmans BAC, Van der Laken CJ, Oerlemans R, Scheffer GL, Jackman AL, Ratnam M, Jansen, G. Antifolates in chronic inflammatory diseases/rheumatoid arthritis: what can we learn from cancer and vice versa? In: “Chemistry and Biology of Pteridines and Folates” (G Jansen & GJ Peters, eds), SHS Publications, Heilbronn, Germany (2007), 29-43.

224