Joost van der Heijden
Targeting DMARD resistance in Rheumatoid Arthritis - Joost van der Heijden
Joost van der Heijden
ISBN nummer: 978 90 8659 253 1
Cover design and lay-out: Esther Beekman, www.estherontwerpt.nl
Photo cover: ‘Schiphol 2006’, by Maarten van Haaff, www.maartenvanhaaff.nl
Printed by: PrintPartners Ipskamp, Enschede
© Copyright: Joost van der Heijden 2008
Targeting DMARD resistance in
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
Johan Willem van der Heijden
geboren te Gouda
The research described in this thesis was performed at the department of
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).
Publication of this thesis was financially supported by: Abbott B.V., Amgen B.V.,
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.
promotoren: prof. dr. B.A.C. Dijkmans
prof. dr. R.J. Scheper
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.
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
Involvement of Breast Cancer Resistance Protein expression on
RA synovial tissue macrophages in resistance to methotrexate and
Submitted for publication 98
Chapter 7 Anti-folate drug combinations for inammatory 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
ABC ATP-binding cassette
AICAR 5-aminoimidazole-4-carboxamide ribonucleotide
AICARTF 5-aminoimidazole-4-carboxamide ribonucleotide formyltransferase
ATRA All-trans retinoic acid
BCRP Breast cancer resistance protein
CCP Cyclic citrunillated peptide
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
Drug concentration required to inhibit cell growth or cytokine production by 50%
compared to non-treated cells
IκK Inhibitor-κB kinase
MDR Multi-drug resistance
MHC Major histocompatibility complex
MMP Matrix metalloproteinase
MRP Multi-drug resistance protein
MTHFR Methylene tetrahydrofolate reductase
NFκB Nuclear factor κB
NSAID Non steroidal anti-inammatory drug
PBL Peripheral blood lymphocyte
PBMC Peripheral blood mononuclear cell
PCR Polymerase chain reaction
RA Rheumatoid arthritis
RF Rheumatoid factor
RFC Reduced folate carrier
SNP Single nucleotide polymorphism
TNF Tumor necrosis factor
TS Thymidylate synthase
List of abbreviations
General introduction and
introduction into the chapters
General introduction and introduction into the chapters
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
last decade, auto-antibodies against citrullinated peptides (anti-CCP antibodies) have
been identified in two third of RA patients, which turned out to be more specific for this
disease (3). These antibodies reflect an abnormal humoral response against citrullinated
self-proteins (4). Interestingly, these antibodies can be detected years before the first
onset of clinical symptoms and antibody levels are correlated with RA disease activity (5, 6).
The question remains whether the occurrence of anti-CCP antibodies are of pathogenetic
relevance in RA.
Conceptually, the onset of joint inflammation in RA is induced by the following events,
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
perpetuation of disease by presentation of (unknown) arthritogenic (auto) antigens.
Several genetic loci are known to be associated with the susceptibility for and the severity
of RA; the most well established being a specific amino-acid sequence in the HLA-DR4
allele, the so called shared epitope, which is involved in antigen presentation (7). Upon
presentation of auto-antigens by appropriate HLA molecules, invading (pre-activated)
T-cells become (further) activated by cell-cell contact or cytokine signalling and start
to produce cytokines. These cytokines in turn stimulate on one hand B-cells to produce
auto-antibodies (e.g. RF and anti-CCP) and on the other hand macrophages and synovial
fibroblasts to produce pro-inflammatory cytokines (like TNFα and IL1-β) and matrix
metalloproteinase’s (MMPs) which provoke cartilage destruction. Activation of joint
osteoclasts eventually leads to bone destruction. Tissue infiltration by inflammatory
cells results in thickening of the synovial membrane (referred to as synovitis) and is
accompanied by neovascularisation (1, 8, 9).
For decades therapy of RA patients consisted of non-steroidal anti-inflammatory drugs
(NSAIDs), reducing the pain and inflammation but not modifying the course of the
disease. The first Disease Modifying Anti-Rheumatic Drugs (DMARDs) were intramuscular
gold (J. Forestier, 1928) and sulfasalazine (N. Svartz, 1938), interfering with the course
and progression of the disease (1,10). The modifying effect of DMARDs is reflected in
improvement of the Disease Activity Score (DAS) (a composite index of the patients
opinion of disease activity, a count of tender and swollen joints by the physician and the
erythrocyte sedimentation rate) (11) and parameters of joint damage as seen on X-rays of
hands and feet.
Corticoids are used since the 1940’s with very good clinical response, but there are
limitations to treatment duration because of the occurrence of side effects upon
prolonged use, like osteoporosis, hypertension and diabetes. Methotrexate (MTX) was first
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:
Figure 1: Schematic view of a normal joint and its changes in RA.
(A) The normal joint. The synovial joint is composed of two adjacent bony ends, each covered with a layer
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.
(B) The arthritic joint. RA is characterized by an inﬂammatory response of the synovial membrane (referred
to as synovitis) that is conveyed by a transendothelial inﬂux and local activation of a variety of mononuclear
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 lining layer becomes hyperplastic and the synovial membrane expands and forms villi; the destructive
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.
Lancet 2007; 370:1861-1874.
General introduction and introduction into the chapters
(a) the rediscovery of MTX in the 1980’s (b) the introduction of combination therapies and
(c) the introduction of biologic agents in the 1990’s. The term biologics refers to a group
of therapeutic agents that specifically target a particular cell or cytokine involved in the
pathogenesis of RA. These highly effective anti-inflammatory agents include infliximab
and adalimumab (both antibodies against TNFα), etanercept (soluble TNFα receptors),
rituximab (antibodies to CD20/B-cells) and abatacept (antibodies to the co-stimulatory
molecule B7 on antigen presenting cells to prevent T-cell activation) (1, 8, 10, 12-14). These
drugs are usually combined with MTX. In the Netherlands physicians agreed with the
government to prescribe biologics only to those patients that sequentially failed on two
regular DMARDs, mainly to reduce drug related costs.
Today, many studies have proven that early and aggressive therapy with (combinations of)
DMARDs and/or biologic agents is beneficial in terms of preservation of functional ability
and slowing down of joint damage progression (12). After remission induction, DMARDs
and/or biologic agents can then be tapered and in some cases even stopped (15).
Despite the current success of biologic therapies, DMARDs have an established place in the
treatment of RA because of their efficacy, convenience, safety and low related drug costs
(16). Currently, the most commonly applied DMARDs are MTX, sulfasalazine, leflunomide,
prednisone and antimalarials like hydroxychloroquine. Their therapeutic efficacy can be
enhanced by combining these DMARDs, however a rationale behind these combinations
is often lacking (17, 18). Sulfasalazine plus MTX is a frequently used combination; however,
this combination does not display additive or synergistic effects over monotherapy (19,
20), unless a third DMARD (hydroxychloroquine or prednisone) is added (21-25).
Observational studies and meta-analyses of treatment efficacy, average treatment
duration and adverse effects for RA-patients consistently demonstrate variable responses
for individual DMARDs and DMARD combinations (26, 26-30). Unfortunately, many
RA patients experience loss of efficacy upon chronic treatment with DMARDs, which
could point to the onset of acquired drug resistance. Galindo-Rodrigues et al showed in
a retrospective practice-based study between 1985 and 1994, including 2296 DMARD
therapies, that after 16 months 50% of treatments had been discontinued because of
inefficacy and/or toxicity and that after 4.5 years 75% of therapies had been discontinued.
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
antimalarials and 25% for sulfasalazine (31) (figure 2). At the time this study was performed
however, TNFα antagonists were not yet available. Despite the initial good response to
MTX for many patients, there is room for improvement of DMARD therapy and a need for
studies that unravel possible mechanisms of DMARD resistance and studies that define
parameters for predicting DMARD efficacy and toxicity.
The issue of drug resistance as a cause of therapy failure and the search for new therapeutic
approaches to circumvent drug-resistance has received much attention in cancer
treatment (32); however it just starts to be appreciated in RA treatment (33-36). Human
beings have intrinsic and inducible mechanisms against daily exposure to hundreds or
even thousands of xenobiotic substances present in our environment and food (32, 37,
38). Most chemically designed therapeutic drugs, including DMARDs, are recognized
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:
(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.
Resistance to biologic agents
While treatment with biologic agents provides great benefit to the majority of RA
patients, some patients experience persistent active disease or loss of efficacy upon
prolonged treatment. Biologic agents are proteins and therefore antibodies can be
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
discontinuations. IM: intramuscular; *study completed in 1994 – no other choices available at that time for
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
Rheumatol 1999; 26(11):2337-2343 and Fleischmann RM et al. J Rheumatol Suppl 2005; 73:3-7.
General introduction and introduction into the chapters
of MDR proteins on inflammatory cells (T-cells, macrophages) in synovial tissue of RA
patients and assessed whether expression of these proteins correlated with clinical
outcome after treatment with MTX or leflunomide.
Besides MTX efflux by MDR transporters (33, 37, 38, 50-52), cellular resistance to MTX
might be conferred by diminished uptake via its cell membrane carrier, the reduced
folate carrier (RFC). Also impaired polyglutamylation by the enzyme folylpolyglutamate
synthethase (FPGS) or alterations in the target enzymes of MTX (a.o. dihydrofolate
reductase) may contribute to a resistant phenotype (53-59).
The second section of this thesis focuses on novel experimental therapeutic strategies to
overcome MTX resistance. In this context, we utilized activated T-cells from RA patients
to test potential anti-inflammatory effects of novel generation antifolate drugs that were
rationally designed to overcome known mechanisms of MTX resistance in cancer patients
(60). Furthermore, we evaluated whether the Folate Receptor β (FRβ), expressed on
synovial tissue macrophages, could be selectively targeted by novel generation antifolate
drugs that display a higher affinity for this receptor than MTX (61, 62).
Finally, in the last section of this thesis we focussed on a novel class of experimental drugs
that may retain therapeutic activity against resistant cells, due to the fact that they are no
substrates for MDR transporters. Such class of compounds include proteasome inhibitors,
which interfere in intracellular protein degradation (63). Bortezomib, a boronic acid
dipeptide, is the clinically used representative of this class of drugs (64-66). Bortezomib
has recently been approved for the treatment of therapy refractory multiple myeloma.
produced against these agents, diminishing clinical efficacy. Wolbink et al showed that
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
plus a biologic agent appeared to be superior over monotherapy with MTX or the biologic
agent (43-48). For this reason, it is recommended to combine a biologic agent with MTX.
Aim and outline of the thesis
This thesis focuses on the mechanisms involving DMARD resistance in the treatment of
RA patients and alternative ways to target RA inflammatory cells in case of resistance to
currently available DMARDs.
From the field of oncology, increased drug efflux has been recognized as one important
mechanism of drug resistance. Cell-membrane proteins responsible for drug efflux belong
to the family of ATP-binding cassette (ABC) transporters of which 49 different proteins
were identified from the human genome project (http://nutrigene.4t.com/humanabc.
htm). Tissue distribution studies showed that these proteins are expressed in tissues
that are heavily exposed to toxic agents, microbial and exogenous compounds, eg. liver,
kidney and intestine (49). Consistently, one of the primary functions of ABC-transporters
is extrusion of toxic substances. Some of these transporters are extensively characterized
while the functional properties of others are still unknown (37, 38, 50). A wide range of
structurally and functionally different drugs can be pumped out of cells by these ABC-
transporter proteins, thereby conferring a so-called multiple-drug resistant (MDR)
phenotype. Therapeutic drugs are frequently extruded in co-transport with gluthatione,
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
hypothesis that these proteins might also provide a cellular defence mechanism against
DMARDs, leading to DMARD-resistance. In the first section of this thesis we evaluated
the potential role of ABC-transporters in conferring resistance to DMARDs. To this end,
we first investigated whether MDR proteins become upregulated on inflammatory model
cells upon chronic exposure to DMARDs in vitro. In addition, we evaluated the expression
Figure 3: Molecular mechanisms of cellular drug resistance.
(1) Diminished drug delivery (2) impaired drug uptake, (3) energy dependent cellular drug extrusion via MDR
transporters, (4) decreased drug activation, (5) drug sequestration or (6) enhanced drug detoxiﬁcation (7)
alterations in the target of the drug or (8) enhanced repair of damage/ impaired capacity for cells to go
Conceptually, bortezomib may elicit potential anti-inflammatory effects by inhibiting the
degradation of the natural inhibitor of the transcription factor NFκB , i.e. IκBα, thereby
inhibiting nuclear translocation of NFκB and transcription of several pro-inflammatory
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.
Introduction into the chapters
In chapter two, a literature review is given that outlines molecular mechanisms that could
be involved in the onset of resistance to DMARDs in RA patients, including methotrexate,
sulfasalazine, hydroxychloroquine, azathioprine and leflunomide. The mechanisms
suggested are based on findings from experimental laboratory studies of specific drug-
uptake and drug-efflux transporters, alterations in intracellular drug metabolism and
genetic polymorphisms of drug transporters and metabolic enzymes. In this chapter we
also discuss strategies to overcome resistance and the current clinical studies aiming to
predict the response to treatments and risk of toxic effects.
In chapter three we determined whether overexpression of MDR proteins contribute to
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
for expression of the MDR-proteins P-glycoprotein (Pgp), Multidrug resistance protein 1
(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
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
resistance on responsiveness to other, non-related DMARDs.
Since we observed (chapter four) that sulfasalazine resistant T-cells in vitro were cross-
resistant to MTX when co-incubated with sulfasalazine, along with the clinical notion
that combination therapy of sulfasalazine and MTX does not show improvement over
monotherapy with MTX or sulfasalazine, we investigated whether exposure of cells
to sulfasalazine provokes intervention with the cellular pharmacology of MTX. For this
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
MTX, the Reduced Folate Carrier (RFC).
In chapter six we described an inventory study of expression of MDR-transporters
on inflammatory cells in RA synovial tissue before and after treatment with MTX or
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
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
cytokines, anti-apoptotic factors and adhesion molecules. Reference: Paramore A and Frantz S. Nat Rev
Drug Discov 2003; 2(8):611-612.
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.
General introduction and introduction into the chapters
1 Smolen,J.S. and Steiner,G. Therapeutic strategies for rheumatoid arthritis, Nat.Rev.Drug Discov.,
2: 473-488, 2003.
2 Smolen,J.S., Aletaha,D., Koeller,M., Weisman,M.H. and Emery,P. New therapies for treatment of
rheumatoid arthritis, Lancet, 370: 1861-1874, 2007.
3 Schellekens,G.A., Visser,H., de Jong,B.A.C., van den Hoogen,F.H., Hazes,J.M., Breedveld,F.C. and
van Venrooij,W.J. The diagnostic properties of rheumatoid arthritis antibodies recognizing a
cyclic citrullinated peptide, Arthritis Rheum., 43: 155-163, 2000.
4 Vossenaar,E.R. and van Venrooij,W.J. Citrullinated proteins: sparks that may ignite the re in
rheumatoid arthritis, Arthritis Res.Ther., 6: 107-111, 2004.
5 Nielen,M.M., van Schaardenburg, D., Reesink,H.W., van de Stadt,R.J., van der Horst-Bruinsma IE,
de Koning,M.H., Habibuw,M.R., Vandenbroucke,J.P. and Dijkmans,B.A.C. Specic autoantibodies
precede the symptoms of rheumatoid arthritis: a study of serial measurements in blood donors,
Arthritis Rheum., 50: 380-386, 2004.
6 Rantapaa-Dahlqvist,S., de Jong,B.A.C., Berglin,E., Hallmans,G., Wadell,G., Stenlund,H., Sundin,U.
and van Venrooij,W.J. Antibodies against cyclic citrullinated peptide and IgA rheumatoid factor
predict the development of rheumatoid arthritis, Arthritis Rheum., 48: 2741-2749, 2003.
7 Bowes,J. and Barton,A. Recent advances in the genetics of RA susceptibility, Rheumatology.
(Oxford), 47: 399-402, 2008.
8 Smolen,J.S., Aletaha,D., Koeller,M., Weisman,M.H. and Emery,P. New therapies for treatment of
rheumatoid arthritis, Lancet, 2007.
9 McInnes,I.B. and Schett,G. Cytokines in the pathogenesis of rheumatoid arthritis, Nat.Rev.
Immunol., 7: 429-442, 2007.
10 O’Dell,J.R. Therapeutic strategies for rheumatoid arthritis, N.Engl.J.Med., 350: 2591-2602, 2004.
11 van der Heijde,D.M., van ‘t Hof,M., van Riel,P.L. and van de Putte,L.B. Validity of single variables
and indices to measure disease activity in rheumatoid arthritis, J.Rheumatol., 20: 538-541, 1993.
12 Goekoop-Ruiterman,Y.P., De Vries-Bouwstra,J.K., Allaart,C.F., van Zeben,D., Kerstens,P.J., Hazes,J.
M., Zwinderman,A.H., Ronday,H.K., Han,K.H., Westedt,M.L., Gerards,A.H., van Groenendael,J.
H., Lems,W.F., van Krugten,M.V., Breedveld,F.C. and Dijkmans,B.A.C. Clinical and radiographic
outcomes of four different treatment strategies in patients with early rheumatoid arthritis (the
BeSt study): a randomized, controlled trial, Arthritis Rheum., 52: 3381-3390, 2005.
13 Elliott,M.J., Maini,R.N., Feldmann,M., Kalden,J.R., Antoni,C., Smolen,J.S., Leeb,B., Breedveld,F.
C., Macfarlane,J.D., Bijl,H. et al. Randomised double-blind comparison of chimeric monoclonal
antibody to tumour necrosis factor alpha (cA2) versus placebo in rheumatoid arthritis, Lancet,
344: 1105-1110, 1994.
14 Ruderman,E.M. and Pope,R.M. Drug Insight: abatacept for the treatment of rheumatoid arthritis,
Nat.Clin.Pract.Rheumatol., 2: 654-660, 2006.
15 van der Kooij,S.M., Goekoop-Ruiterman,Y.P., De Vries-Bouwstra,J.K., Peeters,A.J., van Krugten,M.
V., Breedveld,F.C., Dijkmans,B.A.C. and Allaart,C.F. Probability of continued low disease activity
in patients with recent onset rheumatoid arthritis treated according to the disease activity score,
Ann.Rheum.Dis., 67: 266-269, 2008.
16 Cannella,A.C. and O’Dell,J.R. Is there still a role for traditional disease-modifying antirheumatic
drugs (DMARDs) in rheumatoid arthritis?, Curr.Opin.Rheumatol., 15: 185-192, 2003.
17 Fathy,N. and Furst,D.E. Combination therapy for autoimmune diseases: the rheumatoid arthritis
model, Springer Semin.Immunopathol., 23: 5-26, 2001.
18 Smolen,J.S., Aletaha,D. and Keystone,E. Superior efcacy of combination therapy for rheumatoid
arthritis: fact or ction?, Arthritis Rheum., 52: 2975-2983, 2005.
19 Haagsma,C.J., Russel,F.G., Vree,T.B., van Riel,P.L. and van de Putte,L.B. Combination of
methotrexate and sulphasalazine in patients with rheumatoid arthritis: pharmacokinetic analysis
and relationship to clinical response, Br.J.Clin.Pharmacol., 42: 195-200, 1996.
20 Haagsma,C.J., van Riel,P.L., de Jong,A.J. and van de Putte,L.B. Combination of sulphasalazine
and methotrexate versus the single components in early rheumatoid arthritis: a randomized,
controlled, double-blind, 52 week clinical trial, Br.J.Rheumatol., 36: 1082-1088, 1997.
21 Boers,M., Verhoeven,A.C., Markusse,H.M., van de Laar,M.A., Westhovens,R., van Denderen,J.C.,
van Zeben,D., Dijkmans,B.A.C., Peeters,A.J., Jacobs,P., van den Brink,H.R., Schouten,H.J., van der
Heijde,D.M., Boonen,A. and Van der Linden,S. Randomised comparison of combined step-down
prednisolone, methotrexate and sulphasalazine with sulphasalazine alone in early rheumatoid
arthritis, Lancet, 350: 309-318, 1997.
22 Barrera,P., Haagsma,C.J., Boerbooms,A.M., van Riel,P.L., Borm,G.F., van de Putte,L.B. and Van der
Meer,J.W. Effect of methotrexate alone or in combination with sulphasalazine on the production
and circulating concentrations of cytokines and their antagonists. Longitudinal evaluation in
patients with rheumatoid arthritis, Br.J.Rheumatol., 34: 747-755, 1995.
23 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
Heijde,D.M. and Van der Linden,S. COBRA combination therapy in patients with early rheumatoid
arthritis: Long-term structural benefits of a brief intervention, Arthritis Rheum., 46: 347-356,
24 O’Dell,J.R., Haire,C.E., Erikson,N., Drymalski,W., Palmer,W., Eckhoff,P.J., Garwood,V.,
Maloley,P., Klassen,L.W., Wees,S., Klein,H. and Moore,G.F. Treatment of rheumatoid arthritis
with methotrexate alone, sulfasalazine and hydroxychloroquine, or a combination of all three
medications, N.Engl.J.Med., 334: 1287-1291, 1996.
25 O’Dell,J.R., Leff,R., Paulsen,G., Haire,C., Mallek,J., Eckhoff,P.J., Fernandez,A., Blakely,K.,
Wees,S., Stoner,J., Hadley,S., Felt,J., Palmer,W., Waytz,P., Churchill,M., Klassen,L. and Moore,G.
Treatment of rheumatoid arthritis with methotrexate and hydroxychloroquine, methotrexate
and sulfasalazine, or a combination of the three medications: Results of a two-year, randomized,
double-blind, placebo-controlled trial, Arthritis Rheum., 46: 1164-1170, 2002.
26 Fleischmann,R.M. Is there a need for new therapies for rheumatoid arthritis?, J.Rheumatol.Suppl,
73: 3-7, 2005.
27 Wolfe,F. The epidemiology of drug treatment failure in rheumatoid arthritis, Baillieres Clin.
Rheumatol., 9: 619-632, 1995.
28 de la Mata,J., Blanco,F.J. and Gomez-Reino,J.J. Survival analysis of disease modifying antirheumatic
drugs in Spanish rheumatoid arthritis patients, Ann.Rheum.Dis., 54: 881-885, 1995.
29 Maetzel,A., Wong,A., Strand,V., Tugwell,P., Wells,G. and Bombardier,C. Meta-analysis of
treatment termination rates among rheumatoid arthritis patients receiving disease-modifying
anti-rheumatic drugs, Rheumatology.(Oxford), 39: 975-981, 2000.
30 Aletaha,D. and Smolen,J.S. Effectiveness profiles and dose dependent retention of traditional
disease modifying antirheumatic drugs for rheumatoid arthritis. An observational study,
J.Rheumatol., 29: 1631-1638, 2002.
31 Galindo-Rodriguez,G., vina-Zubieta,J.A., Russell,A.S. and Suarez-Almazor,M.E. Disappointing
longterm results with disease modifying antirheumatic drugs. A practice based study,
J.Rheumatol., 26: 2337-2343, 1999.
32 Szakacs,G., Paterson,J.K., Ludwig,J.A., Booth-Genthe,C. and Gottesman,M.M. Targeting
multidrug resistance in cancer, Nat.Rev.Drug Discov., 5: 219-234, 2006.
33 Jansen,G., Scheper,R.J. and Dijkmans,B.A.C. Multidrug resistance proteins in rheumatoid arthritis,
role in disease-modifying antirheumatic drug efficacy and inflammatory processes: an overview,
Scand.J.Rheumatol., 32: 325-336, 2003.
34 Wollheim,F.A. Drug resistance in rheumatology: an area in search of investigators, Curr.
Rheumatol.Rep., 5: 333-335, 2003.
35 Morgan,C., Lunt,M., Brightwell,H., Bradburn,P., Fallow,W., Lay,M., Silman,A. and Bruce,I.N.
Contribution of patient related differences to multidrug resistance in rheumatoid arthritis, Ann.
Rheum.Dis., 62: 15-19, 2003.
36 Tsujimura,S., Saito,K., Nawata,M., Nakayamada,S. and Tanaka,Y. Overcoming drug resistance
induced by P-glycoprotein on lymphocytes in patients with refractory rheumatoid arthritis, Ann.
Rheum.Dis., 67: 380-388, 2008.
37 Deeley,R.G., Westlake,C. and Cole,S.P. Transmembrane transport of endo- and xenobiotics by
mammalian ATP-binding cassette multidrug resistance proteins, Physiol Rev., 86: 849-899,
38 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,
39 Wolbink,G.J., Vis,M., Lems,W., Voskuyl,A.E., de,G.E., Nurmohamed,M.T., Stapel,S., Tak,P.P.,
Aarden,L. and Dijkmans,B.A.C. Development of antiinfliximab antibodies and relationship to
clinical response in patients with rheumatoid arthritis, Arthritis Rheum., 54: 711-715, 2006.
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.
and van der Horst-Bruinsma IE. Decreased clinical response to infliximab in ankylosing spondylitis
is correlated with anti-infliximab formation, Ann.Rheum.Dis., 66: 1252-1254, 2007.
41 Bartelds,G.M., Wijbrandts,C.A., Nurmohamed,M.T., Stapel,S., Lems,W.F., Aarden,L., Dijkmans,B.
A.C., Tak,P.P. and Wolbink,G.J. Clinical response to adalimumab: relationship to anti-adalimumab
antibodies and serum adalimumab concentrations in rheumatoid arthritis, Ann.Rheum.Dis., 66:
42 Maini,R.N., Breedveld,F.C., Kalden,J.