multidrug resistance proteins in rheumatoid arthritis, role in disease‐modifying antirheumatic...

REVIEW

Multidrug resistance proteins in rheumatoid arthritis, role indisease-modifying antirheumatic drug efficacy and inflammatoryprocesses: an overview

G Jansen1, RJ Scheper2, BAC Dijkmans1

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

Drug resistance is generally accepted as an important cause of treatment failure for patients with neoplastic or

infectious diseases. Molecular mechanisms underlying drug resistance include the action of drug efflux pumps

belonging to the super-family of ATP binding cassette (ABC) proteins, which mediate the cellular extrusion of

a large variety of therapeutic drugs, a phenotype that is referred to as multidrug resistance (MDR). Unlike

neoplastic and infectious diseases, chronic inflammatory diseases have received little attention. The potential

role of ABC transporters in determining the efficacy of anti-rheumatic drugs, notably disease modifying anti-

rheumatic drugs (DMARDs), in patients with rheumatoid arthritis is unclear. Based on knowledge from the

field of oncology and immunology, this review concentrates on the pharmacological role of MDR proteins inthe (clinical) efficacy of several DMARDs, as well as the physiological role of MDR proteins in transporting

signalling molecules important in inflammatory processes.

Key words: rheumatoid arthritis, DMARDs, multidrug resistance, ABC transporters, P-glycoprotein, multidrug resistanceassociated protein, methotrexate, sulphasalazine, chloroquine, review

Rheumatoid arthritis (RA) is a common autoim-

mune disease that is characterized by a chronic

inflammation of the synovial joints, and infiltration

by blood-derived cells (T cells, macrophages),

leading to progressive destruction of cartilage and

bone (1). Current medical therapy of patients with

RA consists of treatment with:

. Non-steroidal anti-inflammatory drugs (NSAIDs),

which reduce pain and inflammation but do not

alter the course of the disease

. Disease modifying anti-rheumatic drugs (DMARDs),

which can influence or modify the course of RA.

Over the past decade ‘biologicals’ have also been

introduced in the treatment of RA (2). These

biologicals are specifically aimed at antibody-guided

blocking of pro-inflammatory cytokines [e.g. tumour

necrosis factor a (TNFa)] that play a major role in

the pathophysiology of RA (3). Novel anti-TNFaagents, such as infliximab (4) or etanercept (5),

displayed marked anti-rheumatic activity, but their

long-term superiority over DMARDs — with known

efficacy and low costs — still needs to be established,

especially in the treatment of early RA. For this

reason, DMARDs or combinations of DMARDs

remain a common treatment option for patients with

RA (6 – 9). However, it is well recognized that with

successive treatment with DMARDs, many RA

patients demonstrate loss of drug efficacy over time.

Whether this is due to the onset of acquired drug

resistance has not been extensively investigated.Meta-analyses of DMARD efficacy for treatment

of RA patients showed that (hydroxy)chloroquine

and sulphasalazine were among the DMARDs with

the relatively shortest duration of efficacy (1 – 2

years), while methotrexate (MTX) had the longest-

lasting efficacy (6 – 7 years) (10 – 13).

Recently, a study by Morgan (14) showed that a

small population of RA patients may be primary

unresponsive to DMARDs, possibly via a multidrug

resistance (MDR)-related mechanism.

This review will further elaborate on the possible

role of MDR proteins in the efficacy of DMARDs in

RA treatment. However, it should be kept in mind

that, as with anti-cancer drugs, DMARD resistance

may be a multifactorial event, not only involving

enhanced drug efflux via specific MDR proteins, but

also other mechanisms, outlined in Figure 1A. In

short, other mechanism of drug resistance can

include:

. Defective drug delivery (pharmacokinetic resis-

tance)

. Impaired drug uptake

. Impaired drug activation

. Enhanced drug detoxification/sequestration

Gerrit Jansen, Department of Rheumatology, Rm 4A42, VU

University Medical Center, De Boelelaan 1117, 1081 HV

Amsterdam, The Netherlands.

E-mail: [email protected]

Received 11 September 2003

Accepted 11 September 2003

Scand J Rheumatol 2003;32:325–336 325

www.scandjrheumatol.dk

# 2003 Taylor & Francis on license from Scandinavian Rheumatology Research Foundation

DOI: 10.1080/03009740310004333

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. Altered (downstream) effects after drug – target

interaction (15).

(Multi) drug resistance

Drug resistance can be an inherent/intrinsic property

of cells, indicating that cells are resistant to specific

classes of drugs without ever been exposed to these

drugs previously. Alternatively, drug resistance

can be acquired, indicating that cells were primary

sensitive, but develop resistance over successive drugtreatments. Drug resistance can be restricted to one

specific class of drugs, e.g. antifolates like MTX,

or can extend to multiple structurally unrelated

drugs, a phenotype designated as MDR (16 – 21).

Molecular mechanisms of cellular drug resistance

can take place at various levels, involving,

for example, impaired cellular uptake, enhanced

metabolism, increased levels of target enzymes,defective apoptotic pathways and/or enhanced cel-

lular extrusion of drugs (15, 22).

This latter mechanism can be mediated by the

up-regulation of specific members of the family of

ATP binding cassette (ABC) transporters (17, 21,

23 – 26). Drug extrusion via these transporters is an

active process requiring ATP as an energy source

(Figure 1B). Data from the human genome projectrevealed that the ABC transporter family includes 48

proteins (25, 27, 28), divided into seven distinct

subfamilies (ABC A–G), some of which are

extensively characterized, while the functional prop-

erties of others are still unknown.

Several review papers have appeared over the past

few years covering specific aspects of ABC trans-

porters in relation to resistance to anti-cancer drugs

(17 – 19, 21, 23 – 26, 29 – 34). ABC transporters that

have an established or putative role in drug

resistance include (Table 1):

. ABCB1 [P-glycoprotein (Pgp), primary MDR

(MDR1)] (16, 35 – 37)

. ABCC1-7, ABCC10/11, also denoted as MDR-

associated protein 1 – 9 (MRP1 – 9) (38 – 49)

. ABCG2, also known as breast-cancer resistance

protein (BCRP) (20, 34, 50 – 53).

Pgp and MRPs are ‘full transporters’ with 12 – 17

transmembrane domains (TMDs) assembling a pore-

forming protein (25). In contrast, ABCG2 is referred

to as a ‘half transporter’ (six TMDs) requiring

homo- or hetero-dimerization to form a functionally

active protein (21, 54).

Although there is overlap in substrate specificity,

Pgp is known to prefer neutral hydrophobic sub-

strates, while MRPs and ABCG2 transport anionic

compounds, mostly as conjugates of glutathione,

Figure 1. (A) Molecular mechanisms of cellular drug resistance. (1) Efficient drug delivery to target cells/tissues is a critical step in the

efficacy of drugs. Drug resistance due to diminished drug delivery is referred to as pharmacokinetic resistance. (2) Cell membrane

translocation of drugs can proceed via various routes, passive diffusion (a), facilitated diffusion (b) or via a carrier-mediated process

(c) (which is the case for MTX). Defective cellular uptake is a common resistance mechanism. (3) Prior to drug activation, cellular

extrusion may take place via specific efflux transporters, e.g. ABC transporters, thereby reducing the intracellular concentration of drug

available for therapeutic activity. (4) Drug activation, e.g. phosphorylation may be required for better intracellular retention and better

inhibition of the target. This process can be counteracted by enhanced drug sequestration (5), e.g. in subcellular compartments (e.g.

lysosomes), or detoxification (6), e.g. dephoshorylation, both of which can lead to drug resistance. Drug targeting (7) should contribute

to drug efficacy by damaging the target and inhibiting its function. (8) Several modes of escape from drug interactions exist, thereby

conferring resistance: they include up-regulation of target levels (e.g. increased DHFR in case of MTX resistance), modified/mutated

targets, enhanced repair of damage, impaired capacity for cells to go into apoptosis, or salvage of the damage by exogenous com-

pounds (e.g. purines and pyrimidines as a bypass for MTX effects (modified from Reference 22). (B) Energy-dependent 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.

326 G Jansen et al


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glucuronides or sulphates (25, 55 – 60). Tissue

distribution studies showed that MDR proteins are

expressed at locations and by tissues that are heavily

exposed to toxic agents, microbials and exogenous

compounds (31, 53, 61, 62). Consistently, one of the

primary functions of MDR proteins is the extrusion

of toxic substances.MDR proteins Pgp and MRP1 have been detected

in immunologically relevant cells [T cells, monocytes,

dendritic cells (DCs) and macrophages] (61, 63 – 73),

where they are thought to play not only a

pharmacological, but also a physiological role in

inflammatory processes (see later sections). Interest-

ingly, ABCG2 has been identified in a side popula-

tion of CD34z haematopoietic progenitor cells (74 –76), although its pharmacological/physiological rele-

vance is still unclear.

MDR proteins and DMARDs/anti-inflammatory drugs

Substrate specificity studies suggest that several

DMARDs, NSAIDs and other anti-inflammatory

drugs (e.g. glucocorticoids) can be transported by,

or inhibit, specific MDR proteins (summarized inTable 2). The putative role of MDR proteins in loss

of efficacy/resistance for these anti-rheumatic drugs

is discussed in greater detail below.

Methotrexate

The folate antagonist MTX is the anchor drug in the

treatment of RA patients. The precise mechanism of

the anti-rheumatic action of MTX has still not been

fully elucidated, but involves the inhibition of keyenzymes in folate metabolism and purine biosynthesis

de novo, both of these are required for DNA

synthesis (77). This inhibition provokes folate

depletion and enhanced adenosine release, leading

to apoptosis (78) and/or interaction with adenosine

receptors, resulting in an anti-inflammatory effect

(79). Cell entry of MTX requires a specific

transporter, the reduced folate carrier (RFC), which

normally internalizes natural folates (80 – 82). Once

taken up intracellularly, MTX is complexed to

polyglutamate forms, which prevents its leakage

from the cells (83, 84). In cancer patients, the most

common mechanisms of acquired resistance to MTX

include impaired RFC transport, elevated levels of

dihydrofolate reductase (DHFR), and/or impaired

polyglutamylation (85 – 87).Originally, the MDR transporter Pgp was pro-

posed to play a role in MTX efflux in a T-cell line

that displayed resistance to supra-pharmacological

concentrations of MTX (88). However, the dominant

mechanism of MTX resistance in this cell line was

defective RFC- mediated uptake of MTX, rather

than Pgp-mediated efflux. Consistent with the notion

that Pgp preferentially transports neutral hydrophic

compounds, rather than a divalent anion like MTX,

there is currently broad consensus that Pgp is not a

major player in MTX efflux (16, 25, 55, 89). Instead

of Pgp, there is compelling evidence that the MDR

transporters MRP1-4 and ABCG2 have the capacity

to extrude non-polyglutamated MTX (90 – 99). Of

note is that ABCG2 appears to be capable of

transporting di- and tri-glutamate forms of MTX

(58).Transport kinetic data from these studies showed

that MTX efflux via MRP1-4 and BCRP is a low

affinity/high capacity process, in contrast to MTX

Table 1. Overview of selected ABC transporters/MDR proteins with known involvement of drug resistance (to anti-cancer drugs).

ABCcode

Commonname

Chromosomelocalization

Size(AA)

Normal tissuedistribution

Immumologicalcells

Preferredsubstrates Refs

ABCB1 P-gp,MDR1

7q21 1279 Many tissues T cells (CD4, CD8)DCs, monocytes

Neutral hydrophobiccompounds

(18, 25, 68, 71,125, 65, 69, 73)

ABCC1 MRP1 16p13.1 1531 Ubiquitous T cells (CD56, CD8,CD4), CD34z, DC, Mw

Anionic conjugates(glutathione, glucoronide)

(25, 38, 61, 70,204, 73, 205)

ABCC2 MRP2 10q24 1545 Liver, intestine,kidney

Anionic conjugates,glutathione

(19, 39)

ABCC3 MRP3 17q21.3 1527 Liver Anionic conjugates (25, 91)ABCC4 MRP4 13q32 1325 Many tissues

(kidney)Nucleoside analogues (39, 41, 93, 113)

ABCC5 MRP5 3q27 1437 Many tissues Nucleoside analogues (39, 43, 113)ABCC6 MRP6 16p13.1 1503 Liver, kidney Glutathione conjugates,

small peptides(45, 206)

ABCC10 MRP7 6p21 1513 Low in alltissues

? (46, 207)

ABCC11 MRP8 16q12.1 1382 Low in alltissues

Fluoropyrimidines (47, 48)

ABCG2 BCRP 4q22 655 Liver, breast,placenta

CD34z (side population) Amphipatic drugs (21, 51, 53, 74)

AA~Number of amino acids, Mw~macrophages. For timely updates on properties of ABC transporters, see http://www.nutrigene.4t.com/humanabc.htm

MDR and rheumatoid arthritis 327


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influx via RFC, which is a high affinity/low capacity

process (80, 82). Apparently, a dynamic balance

exists between the influx and efflux routes in con-

trolling intracellular levels of MTX. In this respect,

MTX exposure time, which allows conversion to

non-effluxable polyglutamated forms, can also be

a determining factor in the efficacy of MTX. This is

illustrated in Table 3, where the efficacy (anti-

proliferative effect) of MTX was evaluated against

Pgp-, MRP1- and BCRP-over-expressing cells com-

pared with controls after either a 72 h continuous

exposure to MTX (permissive for polyglutamyla-

tion), or a short-term (4 h) exposure to MTX

(incomplete polyglutamylation) followed by a 68 h

drug-free period. Following a 4 h exposure to MTX,

only cells over-expressing MRP1 and BCRP dis-

played a high level of resistance to MTX, indicating

that MRP1 and BCRP, but not Pgp, were able to

extrude unpolyglutamated MTX, resulting in lower

intracellular levels and diminished efficacy. Follow-

ing a 72 h exposure to MTX, low levels of resistance

were still noted for the MRP1 and BCRP1, but not

for the Pgp over-expressing cells.

Finally, it is of importance to note that MRP1-3

can also mediate the efflux of natural folates (100).In this respect, diminished expression of MRP1 may

lead to increased intracellular folate pools, which can

antagonize MTX activity (101).

Sulphasalazine

Sulphasalazine (SSZ) is a potent DMARD used as

a single agent and in combination regimens with

MTX, hydroxychloroquine and prednisone (8, 9).

SSZ is an organic anion that, based on structure –

activity relationships, could be a potential substratefor members of the ABCC family of MDR

transporters. In fact, a role for MRP1 in SSZ

transport was indicated from studies by Lightfoot

(102): however, studies from our laboratory (unpub-

lished) did not demonstrate resistance to SSZ in

MRP1-overexpressing cells. This may be caused by

Table 2. Overview of selected ABC transporters/MDR proteins with known physiologic and pharmacologic substrates/inhibitors(DMARDs and other anti-inflammatory drugs).

ABCcode

Commonname

Pharmacologicsubstrate (DMARD/anti-inflammatory drug) Physiologic substrate Inhibitor(s) Refs

ABCB1 P-gp,MDR1

Chloroquine, CSA Steroids, lipids, PAF Verapamil, PSC833 (18, 25, 123, 126, 127)

Prednisone,dexamethasone

LY335979, CsA (106, 120, 190)

ABCC1 MRP1 MTX, Chloroquine LTC4, LTB4, LTE4 MK571, probenecid, (25, 90, 108, 128, 165)Folates NSAIDs, flavonoids, CHQ (158, 183, 208)

ABCC2 MRP2 MTX Bile salts, LTC4, steroids Sulphinpyrazone (25, 90)ABCC3 MRP3 MTX Bile salts, LTC4, steroids Probenecid (91, 92, 94, 95)ABCC4 MRP4 MTX, Azathioprine

(6-MP)LTE4, cAMP, cGMP Probenecid, NSAIDs (93, 113, 114, 135, 136)

ABCC5 MRP5 Azathioprine (6-MP) cGMP, cAMP Nitroprusside (43, 113, 114, 134)ABCC6 MRP6 LTC4 (45)ABCC10 MRP7 LTC4, steroids CSA (46)ABCC11 MRP8 cAMP/cGMP (48)ABCG2 BCRP MTX, SSZ (?) Steroids, chlorophyll

metabolitesGF120918, Ko143 (58, 59, 99, 167, 168)

Fumitremorgin C (20, 26, 185, 186, 188, 209)

E217bG~17b-oestradiol glucuronide.

Table 3. Role of MDR proteins in MTX resistance.

Exposure time MTX

Resistance Factor(#)

ABCB1(##) (Pgp) ABCC1(###) (MRP1) ABCG2(####) (BCRP)

72 h 0.8 1.9 1.74 h (z68 h drug free) 1.1 w80 28

(#)Resistance factor is defined as the ratio of IC50 concentration (MTX concentration resulting in 50% inhibition of cell proliferation) of cellswith over-expression of the indicated ABC transporter versus control cells without over-expression of the ABC transporter.(##)ABCB1/Pgp-overexpressing cells were vinblastin-resistant CEM cells (106).(###)ABCC1/MRP1-overexpressing cells were MRP1 transfected 2008 cells (90).(####)ABCG2/BCRP-overexpressing cells were mitoxantrone-resistant MCF-7/MR cells (50).

328 G Jansen et al


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the fact that SSZ is also an inhibitior of glutathione-

S-transferase (103), the enzyme responsible for

glutathione conjugation prior to MRP1 transport.Recent studies by our laboratory have shown that

acquired resistance to SSZ was accompanied by

induction of ABCG2 (see below).

(Hydroxy)chloroquine

The antimalarial DMARD (hydroxy)chloroquine

has relatively little activity as a single agent, but is

used in combinations with other DMARDs (6, 104,105). Chloroquine has been identified as a substrate,

as well as an inhibitor of Pgp and MRP1 (106 – 108).

Its inhibitory activity suggests its use as a chemo-

sensitizer, as it elevates the intracellular concentra-

tion and efficacy of therapeutic drugs that are

substrates for these MDR proteins (see below).

Leflunomide

Leflunomide is a DMARD with immunosuppressiveactivity due to inhibition of dihydroorotate dehy-

drogenase (DHODH), a key enzyme in pyrimidine

biosynthesis de novo (109, 110). To date, it is

unknown whether leflunomide, or its active meta-

bolite A771726, is a substrate for MDR proteins, or

whether over-expression of specific MDR proteins

confers resistance to leflunomide. Acquired resis-

tance to leflunomide does, however, seem to beassociated with increased activity and/or structural

alterations of the target enzyme DHODH (111).

Azathioprine

Azathioprine is a prodrug of 6-mercaptopurine (6-

MP) and has an established place in RA treatment

(112). 6-MP, along with 6-thioguanine and other

nucleoside analogues, proved to be a potentialsubstrate for MRP4/ABCC4 and MRP5/ABCC5

(41, 43, 113 – 115). Over-expression of MRP4 or

MRP5 is associated with resistance to this class of

compounds. As for MTX, where polyglutamylation

abolished efflux, metabolism of 6-MP to its diphos-

phate and triphosphate metabolites diminished

substrate affinity for MRP4 and MRP5.

Auranofin/aurothiomalate

Although gold preparations belong to the first

generation of DMARDs, they are still prescribed

in some cases of RA. Glennas and colleagues (116,

117) are the only investigators who have studied

mechanisms of acquired resistance to aurothioma-

late. They observed the onset of resistance, but

an underlying mechanism was not revealed. No

evidence for an increase in methallothionein wasobserved (118). Evidence has been presented that

MRP1 and MRP2 can transport glutathione con-

jugates of heavy metal ions (arsenite, cis-platin) (19)

but whether this also includes gold preparationsremains to be established.

Cyclosporin A

In RA treatment, cyclosporin A (CSA) displayedactivity in combination with MTX (119). CSA is well

recognized for its inhibitory interaction with P-gp

(35, 120) (see also below). Data on mechanisms of

acquired resistance to CSA are limited, but surpris-

ingly do not seem to involve up-regulation of Pgp

expression (121, 122).

Glucocorticoids

Glucocorticoids (GC) display DMARD activity.

Given their hydrophobic nature, GCs like pred-

nisone and dexamethasone were identified as sub-strates for Pgp (18, 123). Over-expression of Pgp

reduces the cellular uptake of dexamethasone, which

can be normalized by blocking of Pgp (124).

Altogether, there is ample evidence that several

DMARDs can be substrates for MDR proteins. To

what extent specific MDR proteins actually con-

tribute to clinical DMARD resistance requires

further investigations.

Physiological role of MDR proteins

MDR proteins have received most attention for their

capacity to extrude therapeutic drugs from cells.Less well understood is the putative physiological

role of MDR proteins in transporting signalling

molecules, peptides or small proteins that are

involved in immunological/inflammatory processes

(Table 2). Pgp is expressed on CD4z T cells and

CD14z DCs, where it may mediate interferon-c(IFN-c) and interleukin-12 (IL-12) release (125),

and DC migration to draining lymph nodes (68).Whether these processes involve the Pgp-mediated

transport of lipid molecules, such as platelet-

activating factor (PAF), is the subject of ongoing

studies (126, 127).

An established substrate for MRP1 and other

members of the MRP family is leucotriene C4

(LTC4) (25, 128). Leucotrienes are biologically

active metabolites derived from arachidonic acidthat can activate inflammatory processes by interac-

tion with specific cysteinyl leucotriene receptors,

which are coupled to G proteins and downstream

signalling pathways (129, 130). LTC4 is a glu-

tathione conjugate of LTA4, which explains its

substrate affinity for MRP1 as this protein prefer-

entially transports glutathion conjugates. As for Pgp,

Robbiani (72) has demonstrated an important rolefor MRP1 and LTC4 in antigen-presenting DC



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migration to lymph nodes. MRP1-mediated trans-

port of LTC4 initiated binding to cysteinyl leuco-

triene receptors, which promoted CCL19-dependent

trafficking of DCs to the lymph nodes. MRP12/2

knock-out mice consistently demonstrated a mark-

edly impaired DC mobilization capacity, which

could be restored by exogenous LTC4. Of note,

studies by Wijnholds (131) had shown that MRP12/2

mice had a decreased inflammatory response (ear

oedema) after arachidonic acid, LTC4 or phorbol

ester treatment.

Steroids are another class of biologically active

compounds that are potential physiological sub-

strates for Pgp, MRPs and BCRP (25, 57, 59, 132,

133). A well-known high-affinity substrate for

these MDR transporters is 17b-oestradiol 17b-D-glucuronide (E217bG). Recently, cyclic adenosine

monophosphate (cAMP) and cyclic guanosine

monophosphate (cGMP) were found to be high-

affinity substrates for MRP4 and MRP5 (93, 114,

134, 135). Since cyclic nucleotides play essential

roles as activators of protein kinases and signalling

pathways, one may speculate that MRP4 and MRP5

may (indirectly) contribute to control these processes

by extrusion of cAMP and cGMP.

Finally, Reid (136) reported that MRP4 is a major

efflux transporter for prostaglandins (PG) such as

PGE1, PGE2 and PGA1. Interestingly, several non-

steroidal antiinflammatory drugs (NSAIDs), notably

indomethacin and indoprofen, proved to be potent

inhibitors of PG efflux via MRP4. This suggests

that, beyond inhibition of PG synthesis, part of the

anti-inflammatory effects of these NSAIDs may be

mediated via inhibition of MRP4.In an early report, Salmon and Dalton (137)

hypothesized that MDR proteins, such as P-gp,

could function as efflux transporters for pro-

inflammatory cytokines, such as TNFa. The anti-

inflammatory effect of a DMARD like CSA could

then be compatible with it being an inhibitor of Pgp.

In line with this hypothesis were studies by Drach

(138) and Raghu (139), demonstrating that inhibi-

tion of Pgp blocked the release of Il-2, IL4 and

IFNc. In contrast, however, Eisenbraum and Miller

(140) showed that T cells from Pgp2/2 mice had

unaltered release of IL2, IL4 and IFNc compared

with T cells from wild-type mice. Thus, a direct role

for Pgp in transport of cytokines with a molecular

weight of 10 – 25 kD is still controversial.

An alternative explanation for these observations

may be that Pgp effluxes compounds that control the

release of cytokines via other routes. A few reports

also suggested a role for MRP1 in the transmem-

brane transport of distinct cytokines, e.g. basic

fibroblast growth factor and IL-4 (141, 142).

Vellenga (143) reported a marked increase in the

release of IL-6 from activated monocytes following

inhibition of MRP1 by MK571. Again, as for Pgp,

conclusive evidence for MRP1-mediated efflux of

cytokines will require additional studies, including

competition studies of putative protein substrateswith established MRP1 substrates, such as LTC4.

Rather than for proteins, there is substantial

evidence that Pgp and MRP1 can efflux small

(cyclic) peptides (up to 10 mer) (144, 145).

ANLL (N-acetyl-leu-leu-norleucinal) is a tripep-

tide with Pgp and MRP1 substrate affinity (144,

145). It is an inhibitor of 26S-proteasome activity,

which plays a key role in the activation of nuclearfactor kB (NFkB), the nuclear transcription factor

that mediates the transcription of TNFa (146, 147).

Hence, cellular Pgp and MRP1 expression should be

considered as factors determining the efficacy of

targeting NFkB signaling by peptide-based 26S-

proteasome inhibitors.

In summary, beyond transport of therapeutic

drugs, some MDR proteins have the capacity totransport small signalling molecules/peptides. A

direct role in transporting higher molecular weight

cytokines is still under debate, although (in)direct

effects on cytokine release seem evident.

Regulation of MDR protein expression

Drug-induced up-regulation of MDR protein expres-

sion is well documented, however, mechanisms in

control of the transcriptional regulation of MDR

genes are still relatively poorly understood. There is

accumulating evidence that endogenous or exogen-

ous stress signals (e.g. oxidative stress, toxic drugs,

inflammation, cytokines and growth factors) play acentral role in the transcriptional regulation of Pgp

and MRP1 (148): for most other MDR proteins

this needs to be established. In this respect, several

downstream signal transduction pathways/proteins

(MAPK, JNK) and transcription factors (NFkB,and notably cAMP-responsive transcription factors

AP1 and SP1) have been implicated in the expression

of Pgp and MRP1 (149 – 151).NFkB binding sites were identified in the promo-

ter region of the Pgp gene (152, 153), and actually

appeared to control the regulation of Pgp gene

expression (154). The status/activity of p53, a key

regulatory gene in tumourogenesis, as well as chronic

inflammation (155), also appeared to be of impor-

tance in the regulation of MRP1 expression: wild

type p53 suppresses (156), while mutated p53enhances (157) MRP1 gene transcription. Finally,

a recent study by Assaraf (158) showed that cellular

folate depletion resulted in down-regulation of

MRP1 expression, tentatively as a physiological

response to prevent loss of intracellular folates.

A number of studies showed that cytokines can

have differential effects on MDR protein expression.

In fact, TNFa (159, 160) and IL-1b and IL-6 (148)down-regulated expression of Pgp, whereas IFN-c

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can up-regulate Pgp expression (161). TNFa was

found to up-regulate expression of MRP1, but not

of LRP (162). Effects of cytokines/chemokines onexpression levels of MDR proteins other than Pgp

and MRP1 are currently unknown.

Cell differentiation and maturation are processes

that are associated with changes in MDR protein

expression. For example, an increase in Pgp and

MRP1 expression was noted during monocyte –DC

differentiation, and during DC maturation (71, 73).

Of note are also observations that MDR expressionmay alter as a function of ageing (163). In particular,

Pgp expression in CD4z and CD8z T cells increased

during ageing (164). Hence, clinical studies with RA

patients for the evaluation of MDR protein expres-

sion should include age-matched controls. Taken

together, these studies demonstrate that up/down-

regulated expression of MDR proteins can be either

drug-induced or a response to stress signals, cyto-kines and other inflammatory effects.

MDR protein induction by DMARDs

Induction of MDR proteins during development of

resistance to anti-cancer drugs is well established.With the exception of MTX, it is unknown whether

acquired resistance to DMARDs can also be asso-

ciated with the induction of specific MDR proteins.

In recent studies from our laboratory (165 – 168),

resistance was provoked in vitro in human T cells

(CEM cell line) to the DMARDs choloroquine

(CHQ) and sulphasalazine (SSZ). These two

DMARDs were selected as, in clinical practice, theypresent a relatively more rapid onset of diminished

efficacy than other DMARDs (10, 12, 13). Following

exposure of CEM to stepwise increasing concentra-

tions of CHQ or SSZ for 6 months, appreciable

levels of resistance to CHQ (3.5-fold) and SSZ (6.5-

fold) were noted. Analysis of MDR protein expres-

sion revealed a marked induction of MRP1 in

CHQ- resistant cells and BRCP in SSZ-resistantcells. CHQ-resistant cells retained sensitivity to most

other DMARDs (MTX, CSA, leflunomide and

SSZ), but were highly cross-resistant (w1000-fold)

to the glucocorticoid dexamethasone. In contrast,

SSZ-resistant T cells displayed markedly increased

(13-fold) sensitivity to dexamethasone compared to

parental cells. This may actually reflect the improved

efficacy of glucocorticoids (prednisone) in combina-tion with MTXzSSZ, compared to MTXzSSZ

alone for the treatment of RA patients (9). SSZ-

resistant cells showed unaltered sensitivity to CHQ

and CsA.

Interestingly, SSZ-resistant cells displayed a low

level of cross-resistance to MTX (v2-fold), which

markedly increased (w300-fold) when MTX sensi-

tivity was tested in the presence of the selectiveconcentrations of SSZ. This loss of MTX efficacy

appeared to be due an unexpected and hitherto

unknown inhibition by SSZ of the reduced folate

carrier, the cell membrane transporter for MTX,which abolished cell entry of MTX to confer resis-

tance (169). Finally, an altered release of cytokines

was observed in SSZ-resistant cells, as indicated by

a 3-fold higher secretion of TNFa compared with

control CEM cells.

Collectively, these in vitro results demonstrate that

the onset of DMARD resistance can be accompa-

nied by induction of specific MDR proteins, altereddrug sensitivity and increased TNFa release.

MDR proteins in RA patients under DMARD therapy

A large number of cancer therapy studies have

included an evaluation of MDR protein expression,notably Pgp, MRP1-5 and BCRP, as part of an

assessment of the efficacy of chemotherapeutic drugs

(36, 170 – 173). Other than in a pilot setting, similar

studies have not been carried out for RA patients to

assess the (long term) efficacy of various DMARDs.

In one of four studies published on the putative

role of DMARD resistance and MDR expression,

Maillefert (174) observed a significant increase in thepercentage of Pgp-positive peripheral blood lympho-

cytes in a group of RA patients (n~68, mean disease

duration 10.7 years) treated with prednisone (n~40,

10.7% Pgpz) compared with RA patients treated

with various DMARDs (n~25) and NSAIDs

(n~34), or controls (n~44) (3.3% Pgpz). Pgp

expression did not correlate with gender, disease

duration, past resistances to DMARDs, RF, or C-reactive protein (CRP). Consistent with prednisone

being a potential substrate for Pgp, these results may

suggest a role for prednisone in Pgp induction.

Yudoh (175) studied percentages of Pgp positive

cells in Th1 and Th2 subpopulations of CD4z T

cells of 14 RA patients and eight RA patients non-

responsive to DMARD therapy (sulphasalazine/

bucillamine). The percentage of Pgpz cells wassignificantly higher in Th1 cells of non-responders

versus responders, both at baseline (9.6% versus

3.3%) and two months after therapy (15.4% versus

3.3%). No differences in Pgp expression (2.2 – 2.6%)

were observed in Th2 cells of responders and non-

responders.

Llorente (176) analysed the percentage of periph-

eral blood lymphocytes for Pgp activity (based ondaunorubicin efflux) in 24 controls, eight therapy-

responsive RA patients and eight therapy non-

responsive RA patients. Therapy non-responsive

patients revealed a markedly higher percentage of

Pgp-active cells (34.4%) compared with therapy

responsive patients (3.9%) or controls (v1.5%).

Finally, in a prospective study, Maillefert (177)

analysed Pgp expression (by indirect immunofluor-escence) on CD4z and CD8z T cells, B cells and



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monocytes in a group of RA patients (n~24, mean

age 56 years, disease duration 5.9 years). Pgp

expression was analysed at baseline and 6 monthsafter therapy with one of the following DMARDs:

MTX, sulphasalazine, leflunomide or gold salts. At

this stage, 17 out of 19 evaluable patients displayed

a clinical response. Most significant differences at

baseline and after 6 months therapy were a decrease

in the percentage of Pgpz monocytes in responders:

16% versus 0%, respectively, suggesting that Pgp

expression on monocytes may be a marker ofresponse to DMARDs.

To date, only one study, by Jorgensen (178), has

reported on the expression of Pgp in synovial tissue

of RA patients. Immunohistochemical staining of

Pgp was observed in synovial membranes of five out

of 16 RA patients treated with various DMARDs.

Surprisingly, however, Pgp mRNA was identified

in only one out of these five patients, from whichthe investigators suggested they have identified an

atypical MDR phenotype, distinct from classical

Pgp, that was previously described in a T-cell line

selected for extremely high concentrations of MTX

(179).

In summary, studies on MDR expression on

clinical specimens of RA patients are currently

limited to the analysis of Pgp expression onperipheral blood lymphocytes. Data on expression

of other MDR proteins and in synovial cells are not

readily available.

MDR reversal agents/inhibitors

In cancer chemotherapy, blocking or modulating of

MDR activity has received considerable attention

as a possible strategy to overcome diminished drug

efficacy due to MDR-mediated drug efflux. Inhibi-

tors/modulators for various MDR proteins are now

available (Table 2), which confer in vitro reversal

of resistance to chemotherapeutic drugs transported

by the MDR proteins. Verapamil and CSA are firstgeneration inhibitors of Pgp activity (37, 180, 181).

PSC833 (120), a structural analogue of CSA devoid

of immunosuppressive effects, is a second generation

Pgp inhibitor currently undergoing clinical trials.

Inhibitors of MRP1 include probenecid, which

also inhibits MRP2-4 (25, 90, 93), and MK571, a

leucotriene D4 analogue (182). Interestingly, several

NSAIDs also appeared to be potent inhibitorsof MRP1 (e.g. sulindac and indomethacin) (183)

and MRP4 (e.g. indoprofen, indomethacin) (136).

Recently identified inhibitors of BCRP include

GF120918 (184, 185) and a fumitremorgine analogue

Ko143 (186). Based on in vitro potency to reverse

drug resistance, several MDR inhibitors are cur-

rently being evaluated in a (pre)clinical setting (29,

187, 188) for their ability to modulate clinical drugresistance.

Evaluating the outcome of the various MDR

reversal trials, van Zuylen (189) concluded that in

the cancer chemotherapeutic setting the clinicalbenefit of MDR modulators is rather modest. This

may be due to several reasons:

. Plasma levels of MDR modulators are insufficient

to achieve optimal reversal

. Pharmacokinetic drug interact with some MDR

modulators, requiring dose reductions

. A multifactorial basis for drug resistance rendersreversal of the resistant phenotype incomplete.

Obviously, these issues should be resolved in order

to reveal a greater potential of the MDR inhibitors/

modulators, but recent studies with third generation

MDR reversal agents look promising in this respect

(190). Another novel clinical application of MDR

inhibitors may be in improving oral drug treatment

by temporary inhibition of drug transporters in thegastrointestinal tract (191).

From a rheumatology perspective, lessons can to

be learned for the exploitation of MDR reversal

agents as possible modulators of DMARD resis-

tance. First, in order to utilize the appropriate MDR

modulator, the MDR protein(s) contributing to

efflux of various DMARDs should be identified.

Second, different endpoints may reflect the efficacyof MDR modulators in cancer treatment (restora-

tion of antiproliferative effects) and RA treat-

ment (restoration of anti-inflammatory effects of

DMARDs). These differential effects should not

necessarily be achieved at identical concentrations

of MDR modulators. Ideally, if modulation of

DMARD resistance is effective at lower concentra-

tions of MDR modulators than for anticancer drugs,this would improve the therapeutic window of the

modulators. Third, it is apparent that MDR proteins

have physiological functions in transporting inflam-

matory mediators, e.g. LTC4 via MRP1. Hence,

blocking of MDR proteins may turn out to be a

double-edged sword, as it may reduce the anti-

inflammatory response capacity — as in MRP1 2/2

knock out mice (131).In conclusion, although MDR modulators are

available, their full (in vitro) potential still needs

to be translated into a markedly enhanced clinical

benefit of cancer chemotherapeutic drugs. Explora-

tion of MDR modulators to improve the efficacy of

DMARDs may warrant further attention.

Perspectives

Drug resistance, inherent or acquired, is generally

accepted as a common cause of treatment failure in

the field of infectious diseases and cancer, but has

received relatively little attention in clinical rheuma-tology in relation to DMARD resistance. Expanding

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knowledge on drug resistance has revealed that there

is considerable overlap in molecular mechanisms of

drugs, regardless of whether these drugs have anti-

infectious, anti-cancer or anti-rheumatic effects.

Although drug resistance can be a multifactorial

event, one common mode of cellular drug resistance

is enhanced efflux by several members of the ABC

transporter family, giving rise to the MDR pheno-

type. There is a growing body of evidence that

various ABC transporters are expressed in cell types

that play a role in inflammatory processes, and

which are targets for DMARD therapy. In this

respect it will be important that future studies

categorize in clinical specimens from RA patients

(peripheral blood cells, synovial cells and tissue) the

expression of those ABC transporters that have an

established role in drug resistance. These studies are

now achievable, as the technology required to

analyse mRNA levels of all 48 ABC transporters

is available (192), just as flow cytometric assays are

available to analyse the functional activity of Pgp,

MRP1 and BCRP (193). However, for an assess-

ment of the various MDR transporters in clinical

specimens containing many different cell types,

immunohistochemistry remains the mainstay of the

analytical platform (40, 53).

Other important issues requiring further attention

are:

. The assessing of whether up-regulation of MDR

proteins in RA patients is: a DMARD-induced

phenomenon, with possible variability dependent

on the DMARD used, and/or a disease-related

phenomenon. In other words, is MDR expression/

functional activity a reflection of disease activity

related to the physiological function of some MDR

proteins in transporting inflammatory mediators.

. Patient variability in response to DMARDs may

be envisioned given the identification of a large

variety of Pgp, MRP1 and BCRP polymorphisms

(194 – 200). The functional implications of all these

polymorphisms need to be established. Further-

more, several distinct mutations in the Pgp, MRP1

and BCRP gene have been reported that may

affect transport of MTX (58, 99, 201, 202).

. Studies on drug resistance will have broader

implications in inflammatory diseases rather than

in RA alone, as expression of Pgp was also identified

in blood cells of other autoimmune diseases, e.g.

systemic lupus erythematosus (SLE) (203).

. Along with clinical studies, the use of in vitro

models with acquired resistance to DMARDs

resistance can provide further insight into the

molecular mechanisms of resistance to various

DMARDs, the involvement of MDR proteins

and/or other mechanisms of resistance, and

the design of strategies to overcome DMARD

resistance.

Answering these questions will undoubtedly con-

tribute to better treatment options with DMARDs

for patients with RA or other chronic inflammatorydiseases.

Acknowledgements

Drs Ruud Oerlemans, Willem Lems, Joost van der Heijden and

George Scheffer are acknowledged for critical reading of the

manuscript and helpful suggestions.

This study was supported by a grant (NRF 03I-40) from the

Dutch League against Rheumatism (Nationaal Reumafonds).

Abbreviations

MDR1; Multi Drug Resistance, Pgp; P-glycoprotein, MRP;

Multidrug Resistance associated Protein, MTX; methotrexate,

CsA; cyclosporin A, BCRP; Breast Cancer Resistance Proteins,

ABC; ATP Binding Cassette, GC; Glucocorticoid, cAMP; Cyclic

Adenosine Mono-Phosphate, NFkB; Nuclear Factor kB, DHFR;

Dihydrofolate reductase, FPGS; Folylpolyglutamate synthetase,

RFC; Reduced Folate Carrier, RA; Rheumatoid Arthritis.

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multidrug resistance proteins in rheumatoid arthritis, role in disease‐modifying antirheumatic...

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