Review

Correspondence *Department of Rheumatology, Room 4A42, VU University Medical Center, De Boelelaan 1117, 1081 HV Amsterdam, The Netherlands

Email g.jansen@vumc.nl

Introduction

Despite the current success of biological therapies (inhibitors of tumor necrosis factor [TNF], interleukin-1-receptor antagonists and antibodies to CD20),^{1, 2, 3} disease-modifying antirheumatic drugs (DMARDs) have an established place in the treatment of rheumatoid arthritis (RA) because of their convenience, safety, and low related costs. One common phenomenon associated with chronic DMARD treatment, however, is a gradual reduction in drug efficacy, which could point to the onset of drug resistance. Increases in drug doses might partly regain therapeutic efficacy, but this approach also increases the 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 treatment of cancer and infectious diseases,⁴ but has just started to be appreciated in RA treatment.^{5, 6} Whereas end-point evaluations for anticancer drug resistance usually refer to a loss of the antiproliferative effects on target cells, DMARD resistance might be defined as a loss of ability to block the release of proinflammatory cytokines, in addition to a loss of antiproliferative effects.

This Review describes the current knowledge of the molecular mechanisms of cellular resistance to DMARDs (methotrexate, sulfasalazine, chloroquine, hydroxychloroquine, azathioprine, auranofin, aurothiomalate, ciclosporin, gold, and leflunomide). The suggested mechanisms are mainly based on the results of in vitro laboratory studies and theories about the emergence of DMARD resistance in RA. We also discuss potential strategies to deal with drug resistance.

General features of drug resistance

Human beings have defense mechanisms against daily exposure to hundreds or even thousands of xenobiotic substances present in our environment and food. For this reason, most chemically designed therapeutic drugs, including DMARDs, are recognized by target cells as foreign substances. Sooner or later, immunologic or cellular defense mechanisms will become operative and inactivate the drugs. In a clinical setting, such a process manifests as reduced drug efficacy, for which, at a molecular level, the onset of drug resistance might be an underlying cause.^{7, 8, 9, 10, 11, 12} Cellular drug resistance can be categorized under two main headings: inherent or primary resistance (referring to cells that are already resistant before receiving therapy) and acquired drug resistance (which indicates that cells were initially sensitive to drugs but developed resistance during the course of treatment). In RA treatment, a study by Morgan et al.¹³ showed that 5–10% of RA patients had inherent or primary resistance to DMARDs, which might account for the 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 failure are, however, considered to be the result of various underlying mechanisms, some of which could be specific to the type of DMARD used.

Experimental studies of drug resistance mechanisms

The mechanisms of resistance to DMARDs might be similar to those of resistance to anticancer and antimicrobial drugs. Box 1 outlines several mechanisms of drug resistance for disease-specific target cells; in RA, the causes of resistance could involve cells of the immune system (T lymphocytes and macrophages) implicated in the disease pathophysiology. Possible mechanisms of drug resistance include impaired drug 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 drug delivery is its metabolism to the less active 7-hydroxymethotrexate. For sulfasalazine, the activity of intestinal drug-efflux transporters is a determining factor in variability of plasma drug concentrations.¹⁵

Increased drug efflux has been recognized as an important mechanism of drug resistance. Cell-membrane proteins responsible for drug efflux belong to the family of ATP-binding cassette (ABC) transporters.^{4, 16} A wide range of structurally and functionally different drugs can be pumped out of the cell by these proteins, leading to multidrug resistance. Several DMARDs seem to be among the substrates of ABC transporters.

A summary of reported mechanisms of resistance to various DMARDs is shown in Box 1. On the basis of laboratory and preclinical data from all the DMARDs, resistance mechanisms to methotrexate have been best characterized,^{17, 18} and are shown in Figure 1. A first 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 in almost all immune effector cells,¹⁹ or via folate receptor-, which has a restricted expression on activated macrophages, such as in the synovium.²⁰ Reduced expression of, or alterations in the transport kinetics of, these proteins will diminish cellular uptake of methotrexate, resulting in lower intracellular concentrations of the drug, which will thus not reach thresholds for therapeutic effects. Within the cell, methotrexate is converted to polyanionic polyglutamated forms via the action of the enzyme folylpolyglutamate synthetase, which improves drug retention because the polyglutamated forms are poorer substrates for efflux transporters such as ABCC1–5 or ABCG2.^{21, 22} Thus, cells with a low capacity to retain polyglutamated forms of methotrexate (because of decreased folylpolyglutamate synthetase activity or increased activity of the breakdown enzyme folylpolyglutamate hydrolase) will be more prone to reduced activity because of drug-efflux mechanisms mediated by transporter mechanisms. Polyglutamated forms of methotrexate inhibit several key enzymes in folate metabolism (dihydrofolate reductase and thymidylate synthase) and purine metabolism (aminoimidazole carboxamide ribonucleotide transformylase). Elevated levels of these enzymes or kinetically altered enzymes with lower binding affinities will necessitate higher doses of methotrexate to achieve optimum inhibition.

Figure 1 Mechanisms of pharmacokinetic and cellular resistance to methotrexate.

Metabolism of methotrexate in the liver to partly inactive 7-hydroxymethotrexate reduces plasma concentrations of methotrexate and creates competition for cellular uptake via RFC, which also internalizes reduced folates and folic acid, although with low affinity for the latter. Folate receptors mediate low affinity endocytosis of methotrexate. Quantitative or qualitative alterations in RFCs or folate-receptor expression confer transport-related resistance to methotrexate, as do high levels of exogenous folates competing for cell entry. Intracellularly, methotrexate is metabolized to polyglutamated forms by FPGS, and these are broken down by FPGH. The monoglutamated forms and, to some extent, polyglutamated forms are expelled via transporters ABCC1–ABCC5 and ABCG2. ABCB1 does not transport anionic drugs such as methotrexate. Polyglutamated forms of methotrexate can inhibit several 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 release signaling pathways (such as those mediated by NFB and IKK) all create routes via which methotrexate could suppress RA.^{51, 58, 59} Abbreviations: 5-CH₃-THF, 5-methyltetrahydrofolate; 5,10-CH₂-THF, 5,10-methylenetetrahydrofolate; 7-OH-MTX, 7-hydroxymethotrexate; ABC, ATP-binding cassette, subfamily specific; AICARTFase, 5-aminoimidazole-4-carboxamide ribonucleotide formyltransferase; APRTFase, amidophosphoribosyltransferase; DHFR, dihydrofolate reductase; FA, folic acid; FH₂, dihydrofolate; FH₄, tetrahydrofolate; FPGH, folylpolyglutamate hydrolase; FPGS, folylpolyglutamate synthetase; FR, folate receptor; IKK, inhibitor of IB kinase; LV, leucovorin; MAPK, mitogen-activated protein kinase; MTHFR, methylenetetrahydrofolate reductase; MTX, methotrexate; MTX-PG; polyglutamated forms of methotrexate; NFB, nuclear factor B; RFC, reduced folate carrier; TS, thymidylate synthase; TNF, tumor necrosis factor.

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For the DMARD sulfasalazine, increased drug efflux via ABCG2 has been reported as a potential mechanism of cellular resistance.^{23, 24} Molecular mechanisms of resistance for the antimalarial DMARDs chloroquine and hydroxychloroquine include drug sequestration in lysosomal compartments or drug efflux via ABC transporters ABCB1 (also called MDR1 or P-glycoprotein) and ABCC1.²⁵ With respect to the DMARDS auranofin and aurothiomalate, studies by Glennas et al.²⁶ revealed that upregulated expression of the metal-binding protein metallothionein was the dominant mechanism of acquired resistance to this DMARD. The DMARD azathioprine is a prodrug of the nucleoside analog 6-mercaptopurine. At least two mechanisms could be implicated in diminished azathioprine activity: one relates to variations in metabolism (and toxicity) associated with specific gene polymorphisms of the enzyme thiopurine methyltransferase,²⁷ and in the other, two drug-efflux transporters, ABCC4 and ABCC5, mediate cellular efflux of 6-mercaptopurine to confer resistance.²⁸ Cellular resistance to ciclosporin has been reported in two laboratory studies but the molecular basis for resistance was not disclosed.⁵ Preliminary reports investigating 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 in order 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 isoform of the glucocorticoid receptor and an increased ratio of the isoform to the isoform (the isoform lacks the high-affinity glucocorticoid-binding capacity and acts as a dominant-negative regulator of the 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 the 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 and phenotypic changes that collaterally influence the efficacy of other DMARDs, the release of proinflammatory cytokines, or both. Findings from in vitro studies showed that acquired resistance to chloroquine in T cells is accompanied by a markedly reduced release of TNF and interleukin 8, along with a marked loss of sensitivity to glucocorticoids.²⁵ Conversely, acquired resistance to sulfasalazine in the same T cells is accompanied by a markedly increased sensitivity to glucocorticoids.²³ Hence, upon chronic exposure, DMARDs might exert both beneficial and adverse pleiotropic effects. Such effects might have been unrecognized when the anti-inflammatory effects of specific DMARDs were tested over a short time frame in a laboratory setting.

DMARD resistance or inefficacy in RA

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.^{7, 8, 10, 11, 12, 32, 33} For example, in a 10-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% discontinued treatment as a result of adverse effects.^{7, 33} The percentage of RA patients who had to discontinue DMARD monotherapy for inefficacy was highest for auranofin (37%), sulfasalazine (36%), and the antimalarials chloroquine and hydroxychloroquine (25%), and lowest for methotrexate (9%).⁷ Unfortunately, except for clinical end points, the molecular basis for DMARD inefficacy was not further unraveled in these studies. This lack of findings was probably the result of an insufficient number of clinical samples and small sample sizes of peripheral blood or synovial tissue, which made biochemical and molecular analyses of potential DMARD-resistance-related parameters impossible.

Strategies to overcome DMARD resistance

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, alternating drugs, either in a step-up approach (therapy is started as monotherapy and drug(s) are added in cases of insufficient response) or a step-down approach (therapy is started with multiple drugs but one is stopped at a time), is thought to slow the onset of drug resistance seen with monotherapy. In cases where combination DMARD therapy has proved unsuccessful, addition of a third DMARD has proved to be highly efficacious in certain combinations (e.g. methotrexate plus sulfasalazine plus hydroxychloroquine⁵⁴ or methotrexate plus sulfasalazine plus prednisolone⁵⁵). Whether the superiority of these triple-therapy schedules is explained by minimizing the contribution of resistance-related parameters is not fully clear.²⁵

When a specific mechanism of resistance has been identified for a type of drug, including DMARDs, strategies to overcome resistance might include engagement with, bypassing, or taking advantage of, the mode of resistance. Engagement, for example in the case of drug-efflux transporters, could involve the use of inhibitors specific to these proteins. In cancer chemotherapy, however, this approach has, for various reasons, had limited success.⁴ The lower drug doses given to RA patients might make an engagement strategy more efficacious than in cancer treatment, but the likelihood of this outcome has not yet been established.

Blocking of drug-efflux transporters could raise plasma and intracellular drug concentrations. Zaher et al.¹⁵ showed that ABCG2-knockout mice had markedly altered pharmacokinetics for sulfasalazine; plasma levels were more than 100-fold higher than in control mice. These same raised plasma levels of sulfasalazine could be achieved in normal mice by administering gefitinib, an ABCG2 inhibitor with sulfasalazine. This example suggests that when DMARDs are substrates for drug-efflux transporters, therapy with transporter blockers could modulate plasma levels, reducing the risk of adverse effects.

Accumulated knowledge about resistance mechanisms for methotrexate has led to the rational design of second-generation folate antagonists. Use of these drugs might overcome the issue of methotrexate resistance, because they are more efficiently taken up by RFC and are better substrates for folylpolyglutamate synthetase, which prevents efflux by drug exporters.⁵⁶ Preliminary results in ex vivo experiments indicate that some of these second-generation folate antagonists are potent inhibitors of TNF production in activated T cells of RA patients. Some second-generation folate antagonists might also target folate receptors (see Figure 1). By following this route of entry, they could bypass efflux routes encountered after cell entry via the RFC route. The current status of this research is not, however, advanced enough to predict whether any of the second-generation therapies will be able to replace methotrexate.

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, 57} 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, when 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 might 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 investigation of ways 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.

Acknowledgments

Work described in this Review was supported by grants from the Dutch Arthritis Association and The Netherlands Organization for Scientific Research. JW van der Heijden is a recipient of the 2006 Rheumatology Grant from the Dutch Association for Rheumatology. We apologize to authors whose work could not be cited for space reasons.

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