Methotrexate polyglutamate concentrations are not associated with disease control in rheumatoid arthritis patients receiving long-term methotrexate therapy.код для вставкиСкачать
ARTHRITIS & RHEUMATISM Vol. 62, No. 2, February 2010, pp 359–368 DOI 10.1002/art.27201 © 2010, American College of Rheumatology Methotrexate Polyglutamate Concentrations Are Not Associated With Disease Control in Rheumatoid Arthritis Patients Receiving Long-Term Methotrexate Therapy Lisa K. Stamp,1 John L. O’Donnell,2 Peter T. Chapman,2 Mei Zhang,2 Jill James,2 Christopher Frampton,3 and Murray L. Barclay1 Objective. There are limited data suggesting that methotrexate polyglutamate (MTXGlu) concentrations can guide MTX dosing in patients with rheumatoid arthritis (RA). The aim of this study was to define a therapeutic range of red blood cell (RBC) MTXGlun concentrations (where n refers to the number of glutamate groups), including threshold values for efficacy and adverse effects in patients receiving long-term oral MTX treatment. Methods. A cross-sectional study of 192 patients receiving oral MTX was undertaken. Disease activity was assessed by the swollen and tender joint counts, the C-reactive protein level, and the Disease Activity Score in 28 joints (DAS28). High disease activity was defined as a DAS28 of >3.2. A standardized questionnaire regarding common MTX adverse effects was completed. Results. The MTX dosage was significantly higher in patients in whom the swollen joint count and DAS28 were higher. The MTXGlu4, MTXGlu5, MTXGlu3–5, and MTXGlu1–5 concentrations were significantly higher in patients with high disease activity. After correction for age, the estimated glomerular filtration rate, and the MTX dosage, the association remained significant for MTXGlu5. RBC folate concentrations were significantly higher in the group with high disease activity. There was no association between any MTXGlun concentration and adverse effects. Conclusion. In contrast to other studies, the results of the present study did not show a relationship between the MTXGlun concentration and reduced disease activity in patients with RA who were receiving long-term MTX therapy. However, disease activity was influenced by the RBC folate level, which may be a more important factor than MTXGlun concentrations for disease control. In accordance with the findings of previous studies, we were unable to show a relationship between MTXGlun concentrations and adverse effects. Prospective studies will be important to determine whether there is a role for measuring MTXGlun concentrations in patients receiving long-term treatment with MTX. Methotrexate (MTX) is the gold standard treatment against which newer disease-modifying antirheumatic drugs (DMARDs) are compared, because of its established track record in rheumatoid arthritis (RA). The dosage of MTX required by individual patients varies greatly and is unpredictable. The decision by a clinician to increase the dosage is dependent on the assessment of disease activity, the accepted upper limit of drug dosing, and adverse effects. In patients with RA, both a trend toward a dose-response relationship (1,2) and no association between dose and response have been reported for MTX (3). Serum MTX concentrations have not correlated with disease activity (4). Recent studies suggest a negative correlation between long-chain red blood cell (RBC) MTX polyglutamate (MTXGlun) concentrations ClinicalTrials.gov identifier: ACTRN012606000275561. Supported by Health Research Council of New Zealand and Arthritis New Zealand. 1 Lisa K. Stamp, FRACP, PhD, Murray L. Barclay, FRACP, MD: University of Otago, and Christchurch Hospital, Christchurch, New Zealand; 2John L. O’Donnell, FRACP, FRCPA, Peter T. Chapman, FRACP, MD, Mei Zhang, PhD, Jill James, BN: Christchurch Hospital, Christchurch, New Zealand; 3Christopher Frampton, PhD: University of Otago, Christchurch, New Zealand. Address correspondence and reprint requests to Lisa K. Stamp, FRACP, PhD, Department of Medicine, University of Otago, Christchurch, PO Box 4345, Christchurch 8140, New Zealand. E-mail: firstname.lastname@example.org. Submitted for publication March 29, 2009; accepted in revised form October 9, 2009. 359 360 STAMP ET AL (where n refers to the number of glutamate groups) and disease activity (3,5,6). Studies have also examined whether polymorphisms of genes encoding the enzymes involved in MTX transport and/or the folate pathway may help predict the response and/or adverse effects associated with MTX. However, to date, there is no clear consensus regarding the most important polymorphisms (for review, see ref. 7). Despite its widespread use, the exact mechanism of action of MTX remains unclear. Following administration and absorption, serum MTX concentrations fall rapidly (8), and MTX is transported into a variety of cells including RBCs, white blood cells, hepatocytes, and synoviocytes via the reduced folate carrier (RFC). Intracellularly, glutamate moieties are added by folylpolyglutamate synthetase (FPGS), and MTX is retained as MTXGlun. Terminal MTX glutamates are removed by ␥-glutamyl hydrolase, returning MTX to its monoglutamate form, which is rapidly transported out of the cell by multidrug-resistant proteins. The intracellular concentrations of MTXGlun are therefore related to the balance of activity between these 2 enzymes. Ingested folates are also transported into cells by the RFC and folate receptor and compete with MTX as a substrate for polyglutamation by FPGS. Thus, high concentrations of intracellular folates result in a decrease in MTX polyglutamation (9,10). MTX has several potential adverse effects. The more common, but less serious, adverse effects that often limit a patient’s willingness to continue MTX treatment include nausea and lethargy. The more serious adverse effects, which can limit the dosage or require MTX discontinuation, include hepatotoxicity and myelotoxicity. Because of the inability to predict the occurrence of these adverse effects, patients are required to have regular blood tests. Two small studies in patients with RA demonstrated no clear association between MTXGlun concentrations and adverse effects (5,11). The aim of this study was to determine whether a therapeutic range of MTXGlun concentrations could be defined in order to guide adjustment of oral MTX dosing, including threshold values for efficacy and adverse effects. PATIENTS AND METHODS Ethics approval was obtained from the Upper South B Regional Ethics Committee, New Zealand. Written informed consent was obtained from each patient. Patients and study protocol. This cross-sectional study was undertaken in a single center in Christchurch, New Zealand. Patients ⱖ18 years age with RA, as defined by the American College of Rheumatology (formerly, the American Rheumatism Association) (12), were recruited. Patients were required to have been receiving oral MTX for ⱖ3 months, and the dosage must have been stable for ⱖ1 month prior to study entry. A preference was given to patients receiving MTX monotherapy. Concomitant therapy with nonsteroidal antiinflammatory drugs (NSAIDs) and prednisone was allowed. A change in the MTX dosage, introduction of another DMARD, a change in the oral prednisone dosage, and receipt of an intraarticular steroid injection within 1 month prior to enrollment were not allowed. Clinical assessment. Patient demographic and clinical details were collected on standardized data collection forms. Disease activity was assessed using the swollen joint count (SJC), the tender joint count (TJC), the modified Health Assessment Questionnaire (M-HAQ) (13), the physician’s global assessment of disease activity, and the patient’s assessment of pain and global assessment of disease activity. To avoid interobserver variability, the SJC and TJC were determined by a single trained observer. The physician’s assessment of response to MTX was measured using a 100-point visual analog scale (VAS), where 0 ⫽ excellent response and 100 ⫽ poor response. The Disease Activity Score in 28 joints (DAS28) (14) was calculated, and low disease activity was defined as a DAS28 of ⱕ3.2 (15). The Clinical Disease Activity Index (CDAI) (16) and the Simplified Disease Activity Index (SDAI) (17) were also calculated. A standardized questionnaire related to common MTX adverse effects in the month preceding study entry was developed. Adverse effects were grouped into 3 categories, as follows: 1) gastrointestinal (GI; nausea, vomiting, diarrhea, mouth ulcers, and decreased appetite), 2) central nervous system (CNS; fatigue, loss of concentration, headache, dizziness, blurred vision, sleep disturbance, and weepiness), and 3) other (hair loss, cough, fever, and shortness of breath). Laboratory measurements. Standardized laboratory assessments included a complete blood cell count, liver function tests, and determination of the creatine level, the erythrocyte sedimentation rate (ESR), the C-reactive protein (CRP) level, and the RBC folate level. Estimated creatinine clearance (the estimated glomerular filtration rate [eGFR]) was calculated using the Modification of Diet in Renal Disease study equation (18). MTXGlu terminology and measurement. MTX, the parent drug, contains 1 glutamate moiety and is referred to as MTXGlu1. MTXGlu1 and the products of intracellular glutamation (MTXGlu2, MTXGlu3, MTXGlu4, and MTXGlu5) are collectively referred to as MTX polyglutamates (MTXGlun). The terms MTXGlu3–5 and MTXGlu1–5 refer to the sum of each measured RBC MTXGlun concentration. Trough MTXGlun concentrations were measured by high-performance liquid chromatography, as previously described (19). Results were normalized to an RBC count of 8 ⫻ 1012 cells, so that results were comparable and not confounded by changes in RBC counts between individuals. All samples were analyzed in duplicate, and the mean concentration of each RBC MTXGlun from each sample was used. Statistical analysis. The relationship between the MTX dosage and each disease activity measure was assessed MTX POLYGLUTAMATES IN RA using Spearman’s correlation coefficient. The relationships between MTXGlun concentrations and disease activity measures were first quantified using correlation coefficients. Subsequently, the relationships were further explored using covariate analyses that included variables known to affect disease activity (e.g., age, sex, duration of RA, and prednisone use) as covariates. Differences between the groups with low disease activity and high disease activity in terms of MTXGlun concentrations, demographic and clinical features, and treatment measures were tested using independent t-tests, nonparametric Mann-Whitney U tests, chi-square tests, and Fisher’s exact tests, as appropriate. Further covariate analyses exploring the independent association of MTXGlun concentrations with disease activity were undertaken using age, eGFR, MTX dosage, and RBC folate level as covariates. The relationships between the presence of adverse effects and MTX dosage and MTXGlun concentrations were tested using independent t-tests. Covariate analyses including other factors that might contribute to adverse effects (use of other DMARDS or NSAIDs, duration of MTX treatment, and RBC folate levels) were undertaken to establish the independent association between adverse effects and MTX dosage and MTXGlun concentrations. The association between MTXGlun concentrations and other laboratory measurements was tested using Pearson’s correlation coefficient. Two-tailed P values less than 0.05 were considered significant. RESULTS Demographics. Two hundred patients were recruited between October 2005 and February 2008. An ESR value was not obtained in 8 patients, and these patients were excluded, leaving 192 in the final analysis. Of the 192 patients, 72.9% were women, and the mean age was 60.5 years (range 18–84 years). The mean duration of RA was 10.5 years (range 0.25–53 years). Among the 192 patients, 25.5% had rheumatoid nodules, 63% had radiographic erosions, 80.7% were rheumatoid factor positive, and 76.4% were anti–cyclic citrullinated peptide (anti-CCP) antibody positive. The median dosage of MTX was 15 mg/week (range 5–25), and all but 1 patient received folic acid at a dosage of 5 mg/week, given 3–4 days after the dose of MTX. Patients had been receiving MTX for a median of 3 years (range 0.25–19 years) prior to study entry and had been receiving MTX at the study-entry dosage for a median of 12 months (range 1–240 months). Eighty-six of the 192 patients (44.8%) were receiving NSAIDs, and 20 patients (10.4%) were receiving another DMARD (4 sulfasalazine [SSZ], 9 hydroxychloroquine [HCQ], 5 both SSZ and HCQ, 1 leflunomide [LEF], and 1 both LEF and HCQ). Fifty-nine patients (30.7%) were receiving oral prednisone at a mean dosage of 5.2 mg/day (range 1–20). 361 Relationship between MTX dosage, MTXGlun concentrations, and disease activity. There was a significant association between the MTX dosage and RBC MTXGlu3, MTXGlu4, MTXGlu5, MTXGlu1–5, and MTXGlu3–5 concentrations (P ⬍ 0.0001 for all). However, there was no association between the MTX dosage and the MTXGlu1 or MTXGlu2 concentration (20). The MTX dosage was higher in those patients with a higher SJC (r ⫽ 0.19, P ⫽ 0.006), physician’s global assessment score (r ⫽ 0.3, P ⬍ 0.0001), DAS28 (r ⫽ 0.15, P ⫽ 0.03), physician-rated response to MTX (r ⫽ 0.27, P ⫽ 0.0001), patient’s global assessment score (r ⫽ 0.19, P ⫽ 0.007), CDAI (r ⫽ 0.25, P ⫽ 0.0005), and SDAI (r ⫽ 0.25, P ⫽ 0.0005). There was no association between the MTX dosage and the TJC, M-HAQ score, CRP level, ESR, pain on a VAS, or fatigue on a VAS. Thus, despite increasing dosages of MTX, disease remained active in some patients. RBC MTXGlun profile. MTXGlu1, MTXGlu2, and MTXGlu3 were detectable in all patients, while MTXGlu4 was detectable in 178 (92.7%) of 192 patients, and MTXGlu5 was detectable in 148 (77.1%) of 192 patients. MTXGlu3 was the predominant polyglutamate, accounting for a mean of 35.8% of the total MTXGlun. MTXGlu1 accounted for 21.7%, MTXGlu2 accounted for 20.5%, MTXGlu4 accounted for 14.5%, and MTXGlu5 accounted for 7.5%. Association between the RBC MTXGlun concentration and disease activity measures. Univariate analysis revealed a significant association between MTXGlu4, MTXGlu5, MTXGlu1–5, and MTXGlu3–5 concentrations and the SJC, the physician’s global assessment, the physician’s assessment of response to MTX, the DAS28, the CDAI, and the SDAI, such that higher disease activity was associated with higher MTXGlun concentrations (Table 1). There was also a positive correlation between the MTXGlu3 concentration and the physician’s global assessment score, the DAS28, and the SDAI (Table 1). There was a significant association between MTXGlu1–5 concentrations and the M-HAQ score. There was no association between any other MTXGlun concentration and the M-HAQ, the CRP level, or the patient’s assessment of pain and global assessment of disease activity on a VAS (Table 1). Several variables are known to affect disease activity and outcome measures in RA, including age, sex, duration of RA, smoking, anti-CCP antibody status, concomitant treatment with corticosteroids, NSAIDs, or other DMARDs, and folate status. After adjustment for these variables, patients with a higher TJC, a higher SJC, and a higher DAS28 still had significantly higher 362 STAMP ET AL Table 1. Univariate analysis of RBC MTXGlun concentrations and disease activity variables* RBC MTXGlun SJC MTXGlu1 MTXGlu2 MTXGlu3 MTXGlu4 MTXGlu5 MTXGlu1–5 MTXGlu3–5 ⫺0.12 ⫺0.004 0.13 0.22† 0.28† 0.15‡ 0.21† TJC Physician’s global assessment M-HAQ CRP ⫺0.06 ⫺0.02 0.07 0.09 0.18‡ 0.11 0.13 ⫺0.02 0.10 0.15‡ 0.16‡ 0.13 0.15‡ 0.18‡ 0.09 0.14 0.13 0.08 0.12 0.18‡ 0.13 ⫺0.03 0.13 0.08 0.05 ⫺0.03 0.06 0.05 DAS28 Patient’s global assessment Pain on 100-point VAS Fatigue CDAI SDAI 0.01 0.09 0.15‡ 0.17‡ 0.21† 0.19† 0.19† 0.03 0.09 0.1 0.09 0.03 0.12 0.11 ⫺0.03 0.02 0.12 0.11 0.11 0.09 0.14 0.12 0.19† 0.11 0.04 ⫺0.04 0.14‡ 0.08 ⫺0.05 0.05 0.14 0.18‡ 0.22† 0.17‡ 0.20† ⫺0.06 0.05 0.16‡ 0.19† 0.22† 0.18‡ 0.21† * Values are Spearman’s correlation coefficients. The visual analog scale (VAS) was based on a 0–100 scale. RBC ⫽ red blood cell; MTXGlu ⫽ methotrexate polyglutamate; M-HAQ ⫽ modified Health Assessment Questionnaire; CRP ⫽ C-reactive protein; DAS28 ⫽ Disease Activity Score in 28 joints; VAS ⫽ visual analog scale; CDAI ⫽ Clinical Disease Activity Index; SDAI ⫽ Simplified Disease Activity Index. † P ⬍ 0.01. ‡ P ⬍ 0.05. MTXGlu5 concentrations (P ⬍ 0.05 for all). Similarly, patients with greater fatigue and higher M-HAQ scores had significantly higher MTXGlu2 concentrations (P ⬍ 0.05). Taken together, these data suggest that physicians may increase the MTX dosage in the setting of ongoing disease activity in an effort to improve the drug effect, as would be expected. Patients receiving a higher dosage of MTX and having higher disease activity may therefore be nonresponders to MTX. RBC MTXGlun concentrations in the group with high disease activity and the group with low disease activity. One hundred fourteen patients (59%) were defined as having low disease activity (DAS28 ⱕ3.2), Table 2. and 78 (41%) were categorized as having high disease activity. Other than longer disease duration in the group with high disease activity, there were no significant differences in demographic and treatment variables between these 2 groups (Table 2). The MTXGlu4, MTXGlu5, MTXGlu3–5, and MTXGlu1–5 concentrations were higher in the group with high disease activity compared with the group with low disease activity (Figure 1). In this cohort, we previously showed that age, the eGFR, and the MTX dosage account for most variation in MTXGlun concentrations (20). After correction for these variables, there was no significant difference between the group with high dis- Characteristics of the patients with RA according to the level of disease activity* Characteristic Low disease activity (n ⫽ 114) High disease activity (n ⫽ 78) P Age, mean (range) years No. of men/no. of women eGFR, mean ⫾ SEM ml/minute/1.72 m2 Rheumatoid factor positive Anti-CCP antibody positive Nodules Erosions Duration of RA, mean (range) years Duration of RA ⬎20 years Current smoker NSAIDs Any other DMARD Prednisone Dosage, mean (range) mg/day MTX dosage, median (IQR) mg/week No. of months receiving dosage, median (IQR) 59.61 (18–82) 32/82 76.9 ⫾ 1.4 93 (81.6) 90 (79.6)‡ 26 (22.8) 68 (59.6) 9.15 (0.25–47) 12 (10.53) 23 (20) 55 (48.3) 11 (9.7) 29 (25.4) 5.5 (1–12.5) 15 (12.5–20) 11 (5–24.8) 61.92 (20–84) 20/58 75.7 ⫾ 1.9 62 (80.5)† 56 (72.7)† 23 (29.5) 53 (67.9) 12.5 (0.25–53) 18 (23.1) 12 (15.4) 31 (39.7) 9 (11.5) 30 (38.46) 6.2 (2–20) 17.5 (12.5–20) 12 (4.25–25.5) 0.19 0.71 0.6 0.85 0.26 0.29 0.24 0.03 0.02 0.39 0.24 0.67 0.05 0.43 0.14 0.98 * Except where indicated otherwise, values are the number (%). Low disease activity was defined as a 28-joint Disease Activity Score (DAS28) of ⱕ3.2; high disease activity was defined as a DAS28 of ⬎3.2. RA ⫽ rheumatoid arthritis; eGFR ⫽ estimated glomerular filtration rate; anti-CCP ⫽ anti–cyclic citrullinated peptide; NSAIDs ⫽ nonsteroidal antiinflammatory drugs; DMARD ⫽ disease-modifying antirheumatic drug; MTX ⫽ methotrexate; IQR ⫽ interquartile range. † Only 77 of the patients were assessed. ‡ Only 113 of the patients were assessed. MTX POLYGLUTAMATES IN RA 363 Figure 1. Methotrexate polyglutamate (MTXGlu) concentrations in patients with low disease activity (Disease Activity Score in 28 joints [DAS28] ⱕ3.2) and patients with high disease activity (DAS28 ⬎3.2). Each dot represents an individual patient. Bars show the mean. RBC ⫽ red blood cell. ease activity and the group with low disease activity in the concentration of MTXGlu1 (P ⫽ 0.76), MTXGlu2 (P ⫽ 0.47), MTXGlu3 (P ⫽ 0.38), MTXGlu4 (P ⫽ 0.08), MTXGlu1–5 (P ⫽ 0.11), or MTXGlu3–5 (P ⫽ 0.08). However, MTXGlu5 concentrations remained significantly higher in the group with high disease activity (P ⫽ 0.02). MTXGlu5 was undetectable in 25% of the group with low disease activity compared with 19% of the group with high disease activity (P ⫽ 0.31). When patients were grouped according to broad ranges of each MTXGlun concentration, similar percentages of patients were classified as having low disease activity, irrespective of the MTXGlun concentration (Figure 2). It has been suggested that patients in whom the MTXGlu3–5 concentration is ⬎60 nmoles/liter are more likely to have a good response to MTX (3). Among our patients with a DAS28 of ⱕ3.2, 43% had an MTXGlu3–5 concentration of ⱕ60 nmoles/8 ⫻ 1012 RBCs, and 57% had an MTXGlu3–5 concentration of ⬎60 nmoles/8 ⫻ Figure 2. Percentage of patients with low disease activity (Disease Activity Score in 28 joints [DAS28] ⱕ3.2) according to ranges of each methotrexate polyglutamate (MTXGlu) concentration. RBC ⫽ red blood cell. 364 STAMP ET AL Table 3. Classification of the patients according to the DAS28 and RBC MTXGlu3–5 concentrations* DAS28 ⱕ3.2 (n ⫽ 114) MTXGlu3–5 concentration Low High DAS28 ⬎3.2 (n ⫽ 78) MTXGlu3–5 concentration Low High No. (%) of patients MTXGlu3–5 concentration, mean (range) 49 (43) 65 (57) 37.58 (3.8–59.2) 100.11 (62.1–247.7) 24 (31) 54 (69) 41.0 (10.4–59.9) 113.2 (60.1–283.9) Interpretation Responders Possibly overtreated Possibly undertreated Resistant * Low and high concentrations of methotrexate polyglutamate (MTXGlu) were defined as ⬍60 nmoles/ 8 ⫻ 1012 red blood cells (RBCs) and ⬎60 nmoles/8 ⫻ 1012 RBCs, respectively. DAS28 ⫽ Disease Activity Score in 28 joints. 1012 RBCs. Among patients with a DAS28 of ⬎3.2, 31% had an MTXGlu3–5 concentration of ⱕ60 nmoles/8 ⫻ 1012 RBCs, and 69% had an MTXGlu3–5 concentration of ⬎60 nmoles/8 ⫻ 1012 RBCs (Table 3). RBC folate concentrations and disease activity. The mean ⫾ SEM RBC folate concentration was higher in the group with high disease activity compared with that in the group with low disease activity (786.9 ⫾ 31.2 nmoles/liter versus 664.2 ⫾ 27.4 nmoles/liter; P ⫽ 0.002). Current smokers had lower mean ⫾ SEM RBC folate concentrations compared with current nonsmokers (611.9 ⫾ 44.7 nmoles/liter versus 743.6 ⫾ 24.5 nmoles/ liter; P ⫽ 0.008). After correction for RBC folate level in addition to age, eGFR, and MTX dosage, the group with high disease activity still had higher MTXGlu5 concentrations (P ⫽ 0.046). However, the remaining MTXGlun concentrations were not significantly different between the group with high disease activity and the group with low disease activity (data not shown). Association between MTX dosage, RBC MTXGlun concentration, and MTX adverse effects. One hundred forty-one patients (73.4%) reported ⱖ1 adverse effect, 118 (61.4%) reported ⱖ1 CNS-related adverse effect, 81 (42.2%) reported ⱖ1 GI-related adverse effect, and 53 (27.6%) reported ⱖ1 other adverse effect. There was no association between the MTX dosage and the presence of any adverse effect (P ⫽ 0.53), a CNS-related adverse effect (P ⫽ 0.31), a GIrelated adverse effect (P ⫽ 0.70), or other adverse effect (P ⫽ 0.39). There was no association between RBC MTXGlun concentrations and the presence of any adverse effect, GI-related adverse effect, or CNS-related adverse effect. There was a trend toward association between “other” adverse effects and higher RBC MTXGlu3 (P ⫽ 0.07), MTXGlu4 (P ⫽ 0.052), and MTXGlu3–5 (P ⫽ 0.07) concentrations. Allowing for factors that may contribute to adverse effects, such as concomitant treatment with other DMARDs or NSAIDs and the duration of time receiving MTX and folate, there was still a trend toward an association between the presence of “other” adverse effects and higher MTXGlu3 (P ⫽ 0.08) and MTXGlu4 (P ⫽ 0.08) concentrations. There was no association between any other MTXGlun concentrations and the presence of any adverse effect, GI-related adverse effect, or CNS-related adverse effect (data not shown). Many patients experience adverse effects early in the course of therapy, necessitating discontinuation of MTX. We therefore examined the relationship between the presence of adverse effects and MTXGlun concentrations in 35 patients receiving MTX for ⱕ12 months. There were no associations between any MTXGlun concentration and adverse effects. There was no association between the RBC folate concentration and the presence of any adverse effects (P ⫽ 0.27), CNS-related adverse effects (P ⫽ 0.17), GI-related adverse effects (P ⫽ 0.29), or other adverse effects (P ⫽ 0.34). Relationship between MTXGlun concentrations and laboratory variables. There was a significant negative correlation between hemoglobin and MTXGlu1–5 (P ⫽ 0.03), MTXGlu2 (P ⫽ 0.002), and MTXGlu3 (P ⫽ 0.04) concentrations. There was a positive correlation between the mean cell volume and the MTXGlu1–5 (P ⫽ 0.03) and MTXGlu1 (P ⫽ 0.003) concentrations and a trend toward a correlation with MTXGlu3 (P ⫽ 0.07). There was no association between any MTXGlun concentration and the platelet, neutrophil, or lymphocyte count (data not shown). There was no association between any MTXGlun concentration and the aspartate MTX POLYGLUTAMATES IN RA aminotransferase, alanine aminotransferase, or serum albumin concentration (data not shown). DISCUSSION MTX remains the “anchor” drug in the management of RA. Although it is evident that early intensive DMARD therapy can prevent joint damage, a key challenge that remains is determining whether an individual patient will respond to a particular drug, and at what dosage. In practice, patients are begun on a low dosage of MTX (7.5–10 mg/week), with upward titration according to response until the maximum dosage is reached (20–25 mg/week) (21). Such an approach may be a waste of time in nonresponsive or MTX-resistant patients. The ability to perform a blood test to determine whether further dosage escalation will provide therapeutic benefit would be a major advance. Such an advance would aid decisions regarding dosage increases for MTX monotherapy versus changing to alternative or combination DMARD or biologic therapies. It has been suggested that RBC MTXGlun concentrations can fulfill such a role and help guide MTX dosing in RA. The lack of an association between MTXGlun concentrations and reduced disease activity in this crosssectional study in patients receiving long-term MTX treatment was surprising and contrasts with earlier reports. Somewhat unexpectedly, we observed a positive correlation between MTXGlu5 and the SJC, TJC, M-HAQ score, and DAS28, which persisted despite correction for age, sex, duration of RA, smoking, antiCCP antibody status, concomitant treatment with steroids, NSAIDs or other DMARDS, and RBC folate status. The clinical significance of these results is uncertain, but they suggest that there is a group of patients in whom disease remains active despite higher MTX dosages and higher RBC MTXGlu5 concentrations. It would seem logical that the MTX dosage had been increased in order to increase the drug effect in these patients with persistently active disease that failed to respond to MTX at lower dosages. This is supported by the findings that MTX dosages and MTXGlun concentrations were higher in patients with more active disease, perhaps defining an MTX-resistant population. This reflects a confounding-by-indication scenario that is a consequence of MTX as the first-line DMARD in our practice, unless there is a specific contraindication. The results, therefore, reflect our local practice of increasing the MTX dosage to the maximum prior to the introduction of alternative DMARDs. The presence of patients 365 with high disease activity and high MTXGlun concentrations is likely to mask a positive association between MTXGlun concentrations and disease control in those patients in whom disease does not respond to MTX. We acknowledge that the cross-sectional study design has limitations. It dictated that many patients had been receiving MTX, often at stable dosages for long durations. The population studied was heterogeneous, and management was under the direction of 3 different rheumatologists who made therapeutic decisions based on perceptions of disease activity and patient choices rather than a predefined treatment protocol. Such a study design does not enable us to address the question of whether increasing the MTX dosage, thus increasing MTXGlun concentrations, results in additional disease control. A prospective study would enable examination of both the response to MTX and the occurrence of the more severe adverse effects that necessitate discontinuation of therapy. Such a study would enable comparisons between different dosing strategies and responses to MTX therapy but would be lengthy and logistically difficult. However, clinicians are frequently faced with patients presenting with active disease who have already been receiving treatment for a period of time. The key clinical question in this “real life” setting is whether to increase the dosage of MTX or add or change to another DMARD. In this regard, a cross-sectional study design examining the relationship between MTXGlun concentrations and disease activity, such as the design used in this study, provides useful information. It is possible that the duration of MTX treatment prior to measurement of the MTXGlun concentration may be an important factor. In a study of 48 patients with RA who were commencing MTX therapy, responders had higher MTXGlun concentrations at 6 months compared with nonresponders (40 ⫾ 18 nmoles/liter versus 27 ⫾ 6 nmoles/liter), although significance could not be determined because of the low number of nonresponders (11). Patients with RBC MTXGlun concentrations of ⬍20 nmoles/liter at 3 months were more likely to have had a smaller change in the DAS28. Another study of 40 patients with RA who were commencing MTX therapy showed that “erythrocyte (Ery)MTX” concentrations were higher in responders compared with nonresponders (P ⫽ 0.013) (22). Thus, in early RA, it has been shown that higher MTXGlun concentrations are associated with lower disease activity, but this relationship may wane as disease becomes established due to alterations in the biologic process of RA over time (2). In patients receiving long-term MTX therapy, the 366 role of measuring the MTXGlun concentration seems less clear. In a study of 65 patients receiving MTX for ⱖ2 months, erythrocyte MTX concentrations were higher in responders compared with nonresponders (60.92 ⫾ 19.1 versus 21.47 ⫾ 10.67; P ⫽ 0.0001). In that study, the total duration of therapy with MTX was not reported, and response was based on the physician’s global assessment including history, joint examination, and a comparison with the patient’s condition at an earlier time point (5). In a cross-sectional study of 108 patients with RA who were receiving MTX for ⱖ3 months (median 65 months, range 3–266 months), higher RBC MTXGlu3 concentrations were associated with lower TJCs (P ⫽ 0.04), lower SJCs (P ⫽ 0.021), and a lower score for the physician’s global assessment (P ⫽ 0.001) but not with the ESR or the M-HAQ score. In addition, MTXGlu concentrations of ⬎60 nmoles/liter were associated with a 14-fold higher likelihood of a VAS score ⱕ2 cm for the physician’s assessment of the response to MTX (3). In a study of 226 patients with RA who were receiving MTX for ⱖ3 months (median 51 months, interquartile range 19–97 months), patients with MTXGlu concentrations of ⬍60 nmoles/liter were 4-fold more likely to have a poor response (as defined by the physician’s assessment of the patient’s response to MTX) (6). The cross-sectional nature of this study means that it is not possible to determine whether those patients with a DAS28 of ⱕ3.2 and an MTXGlu3–5 concentration of ⬎60 nmoles/liter may have achieved a similar level of disease control at lower MTXGlun concentrations. Although the MTX dosage is generally increased until a response is achieved, it is possible that some patients may have responded to lower dosages. Dosage escalation typically occurs at 2–6-week intervals, and our previous work has shown that it takes much longer than this for steady-state concentrations to be achieved (19); therefore, the dosage may have been increased unnecessarily in some patients. In all of the cross-sectional studies to date, including the present study, the minimum duration of MTX therapy has been 3 months, with the dosage being stable for ⱖ1 month. Given that it can take up to 6 months to reach steady state after commencing MTX therapy and after dosage adjustments, it is likely that steady-state concentrations may not have been achieved in some patients. However, using the same cohort of patients, we have shown that a longer duration of therapy with MTX was associated with higher RBC MTXGlu4 and MTXGlu5 concentrations only in a univariate analysis, and this association did not persist in STAMP ET AL the multivariate model including age, dosage, and renal function (20). Our data suggest that there is a group of patients (31%) with a high DAS28 and low MTXGlun concentrations in whom a dosage increase could be considered. If such an approach were undertaken, we would anticipate that ⬃50% of patients may experience an additional reduction in disease activity. In our cohort, 41% of patients had high disease activity. It was our preference to enroll patients receiving MTX monotherapy, in order to minimize confounding from the use of other DMARDs. Although there is no disagreement that low disease activity and remission are the desired outcomes, such outcomes are not achieved in all patients. A variety of factors contribute to ongoing disease activity, including patient treatment choices and availability of alternative DMARDs. In addition, some patients may have been in the process of receiving dosage escalations or, alternatively, may have presented with a flare of arthritis after previously having had good disease control. We have shown that patients with high disease activity have significantly higher RBC folate concentrations than those with low disease activity. This is consistent with results of a previous study in which reduced RBC folate levels were associated with improvement in disease control in patients with RA who were beginning therapy with MTX (11). Our data suggest that RBC folate levels may be a more important factor than MTXGlun concentrations for disease control in patients receiving MTX. This possibility warrants further investigation. In patients with a poor response to therapy, reduced drug compliance should be considered. Some laboratory results, such as an increase in the mean red cell volume (MCV), may give an indication of compliance, in that the MCV should rise gradually after starting treatment with MTX (23). It has been suggested that MTXGlu3–5 concentrations can also be used to help determine compliance. However, we have previously shown that many weeks or months are required for MTXGlu3–5 to reach steady state and to be eliminated (19). Thus, only patients who are consistently noncompliant over many weeks may exhibit low concentrations. Consistent with previous reports (3,5), we have shown no association between MTXGlun concentrations and MTX-related adverse effects. Because there is no validated questionnaire for the adverse effects of MTX, we developed a questionnaire, using expert opinion. The questionnaire addressed the presence or absence of well-recognized but often underappreciated and under- MTX POLYGLUTAMATES IN RA reported adverse effects of MTX. We acknowledge that many of these adverse effects are nonspecific, and that placebos may be associated with such adverse effects. The majority of the patients in this study had received MTX for several years. Therefore, the crosssectional nature of the study would have excluded many patients who could not tolerate MTX and discontinued treatment early, thereby making it more difficult to detect a relationship between MTXGlun concentrations and adverse effects and factors that predict adverse effects. A prospective study is needed to determine the relationship between MTXGlun concentrations and serious adverse effects such as myelotoxicity, pneumonitis, and/or hepatotoxicity, which usually necessitate MTX discontinuation or dosage reduction. In addition, it is not clear whether RBC MTXGlun concentrations reflect accumulation in other tissues such as liver or bone marrow. In this regard, Ahern et al examined liver biopsy specimens obtained from 18 patients with RA who were receiving MTX and observed increased concentrations of MTX, MTXGlu2, and 2,4 diamino-N10methylpteroic acid (another MTX metabolite) in 3 patients with hepatic fibrosis (24). Using the same questionnaire that was used in this study, a small study of 18 patients with inflammatory bowel disease demonstrated higher MTXGlu5 concentrations in patients experiencing ⱖ1 MTX-related adverse effect, and MTXGlu4 and MTXGlu5 concentrations were higher in those experiencing GI adverse effects (25). It is possible, due to the small number of patients, that this was a Type I error. Alternatively, there may be differences in the perception or pattern of adverse effects between different patient groups. In summary, the results of our cross-sectional study do not support earlier findings of a correlation between MTXGlun concentrations and the control of RA disease activity. This result was unexpected but may reflect differences in established versus early disease, selection of patients tolerant to MTX, dosage increases in MTX-resistant patients, RBC folate status, differences in intracellular drug metabolism, or other as-yetundetermined factors. The best way to clarify this situation would be to conduct a long-term prospective study, serially examining the relationships between MTX dosage, MTXGlun concentrations, RBC folate status, and effects/adverse effects after commencing treatment. Such a long-term study may help explain the apparent differences in findings between studies, the optimal time for measuring MTXGlun concentrations after commencing MTX therapy, and the relationship between the MTXGlun con- 367 centration and loss of disease control in patients receiving MTX. Furthermore, such a study may help to determine whether there is clinical benefit in increasing the dosage of MTX in patients who are not responding to treatment. Careful consideration will need to be given to the study design, given the long time required for MTXGlun concentrations to reach a steady state and the need for rapid disease control. Although we have shown no relationship between MTXGlun concentrations and adverse effects, a prospective study would clarify whether there is a relationship between MTXGlun concentrations and serious MTX-related adverse effects that necessitate MTX discontinuation. Finally, RBC folate status has been shown to be an important influence on MTX efficacy. Further studies should help to address the optimal folate regimen and RBC folate levels with concomitant MTX therapy. ACKNOWLEDGMENT We gratefully acknowledge the assistance of Jan Ipenburg, Rheumatology Clinical Nurse Specialist, in assisting with patient data collection. AUTHOR CONTRIBUTIONS All authors were involved in drafting the article or revising it critically for important intellectual content, and all authors approved the final version to be published. Dr. Stamp had full access to all of the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis. Study conception and design. Stamp, O’Donnell, Chapman, Frampton, Barclay. Acquisition of data. Stamp, O’Donnell, Chapman, Zhang, James. Analysis and interpretation of data. Stamp, O’Donnell, Chapman, James, Frampton, Barclay. REFERENCES 1. Furst DE, Koehnke R, Burmeister LF, Kohler J, Cargill I. 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