Rheumatology 2002; 41: 741-749
© 2002 British Society for Rheumatology
Original Papers |
Methotrexate in the treatment of rheumatoid arthritis. II. In vivo effects on bone mineral density
The Royal National Hospital for Rheumatic Diseases, Upper Borough Walls, Bath BA1 1RL,
1 Medical Research Council Environmental Epidemiology Unit, Southampton General Hospital, Southampton SO16 6YD,
2 Department of Medicine, Addenbrooke's Hospital, Cambridge CB2 2QQ and
3 Bone Research Group, Department of Pharmacy and Pharmacology, University of Bath, Claverton Down, Bath BA2 7AY, UK
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Abstract |
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Objective. To determine the effect of methotrexate (MTX) on bone mineral density (BMD) in rheumatoid arthritis (RA).
Methods. One hundred and sixteen non-steroid-treated RA subjects (90 women) were studied in a prospective, longitudinal, non-randomized study. Subjects started MTX (n=36) or sulphasalazine (n=23) or continued long-term (>5 yr) treatment with MTX (n=28) or other disease-modifying anti-rheumatic drugs (n=29). BMD was estimated at entry and after 1 yr. Markers of bone turnover were measured at entry and at 1 yr, and additionally at 3 and 6 months in those starting treatment. Bone biopsies were taken before and after MTX treatment in four subjects. The primary outcome was change in BMD Z score and secondary outcomes were changes in bone turnover markers and bone formation by histomorphometry.
Results. Univariate analysis of covariance found that MTX at baseline was associated with reduced BMD at the femoral neck. However, femoral neck BMD was also associated with radiological damage score for the hand. Multivariate analysis and discriminant analysis of the subset of post-menopausal women showed that reduced bone density associated with MTX was due to confounders such as disease activity. There was no adverse effect of MTX on bone turnover markers or on measures of bone formation in biopsies.
Conclusions. No adverse effect of low-dose MTX (mean 10 mg/week) on bone formation in RA was found.
KEY WORDS: Rheumatoid arthritis, Methotrexate, Osteoporosis, Bone mineral density, Bone turnover.
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Introduction |
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The drug methotrexate (MTX) competitively inhibits the reduction of tetrahydrofolate by dihydrofolate reductase, thus inhibiting the synthesis of DNA and RNA [1]. MTX is a chemotherapeutic agent when used in high doses of 100–1000 mg/m2. In low doses of 5–25 mg per week, MTX has many anti-inflammatory and cytokine-modulating properties which are thought to contribute to its beneficial effect in the treatment of rheumatoid arthritis (RA) and other chronic inflammatory conditions [2].
A syndrome of bone pain, radiographic osteopenia and fractures (MTX osteopathy) has been reported with short-term [3] and long-term use up to 5 yr [4] in children receiving high-dose MTX as chemotherapy. There have also been case reports of fragility fractures occurring in adult patients on long-term, low-dose MTX for RA or psoriatic arthritis [5–8].
However, Buckley et al. [9] found no effect of MTX on bone mineral density (BMD) in adult patients with RA in a study designed primarily to assess the effects of calcium and vitamin D on the skeleton. Similarly, a study of children with RA found that MTX had no adverse effect on the growing skeleton [10].
A number of studies have shown that patients with RA, even those in the early stages of the disease, have significantly reduced BMD and bone formation rates when compared with age- and sex-matched controls that is independent of corticosteroid use [11–13]. It would be undesirable to use a drug that might further compromise the structural integrity of the skeleton. For this reason, we performed a study to assess the long- and short-term skeletal effects of MTX in patients with RA. We used bone densitometry, histomorphometry and biochemical indices of bone formation and resorption to measure bone turnover.
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Patients and methods |
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Patients
The study was approved by the local ethics committee and had an observational, pragmatic design. The subjects gave written consent and were Caucasian, aged 18–80 yr, attended the Royal National Hospital for Rheumatic Diseases, Bath, UK, and fulfilled the American College of Rheumatology 1987 revised criteria for RA [14]. Exclusion criteria were current oral corticosteroid therapy for any disease, previous maintenance corticosteroids, intravenous or intramuscular pulse corticosteroid treatment within the previous 3 months, and treatment (past or present) with any bone-active treatment except female hormone replacement therapy. Prohibited treatments included bisphosphonates, calcitonin, anti-epileptic medication, cyclosporin and testosterone replacement therapy. Short, reducing courses of oral corticosteroids for reversible airways disease (at least 6 months earlier) were permitted. All patients who attended the out-patients department between April 1995 and March 1996 and who fulfilled the entry criteria were invited to participate. Two hundred and thirty-one patients agreed to be telephoned to discuss the study, and 138 agreed to participate. Twenty-two were excluded at the initial visit, leaving 116 subjects.
There were four study groups: (i) the ‘continue MTX’ group (n=28) had been taking MTX continuously for at least 5 yr; (ii) the ‘continue DMARD’ group (n=29) had been taking a different disease-modifying anti-rheumatic drug (DMARD) for at least 5 yr and had never had MTX; (iii) the ‘start MTX’ group (n=36) were to start treatment with MTX; and (iv) the ‘start SPZ’ group (n=23) were to start treatment with sulphasalazine (SPZ). No subjects who started treatment had received MTX in the past, although some had been on other DMARDs (two in the group starting SPZ and 11 in the group starting MTX). The decisions regarding when to start treatments and which drug to use were taken by the clinicians responsible for each case, and all patients who met the selection criteria were invited to take part.
The primary outcome was change in BMD and secondary outcomes were changes in bone turnover markers and bone formation, measured by histomorphometry.
Clinical assessment and markers of bone turnover
There were two baseline visits, 2 weeks apart, for all subjects and before any new treatment was started, and the results from these were averaged. Subjects starting either MTX or SPZ were seen at 3 and 6 months, and the final visit for all subjects (including those continuing treatment) was at 12 months, except for the subjects with early RA (disease duration <2 yr), who were seen additionally at 24 months. A validated questionnaire was modified and administered to each subject on study entry to determine both past and present physical activity and calcium intake [15]. These data were used in the statistical analysis (see below, Statistical analysis).
RA disease activity was assessed at each visit using a modified disease activity score (DAS) calculated from a 28-joint count (swollen and tender joints), the erythrocyte sedimentation rate (ESR) and patient assessment of disease activity [16]. Function was recorded using the Health Assessment Questionnaire (HAQ) modified for British patients [17]. Bone turnover markers were also measured at each visit and analysed by enzyme-linked immunosorbent assay. Bone formation was assessed by measurement of osteocalcin (OC) (Novocalcin; Metra Biosystems, San Diego, CA, USA) and bone-specific alkaline phosphatase (BALP) (Alkphase-B; Metra Biosystems) in serum. Bone resorption was assessed by measurement of deoxypyridinoline (Dpd) (Pyrilinks-D; Metra Biosystems) in a fasting second-void urine sample and corrected for urinary creatinine. The coefficients of variation for the different assays were 6.9% (OC), 4.8% (BALP) and 5.5% (Dpd).
Bone mineral density
BMD of the lumbar spine (L1–4), total hip (and hip subregions: neck of the femur, trochanteric, intertrochanteric and Ward's triangle) and total forearm (and subregions of the ultra-distal, mid-portion and proximal regions) were measured at baseline and at 1 yr with a QDR 4500 (Hologic Inc., Bedford, MA, USA) bone densitometer. The coefficient of variation of the phantom measurement over the study period was 0.51%; typical precision errors for the sites scanned are 1.0% (lumbar spine), 1.6% (femoral neck) and 1.0% (forearm) [18].
Measurement of RA disease impact
Radiographs of hands and wrists were obtained at the start of the study and at 1 yr. As the preliminary analysis showed significantly reduced forearm BMD in the subjects treated with MTX for >5 yr compared with those on other DMARDs, elbow radiographs were obtained at 1 yr in those who agreed. Larsen scores [19] were assigned to each radiograph by a single radiologist who was blinded to the patient groups.
Histomorphometry
Full-thickness, double tetracycline-labelled transiliac crest bone biopsies were taken under local anaesthesia from four subjects with recent-onset RA (<12 months) before starting MTX and after 1 yr of treatment. All were pre-menopausal women and had taken demethylchlortetracycline following a described protocol [13]. Measurements of cancellous bone area, osteoid perimeter, osteoid width, mean wall width, resorption cavity indices, mineral apposition rate and mineralizing perimeter were made. Inter-observer and intra-observer variability were calculated for each measurement prior to analysis of the study biopsies, and all measurements were performed by a single observer. From the direct measurements, the following derived indices of bone formation were calculated: adjusted apposition rate; bone formation rate; activation frequency; and formation period. All histomorphometric data are described according to American Society for Bone and Mineral Research nomenclature [20].
Statistical analysis
The preliminary analysis was performed using Statview (SAS Institute, Cary, NC, USA). Two-way comparisons were done by paired or unpaired t-tests for single comparisons as appropriate. When repeated measures were being compared, analysis of variance (ANOVA) was used. Four-way comparisons were done by ANOVA with the Bonferroni test for multiple comparisons. When the assumptions needed for ANOVA, such as normality and homogeneity of variance, were violated, either Friedman's non-parametric test was used or the data were log-transformed prior to ANOVA. Multivariate analysis of covariance, analysis of covariance and a discriminant analysis were performed on the data for the subset of post-menopausal women, as this was the group most at risk of osteoporotic fracture. The following potentially confounding factors (covariates) were used in the analyses: duration of RA (months); disease activity (DAS); body mass index (kg/m2); time since menopause (yr); Larsen baseline hand and wrist X-ray scores; changes in the hand and wrist X-ray scores; and elbow X-ray score.
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Results |
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The characteristics of the subjects are shown in Table 1
and the menopausal status of the women in Table 2. The subjects continuing MTX were well matched with those continuing other DMARDs for age, body mass index, duration of disease and measures of disease activity. However, the groups did differ in the number of previous DMARDs used (Table 1). In the ‘continue DMARD’ group, current therapy was as follows: intramuscular gold, n=8; penicillamine, n=8; oral gold, n=4; sulphasalazine, n=4; hydroxychloroquine, n=4; and azathioprine, n=1. No subject was on combination therapy. The subjects starting MTX had longer average disease duration than those starting SPZ, but there were no differences in the baseline HAQ between the four groups. There were no significant differences in RA disease activity (swollen joint count, tender joint count, ESR or DAS) between the groups continuing MTX or another DMARD at baseline or at 1 yr. The subjects starting either MTX or SPZ had significantly higher mean DAS than the groups continuing treatment at baseline, but there was no difference between the groups starting MTX and SPZ at baseline (Table 1). Both groups starting a DMARD showed a significant reduction in DAS over the course of the study, with no significant difference between the treatments. Mean baseline Larsen scores of hand radiographs were significantly higher in subjects on long-term treatment than in subjects starting either MTX or SPZ. There was no difference in wrist or hand Larsen score at baseline between those starting MTX or SPZ.
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The majority of women in each group were postmenopausal but very few were current users of hormone replacement therapy (Table 2
). Hormone status was not measured and women were classified as peri-menopausal if they were within 5 yr of the cessation of regular menses. Women were classified as post-menopausal if they were >5 yr after the onset of menopausal symptoms. There was one death during the study period (a myocardial infarction in a 78-yr-old woman within 3 months of starting SPZ). Another five subjects who started SPZ stopped the drug (two due to a rash and three due to ineffectiveness). None of the subjects starting MTX discontinued the drug.
Unadjusted bone mineral density
Results are presented as BMD and Z scores. The Z score is the BMD expressed as the number of standard deviations above (positive value) or below (negative value) the mean value of an age- and sex-matched reference population [21]. Table 3 shows the baseline unadjusted BMD and Z scores, and Table 4 the percentage changes in BMD from baseline to 1 yr; these tables indicate where significant between-group differences occurred.
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The Z scores were near zero in the groups starting either MTX or SPZ, with large standard deviations, reflecting the normal distribution of this measurement in this population.
The group continuing MTX had significantly lower baseline BMD Z scores at the proximal third, mid-portion and total forearm sites when compared with all the other groups (Table 3B).
For the annual change in BMD, the greatest losses were seen in the group ‘start MTX’, at the neck of femur, lumbar spine (Table 4A) and forearm sites (Table 4B).
Subjects who had early RA (disease duration <2 yr) were followed over 2 yr and had an annual BMD measurement. No statistical evidence of between-group differences in BMD was apparent during a 2-yr observer period in early RA subjects (MTX, n=16; SPZ, n=13) (data not shown).
Regression analyses
In this analysis, all four treatment groups were considered together and significant between-group differences were found. The results for the BMD Z scores are presented, although absolute BMD measurements were also analysed.
Baseline Z score
Univariate analysis of covariance was performed to assess the between-group differences in the baseline BMD Z score for each site, adjusting for important confounders (see Patients and methods). The differences were found to be significant only at the neck of femur site (P=0.027), where MTX treatment was associated with a lower Z score. However, the Z score for the neck of the femur was also associated significantly with disease severity measured by the baseline hand X-ray score. There were also statistically marginal differences between treatment groups in the baseline Z scores for the mid-portion of the forearm and total forearm, which were also significantly associated with disease duration and the number of years after the menopause, respectively.
Annual change in Z score
When adjustment was made for important differences between groups, the univariate ANOVA for the change in BMD Z score for each of the sites found significant differences between groups at the following sites: neck of femur (P=0.004); the proximal third of the forearm (P=0.0001); the mid-portion of the forearm (P=0.001); and total forearm (P=0.002). Changes in all these sites were significantly associated with both baseline values of BMD Z score and measures of disease severity. The changes in the BMD Z scores for the proximal third and the mid-portion of the forearm were additionally associated with body mass index and years since the menopause. After adjustment, the group continuing MTX had less reduction in BMD than did those on other DMARDs, and the groups starting treatment with either MTX or SPZ lost least bone in the neck of the femur. In the total forearm, the group starting SPZ had the greatest mean loss of BMD.
In a discriminant analysis, the variables were analysed at the same time rather than sequentially, as in a regression analysis. This is a useful method as many of the factors contributing to bone loss in RA are not independent; for example, disease duration and X-ray score were associated in our study. We used discriminant analysis to determine which of the BMD Z scores were most important in differentiating between groups, whilst accounting for confounding factors. A direct multiple discriminant analysis was performed in the post-menopausal female subjects using the multivariate baseline BMD and change in BMD profile, and included body mass index, years after the menopause, the baseline indices of disease severity, disease duration and disease activity as predictors for treatment group membership.
Three discriminant functions were calculated; the first two accounted for 77.6 and 16.6% of the between-group variability respectively (Table 5) and are presented here. Figure 1 illustrates the distribution of the discrimination scores according to treatment. The first discriminant function maximally separates patients continuing treatment from those starting treatment. The second discriminant function separates those receiving MTX from those on other DMARDs. The relative positioning of groups in the classification plot depicts the differential effect of duration of treatment according to type of treatment.
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The matrix of coefficients between predictors and discriminant functions is shown in Table 5
. The best predictors for distinguishing between subjects continuing treatment and subjects starting treatment were disease severity (baseline hand X-ray score) and disease duration. Higher baseline OC levels and lower baseline Z scores for the proximal third and mid-portion of the forearm were also predictive for the first discriminant function. However, the total forearm and mid-portion Z scores were positively associated with the secondary discriminant function, suggesting that the group on long-term MTX were more likely than those on other DMARDs to have an increase in the BMD in these forearm sites. Also, in those starting treatment, SPZ treatment was more likely to be associated with large negative changes in forearm BMD Z score than was MTX. Furthermore, the proximal third and total forearm changes in BMD were strongly correlated with elbow X-ray score, suggesting that local disease activity had contributed to bone loss at these sites. In conclusion, changes in BMD Z score at different sites weakly discriminated the treatment groups. The most important contributors were the mid-portion and total forearm sites. We found no indication that the losses were associated with MTX treatment. When the model was applied back to the data, it correctly categorized 97.7% of cases (only one case was incorrectly assigned), suggesting it was robust.
Bone turnover markers
There were no significant differences between baseline and later measurements in any group for OC, BALP or Dpd corrected for creatinine (data not shown).
Histomorphometry
Table 6 shows results of the static and dynamic measurements for the four subjects together with normal values for pre-menopausal women [22]. Analysis of the results by the paired t-test found no significant difference between the pre- and post-treatment biopsies for any of the direct measurements, including the resorption cavity measurements (data not shown). The only significant difference found for the derived indices was the formation period, which was a mean of 8.4 days longer in the post-treatment biopsies (P=0.046). As we found no significant treatment effect on the direct measurements of bone formation, we do not think this result is clinically relevant and may be related to the small number of subjects. MTX therapy was not associated with reduced formation or increased resorption of cancellous bone in these patients.
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Discussion |
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TopAbstractIntroductionPatients and methodsResultsDiscussionReferences |
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We report the results from a prospective study of 116 non-steroid-treated RA subjects in whom the influence of MTX on bone formation, and thus BMD, was studied. The study was observational, comparing the effect of MTX with SPZ in the patients starting therapy and comparing MTX with all other DMARDs in those patients on long-term (>5 yr) treatment. This study design was chosen because it would have been unethical to use a placebo-controlled design in subjects with early RA [23]. Patients were not randomized to treatment with MTX or SPZ, as the benefits of randomization would have been compromised by non-participation and thus smaller sample sizes, reducing the power of the study. Both MTX and SPZ are used as first-line DMARDs in RA, and we felt our pragmatic approach to be justified as it reflected current rheumatological practice in the UK. Analysis of the data was limited more by sample size than lack of randomization. Subject numbers reflected the strict entry criteria we applied, permitting no steroid use. All suitable subjects attending the Royal National Hospital for Rheumatic Diseases during the 12 months of recruitment were approached, and a larger study would therefore have necessitated a multicentre design.
Initial analysis of the unadjusted data revealed significantly lower baseline BMD and Z scores at all forearm sites in the group continuing MTX, whereas for the annual change in BMD the greatest losses were seen in the group starting MTX (at several sites, including the forearm). Whilst these results suggested that the use of MTX was associated with increased bone loss, baseline differences between the treatment groups, in factors such as disease duration and in the number of previous DMARDs used, may have influenced the results. For example, 21 subjects in the group starting MTX had failed a previous DMARD compared with only five subjects in the group starting SPZ. Prior to 1991, when those continuing on MTX had started it, the drug was reserved for second- or third-line DMARD use, and so subjects in this group may have had arthritis that was more aggressive and difficult to treat than those taking other DMARDs.
A more detailed statistical analysis was undertaken in the subgroup of post-menopausal women, as they constituted the majority of subjects in each group and were most at risk of developing osteoporosis. The multivariate analysis, which took into account the subjects' age, duration and severity of disease (assessed by radiographic damage and DAS) and years since the menopause, confirmed that many of the differences in the unadjusted data were due to factors other than MTX. However, significant differences remained at several sites: the femoral neck (baseline and annual change) and the proximal third and mid-portion of the forearm and total forearm (annual change only).
The discriminant analysis was carried out as many of the variables that affect bone mass in RA are not truly independent, and this model allowed them to be studied at the same time, not entered sequentially, as in multiple regression analysis. The proximal forearm BMD change was no longer significant as it was found to correlate strongly with elbow disease (X-ray score), suggesting that local inflammatory joint disease was a more important determinant of bone loss at this site than MTX therapy.
A strength of the study is that we only recruited subjects who had never been on oral corticosteroids for RA, to avoid the strong confounding effects on BMD associated with this medication [24]. All joint assessments, analysis of markers of bone turnover and histomorphometric measurements were made by one observer (NJM). The number of bone biopsies obtained was small and subtle effects of MTX may have been missed in such a small sample. However, this is the first histomorphometric report of the effect of low-dose MTX on trabecular bone, and it is reassuring that no adverse effects were seen. Histomorphometry gives information at the tissue level, including connectivity, which cannot be deduced from bone density or markers of bone turnover. We do not think that the increased bone formation period seen in the post-treatment biopsies is clinically relevant.
Our results are in keeping with other prospective studies in which adverse effects on lumbar spine and hip BMD were found to be due to corticosteroid rather than MTX treatment [13, 14]. Fragility fractures have been reported at the distal tibial site in subjects using low doses of MTX [5–8], and it has been suggested that MTX has a predominately adverse effect on cortical bone [7]. To our knowledge, there have been no prospective studies of distal tibial BMD in MTX-treated subjects, and no studies other than the present one with such detailed examination of the forearm. Our findings suggest that where reduced BMD occurs in a patient on MTX for RA, it is more likely to be due to local disease activity and adjacent joint damage rather than a toxic effect of MTX. In support of this, no effect of MTX on vertebral BMD was found in a 2-yr prospective study of 40 female RA subjects, in which those starting MTX were compared with those starting other DMARDs [25]. As in our study, RA disease activity had a greater effect on BMD than individual DMARDs, including MTX.
A recent paper compared 30 RA subjects who were taking MTX at a mean weekly dose of 10 mg for a mean of 6 yr with 30 RA subjects who had never had MTX [26]. Corticosteroids were being taken by 14 of the MTX group (mean daily dose 8.5 mg) and 10 of the comparator group (mean daily dose 5 mg). BMD of the hip, lumbar spine and forearm was measured once in a cross-sectional design. As in the present study, no adverse effect of low-dose MTX on BMD was found, despite the higher mean daily dose of corticosteroids in the MTX group. The majority of the subjects were post-menopausal women (60% in the MTX group and 90% in the comparator group). The authors acknowledge that the cross-sectional design of their study was a limiting factor, and we would add that the inclusion of those on corticosteroids is an additional confounding factor, which we avoided.
The results we have reported here suggest that, at the doses used in this study (mean weekly dose of MTX 10 mg, maximum 15 mg), MTX is safe in RA with respect to bone health. However, increasingly, higher doses of MTX (up to 30 mg per week) are being used in RA and our results should not be extrapolated to patients so treated. In vitro data we have obtained using MTX in cultures of osteoblast-like cells showed inhibition of osteoblast proliferation, which was greater with higher doses [27]. Nevertheless, we found that MTX at the lower doses used in this study had no adverse effect on bone density or turnover in patients with RA.
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Acknowledgments |
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We would like to thank Dr G. Evison, who scored the radiographs, and Dr J. Tobias for critical reading of the manuscript and helpful comments. NJM was supported by a Clinical Research Fellowship (M0530) funded by the Arthritis Research Campaign, UK.
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Notes |
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Correspondence to: N. J. Minaur, 66 Third Avenue, Bath BA2 3NZ, UK.
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References |
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