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Rheumatology Advance Access originally published online on January 25, 2006
Rheumatology 2006 45(6):761-764; doi:10.1093/rheumatology/kei280
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© Published by Oxford University Press on behalf of the British Society for Rheumatology 2006.

The effect of infliximab on bone metabolism markers in patients with rheumatoid arthritis

E. Torikai, Y. Kageyama, M. Takahashi, M. Suzuki, T. Ichikawa, T. Nagafusa and A. Nagano

Department of Orthopaedic Surgery, Hamamatsu University School of Medicine, Hamamatsu, Japan.

Correspondence to: E. Torikai, Department of Orthopedic Surgery, Hamamatsu University School of Medicine, 1-20-1 Handayama, Hamamatsu, Japan. E-mail: eiji{at}hama-med.ac.jp


   Abstract
Top
 Abstract
Introduction
 Materials and methods
 Results
 Discussion
 References
 
Objective. The aim of this study was to evaluate urinary excretion of N-telopeptide of type I collagen (NTX) and deoxypyridinoline (DPD), markers of bone resorption, and serum bone alkaline phosphatase (BAP) level, a marker of bone formation and an early marker of osteoblast differentiation, in patients with rheumatoid arthritis (RA) treated with infliximab.

Methods. Seventeen male and female patients (age 60.7±2.53 yr; mean disease duration 12.9±3.01 yr; Steinbrocker's class II–IV) with RA, diagnosed according to the criteria of the American College of Rheumatology (ACR), took part in the study between March 2003 and January 2005. None of the patients had a history of oestrogen replacement therapy. All patients were treated with infliximab combined with methotrexate. Infliximab was infused intravenously at 3 mg/kg at baseline, 2 and 6 weeks, then every 8 weeks. To evaluate disease activity, ESR, CRP, the numbers of swollen and tender joints, modified Stanford Health Assessment Questionnaire (mHAQ) score and ACR score were measured. Levels of NTX and DPD in urine and BAP in serum were measured in all patients.

Results. ESR, CRP, the number of swollen joints and tender joints, and mHAQ score had decreased significantly 6 weeks after initial treatment and were still low 6 months after initial treatment. NTX levels had decreased significantly 6 weeks after the initial treatment and were still low 6 months after initial treatment. DPD levels had decreased 6 months after initial infusion. Mean serum BAP level did not differ significantly among the three time points. NTX levels were statistically corresponding with the number of swollen joints and mHAQ scores. DPD levels were statistically lower corresponding with ESR.

Conclusion. Infliximab therapy may inhibit generalized bone loss in patients with RA. NTX is a more sensitive marker than DPD.

KEY WORDS: Infliximab, Bone metabolism marker, NTX, Rheumatoid arthritis


   Introduction
Top
 Abstract
 Introduction
Materials and methods
 Results
 Discussion
 References
 
Rheumatoid arthritis (RA) is a chronic inflammatory disease that increases the risk of developing bone loss, resulting in juxta-articular and generalized osteoporosis [1, 2]. Generalized bone loss may be influenced by immobility, the inflammatory process and treatments such as corticosteroids. Juxta-articular bone loss is probably due to local release of inflammatory agents such as cytokines [3] derived from rheumatoid synovium [4–7] and articular immobility [1, 6–9]. Therefore, the mechanisms responsible for causing joint damage and functional impairments in RA are complex. Of many pro-inflammatory mediators, interleukin 1 (IL-1) and tumour necrosis factor {alpha} (TNF-{alpha}) are believed to play an essential role in the pathogenesis of RA [10, 11]. We set out to show that infliximab, which is a chimeric human–mouse monoclonal antibody that neutralizes soluble and membrane-bound TNF-{alpha}, and has been shown to inhibit bone destruction [12, 13], is highly effective in relieving signs and symptoms in patients with active RA.

It is now possible to assess bone turnover using a range of biochemical markers to reflect bone resorption and formation [14]. Deoxypyridinoline (DPD) and N-telopeptide of type I collagen (NTX) have been validated as useful markers of bone resorption, and bone alkaline phosphatase (BAP) as a useful marker of bone formation and an early marker of osteoblast differentiation.

In this study we administered infliximab combined with methotrexate (MTX), using a routine infusion method, to patients with RA. The aim of the present study was to evaluate urinary excretion levels of NTX and DPD and serum BAP levels as bone metabolism markers in patients with RA treated with infliximab.


   Materials and methods
Top
 Abstract
 Introduction
 Materials and methods
Results
 Discussion
 References
 
Subjects
Seventeen patients (15 females and two males; age 60.7±2.53 yr; mean disease duration 12.9±3.01 yr; Steinbrocker criteria: class II 3, class III 9, class IV 5) with RA, diagnosed according to the criteria of the American Collage of Rheumatology (ACR), took part in this study between March 2003 and January 2005. For all patients, MTX alone was not effective for more than 6 months and patients were naive with respect to infliximab at the commencement of the study. None of the patients had a history of hormone (oestrogen) replacement therapy or had used any other bone-sparing drugs or calcium supplements. Thirteen patients received a constant dose of prednisolone equivalent to 10 mg or less per day (mean±S.E., 5.70±0.71 mg; median dose 5.0 mg/day) at a stable dosage throughout the present study. Infliximab was administered by intravenous infusion at a dose of 3 mg/kg at the baseline, then at 2 and 6 weeks, and then every 8 weeks. MTX was administered orally at 4 ~ 10 mg/week (mean±S.E. 6.35±0.58 mg/week; median dose 6.0 mg/week). All patients underwent general and physical examinations and routine blood and urinary analysis at baseline, 6 weeks and 6 months after the initial treatment. To evaluate disease activity, ESR, CRP, the number of swollen joints and tender joints, modified Stanford Health Assessment Questionnaire (mHAQ) score and ACR score were measured. Then, the levels of NTX and DPD in urine and BAP in serum were measured in all patients, as described below. Informed consent was obtained from all patients. The ethical committee of Hamamatsu University School of Medicine authorized this work.

Measurement of urinary NTX, urinary DPD and serum BAP
Urine samples were collected at approximately the same time in the morning after the patients’ first overnight stay, and stored at –70°C until assayed. NTX levels were measured using an enzyme-linked immunosorbent assay (ELISA; Osteomark; Ostex International, Seattle, WA, USA) with a monoclonal antibody against the N-telopeptide to the helix intermolecular cross-linking domain of type 1 collagen. DPD levels were determined with commercially available pyridinium cross-links using an HPLC kit (enzyme immunoassay; Metra Biosystems, Mountain View, CA, USA). Serum BAP was measured using an AlkaphaseB kit (enzyme immunoassay; Metra Biosystems).

Statistical analysis
All data are presented as mean±S.E. Pretreatment and post-treatment measurements were compared using the Wilcoxon signed ranks test. Single regression analysis was used to examine the cross-correlation of change rate between markers of bone metabolism and clinical factors in patients with RA. P values less than 0.05 were considered significant. Analysis was performed on a Macintosh computer using the StatView 5.0 software package.


   Results
Top
 Abstract
 Introduction
 Materials and methods
 Results
Discussion
 References
 
Measurements of disease activity
The patients improved clinically after receiving infliximab, and at 6 months after the initial treatment 76% of patients demonstrated an ACR 20 response, 29% an ACR 50 response and 12% an ACR 70 response. CRP levels decreased from 4.29±2.37 mg/dl before treatment to 1.86±0.63 mg/dl (P<0.01) and 1.84±0.79 mg/dl (P<0.05) 6 weeks and 6 months after the initial infusion, respectively. ESR levels also decreased, from 66.7±9.1 mm/h before treatment to 44.4±7.8 mm/h (P<0.05) and 43.9±8.8 mm/h (P<0.01) 6 weeks and 6 months after the initial infusion, respectively. In addition, the mHAQ score, the number of tender joints at 6 weeks and 6 months, and the number of swollen joints at 6 weeks after the initial therapy all decreased significantly (Table 1).


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  		TABLE 1. Parameters of disease activity and metabolism markers during therapy with infliximab (mean±S.E.)

 
Urinary NTX levels
The mean NTX levels decreased significantly from 80.1±15.7 nM bore collagen equivalent/mM Cr before treatment to 52.8±9.1 nM BCE/mM Cr (P<0.01) and 52.5±7.5 nM BCE/mM Cr 6 weeks and 6 months (P<0.01) after the initial treatment, respectively (Table 1).

Urinary DPD levels
The mean DPD levels were 9.22±0.85 nM/mM Cl before treatment, 8.12±0.85 (P = 0.062) nM/mM Cl at 6 weeks and 7.38±0.65 (P<0.05) nM/mM Cl 6 months after the initial infusion, respectively (Table 1). Thus, there was no significance decrease in DPD level at 6 weeks but a significant decrease 6 months after the initial infusion, compared with pretreatment values.

Serum BAP levels
The mean serum BAP levels were 34.6±19.6 U/l before treatment, 31.9±15.1 U/l at 6 weeks and 32.5±17.3 U/l 6 months after the initial treatment, respectively; there were no significant differences among the three time points (Table 1).

Correlation between bone resorption markers and disease activity
NTX levels were statistically corresponding with the number of swollen joints (r = 0.655, P<0.01) and mHAQ scores (r = 0.487, P<0.05). DPD levels were also statistically lower, corresponding with ESR (r = 0.234, P<0.01) (Table 2).


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  		TABLE 2. Correlation between the bone resorption markers and the disease activity in patients treated with infliximab

 

   Discussion
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
References
 
NTX and DPD have been validated as useful markers of bone resorption, and BAP as a useful marker of bone formation and an early marker of osteoblast differentiation. Elevated concentrations of bone metabolism markers can reflect several pathophysiological processes that occur in RA, including activity of the inflammatory process or a high rate of bone turnover, as occurs in osteoporosis, osteoarthritis and erosive joint destruction.

In the present study, disease activity ESR, CRP, the numbers of swollen joints and tender joints, and mHAQ score decreased rapidly after the initial treatment; they were significantly decreased 6 weeks after the initial treatment and were still low 6 months after the initial treatment. The same can be said of the bone resorption marker NTX. NTX levels were significantly decreased 6 weeks after the initial treatment and were still low 6 months after the initial treatment, whereas DPD levels were decreased significantly for the first time 6 months after the initial infusion. The NTX level was statistically associated with the number of swollen joints and mHAQ score. The DPD level was statistically associated with ESR. Several studies [15–17] have indicated that weekly low-dose MTX therapy improves markers of bone resorption and might also have a bone-protective effect by controlling disease activity. Vis et al. [18] have suggested that the same theory could be applied to patients with RA treated with infliximab. However, in our study there were two patients whose decrease in RA activity did not parallel the decrease in the markers of bone resorption. In one patient, NTX levels decreased from 50.1 nM BCE/mM Cr before treatment to 35.1 nM BCE/mM Cr 6 weeks after the initial treatment (a decrease of 30%), and in another patient the NTX level decreased from 59.1 nM BCE/mM Cr before treatment to 27.3 nM BCE/mM Cr 6 weeks after the initial treatment (a decrease of 52%); in both patients CRP and ESR levels remained high. These results may support reports [19, 20] in which the suppressive effect on bone destruction was seen in the non-responder RA group in the infliximab therapy.

The mechanisms causing joint damage and functional impairment in RA are complex and involve the association of many pro-inflammatory mediators and degradative enzymes. The proinflammatory cytokines TNF-{alpha}, IL-1 and IL-6 have been suggested to play an important role in the pathogenesis of RA [10, 11]. Among RA patients, the markers of cartilage and collagen degradation exhibit positive correlations with levels of TNF-{alpha} and IL-6, suggesting that these cytokines are associated with cartilage and bone degradation by synovial inflammation. The bone loss in RA may occur as a result of increased production of receptor activator of nuclear factor {kappa}B ligand (RANKL) in the bone marrow, which activates osteoclasts to resorb bone and cause erosions [21, 22]. Furthermore, when osteoblasts are stimulated by TNF-{alpha}, it guides the expresion of RANKL. TNF-{alpha} directly activates the macrophages, which are the antecedents of osteoclasts, and finally guides the differentiation of macrophages into osteoclasts [21, 23–25]. Thus, the inhibition of TNF-{alpha} by infliximab may decrease the production of RANKL and prevent macrophages from differentiating into osteoclasts, and subsequently may result in a decrease in urinary NTX levels.

Although NTX levels showed a significant rapid on bone resorption markers, which were decreased significantly 6 weeks after the initial treatment, DPD levels were not significantly decreased 6 weeks after the initial treatment. Greenspan et al. [26] and Ravn et al. [27] have indicated that NTX is a more sensitive marker than DPD when measuring bone-derived type I collagen fragment. Furthermore, NTX changes earlier than any other marker of bone resorption after therapy. Because we examined subjects comparatively soon after the initial treatment with infliximab, DPD showed little significant decrease at 6 weeks after therapy.

BAP levels were not significantly changed in the present study. In RA, there was conflicting data about the effect on bone formation by the administration of DMARDs. Data on both increased and decreased changes in the levels of bone formation markers in patients with RA after treatment with DMARDs have been reported [28, 29]. Although in the present study bone formation was not affected by the blockade of TNF-{alpha} activity, because osteoblast activity may change long after the initial treatment with infliximab, long-term follow-up is needed. Several studies support a delayed effect on bone formation. Reduction in bone formation was seen only several weeks after antiresorptive therapy with bisphosphonates in post-menopausal women or with MTX in RA [30].

In conclusion, the present study showed that infliximab treatment for RA not only reduces clinical disease activity, as already demonstrated in recent trials, but may also ameliorate bone resorption. Urinary NTX may be a more specific marker of bone-derived type 1 collagen fragments than urinary DPD. It should be noted that our study design has some limitations. For example, it was not conducted in a longitudinal fashion, patients were not classified by sex, and in this study steroid use might have had some effect on bone metabolism markers in patients with RA, although the amount of steroid had been fixed during the treatment with infliximab combined with low-dose MTX or by low-dose MTX alone. Therefore, further longitudinal studies with a large number of subjects may be needed to clarify the pathogenesis of RA-related bone loss.

The authors have declared no conflicts of interest.


   References
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

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Submitted 30 May 2005; revised version accepted 29 November 2005.
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